(en)An improved method (process) and medicinal composition is provided to treat herpes, pseudomonas, hepatitis, staph (staphylococci) and other infectious diseases The inexpensive medicinal composition can be self-administered and maintained for a prescribed time. The attractive medicinal composition can comprise a quaternary ammonium salt surfactant, a skin protectant and an alcohol. The quaternary ammonium salt surfactant can comprise benzalkonium halide, preferably benzalkonium chloride. The skin protectant can comprise Allantoin. The alcohol can serve as a pain reliever and can comprise benzyl alcohol. The medicinal composition can also include other compounds, additives, herbal extracts and/or carriers.

1.ApplicationNumber: US-24840208-A
1.PublishNumber: US-2009191288-A1
2.Date Publish: 20090730
3.Inventor: SQUIRES MERYL J.

4.Inventor Harmonized: SQUIRES MERYL J(US)

5.Country: US
6.Claims:
(en)An improved method (process) and medicinal composition is provided to treat herpes, pseudomonas, hepatitis, staph (staphylococci) and other infectious diseases The inexpensive medicinal composition can be self-administered and maintained for a prescribed time. The attractive medicinal composition can comprise a quaternary ammonium salt surfactant, a skin protectant and an alcohol. The quaternary ammonium salt surfactant can comprise benzalkonium halide, preferably benzalkonium chloride. The skin protectant can comprise Allantoin. The alcohol can serve as a pain reliever and can comprise benzyl alcohol. The medicinal composition can also include other compounds, additives, herbal extracts and/or carriers.

7.Description:
(en)CROSS REFERENCES TO RELATED APPLICATIONS
This application is a continuation-in-part of U.S. patent application Ser. No. 11/202,986 filed on Aug. 12, 2005, U.S. Patent Application Publication No. US 2006/0024393A1, published Feb. 2, 2002, which in turn is a continuation of U.S. patent application Ser. No. 10/084,759 filed on Feb. 26, 2002, now U.S. Pat. No. 7,071,233 B2, issued on Jul. 4, 2006, which in turn is a continuation of U.S. Ser. No. 08/824,041 filed on Mar. 26, 1997, now U.S. Pat. No. 6,350,784 B1, issued Feb. 6, 2002, which in turn is a continuation-in-part of U.S. Ser. No. 08/646,988 filed on May 8, 1996, now U.S. Pat. No. 6,355,684 B1, issued Mar. 12, 2002, which in turn is a continuation-in-part of U.S. Ser. No. 08/600,217, filed on Feb. 12, 1996, now U.S. Pat. No. 6,348,503 B1, issued Feb. 19, 2002.


BACKGROUND OF THE INVENTION
The present invention relates to medicinal compositions, and more particularly, to medicinal treatments and preventions to treat or alleviate cold sores, rashes or other skin conditions resulting from herpes, pseudomonas, staphylococcus (staph), hepatitis, or other infectious diseases.
Cold sores, also known as fever blisters, are clusters of small blisters on the lip and outer edge of the mouth. They are called cold sores or fever blisters because they often occur during a viral illness, such as a cold. Cold sores are small, painful, fluid-filled blisters on the mouth or nose. The skin around the blisters is often red and inflamed. The blisters can break open, weep a clear fluid, and then scab over after a few days. Cold sores usually appear on the lips. Occasionally, they occur on the nostrils, chin or fingers. Sometimes, cold sores can occur inside the mouth, but typically on the gums or hard palate or roof of the mouth.
Cold sores are caused by a viral infection or a virus known as herpes simplex type I (HSV-1) that attacks the skin and nervous system. Cold sores are the most common manifestation of a herpes simplex virus infection. Fever blisters are caused more often by the herpes simplex virus type 1 (HSV-1) than herpes simplex virus type 2 (HSV-2). HSV-1 is different from herpes simplex type II (HSV-2), which is the virus that causes the sexually transmitted disease known as genital herpes. Herpes simplex virus type 2 is usually responsible for genital herpes. However, either type of the herpes virus can cause sores in the facial area or on the genitals.
After the first episode of cold sores or fever blisters, the herpes virus lies dormant in the nerves or skin around the original area until something sets the virus off into another eruption. Herpes simplex virus can be reactivated in response to various stimuli including UV radiation from sun exposure, stress, a cold, illness, or dental work. Fever and menstruation can also trigger a recurrence of cold sores.
Cold sores or fever blisters are contagious and can be spread by direct contact with infected saliva or droplets in the breath, or by skin to skin contact. Cold sores can be spread through coughing, sneezing, direct contact with a cold sore, or touching contaminated fluid—such as from kissing an infected person and touching the person's saliva. Cold sores can also be spread from one person to an area of someone else's body by touching an active cold sore or sharing things that a person who has a cold sore has used. Shared eating utensils, razors and towels can spread the infection of cold sores. A parent who has a cold sore can spread the infection to their child. The greatest risk of infection of cold sores is from the time the blisters appear until they have completely dried and crusted over. There is a possibility of spreading the virus from cold sores even after the skin has healed.
People with cold sores should avoid close contact with infants, anyone who has eczema (atopic dermatitis) or people with a suppressed immune system, such as people with cancer, AIDS or an organ transplant. These people are at higher risk of more severe infection.
If the sore appears on the soft tissues inside the mouth, it may be a canker sore. Cold sores or fever blisters and are quite different from canker sores, a condition people sometimes associate them with. Cold sores are caused by the herpes simplex virus and they're contagious. Canker sores, which aren't contagious, are ulcers that occur in the soft tissues inside your mouth, places where cold sores don't occur. Cold sores are common occurrences although cold sores may not be completely curable or prevented, medicinal treatments can help reduce their frequency and limit the duration of their occurrence.
Complete healing of cold sores or fever blisters can take 7 to 10 days or more. For some people, however, cold sores can be painful. Initial symptoms can include mouth soreness, fever, sore throat, or swollen lymph glands. Small children sometimes drool before a cold sore appears. After the blisters develop, cold sores usually break open, weep clear fluid, and then crust over and disappear after several days to a week.
Cold sores follow the usual course of herpes simplex virus infections in that the first outbreak is usually more painful and lasts longer than recurrent infections. The first time the skin in or around the mouth comes in contact with the herpes simplex virus; the outbreak occurs inside the mouth on the gums, tongue, and throat. This is called gingivostomatitis. This first infection occurs most often in childhood, and the highest incidence of infection occurs between 6 months and three years of age. Children get pain, fever, swollen lymph nodes, and can have difficulty swallowing. These symptoms last for about a week and resolve spontaneously. Children with gingivostomatitis are at risk for dehydration if the pain keeps them from drinking fluids.
Once a person has been exposed to the herpes simplex virus, the virus remains in the cells in the body and can reactivate at any time. Reactivation can be triggered by trauma to the skin, menstruation, sun exposure, stress, fever, and other causes. This reactivation produces the lesions we know as cold sores or fever blisters. Fever blisters are most often seen on the border of the lip and consist of three to five vesicles. Over the next three to five days, the vesicles can become pustular, ulcerative, and then crust over. Symptoms are usually most severe 8 hours after the outbreak. Many people have about two outbreaks per year, but some people have greater than six outbreaks per year.
Recurrent infections of cold sores are often preceded by a prodrome, symptoms that appear before the outbreak occurs. Common prodromal symptoms for cold sores or fever blisters are pain, tingling, and burning. A herpes prodrome can last from two hours to two days. The first symptoms that can appear during an outbreak of cold sores may include tingling, burning, or itching in the area around the mouth or nose. This first portion of the outbreak is known as the prodromal stage or period. Within a few hours to days, the area may become reddened and develop small fluid-filled blisters. Several of these small blisters may even come together and form one large blister.
Signs and symptoms of cold sores can include: small, painful, fluid-filled blisters on a raised, red, painful area of the skin. Pain or tingling, called the prodrome, often precedes the blisters by one to two days. Signs and symptoms may not start for as long as 20 days after exposure to the herpes simplex virus, and usually last seven to 10 days. The blisters form, break and ooze. Then a yellow crust forms and finally sloughs off to uncover pinkish skin that heals without a scar.
Once a person has had an episode of cold sores, the virus lies dormant in the nerve cells in the skin and may emerge again as an active infection at or near the original site. The person may experience an itch or heightened sensitivity at the site preceding each attack.
The herpes simplex virus that causes cold sores cannot be cured. If sores develop regularly, treatment can reduce the number and severity of outbreaks. Herpes simplex infection of the eye can cause scarring of the cornea and is a leading cause of blindness in the United States.
Herpes simplex virus (HSV) commonly referred to as “herpes virus” or “herpes,” is an infectious disease which also has reached crisis proportions nationally with estimated numbers of infected people at 70%-80% of our population as reported by the American Societal Health Association (ASHA) and growing annually by 500,000 people. There are two common types of herpes: herpes simplex virus 1 (HSV 1) and herpes simplex virus 2 (HSV 2). Herpes enters the human body through minuscule breaks in the epidermal tissue usually by contact with an infected host and is marked by eruption of one or more vesicles, usually in groups, following an incubation period of approximately four days. Typically the course of the infectious outbreak initiates with the prodromal stage; advancing to vesicular eruption; followed by ulceration; coalescing; resolution; and the latency period. The outbreak can last for several weeks and on average lasts two-three weeks. In some immune compromised individuals the outbreak can last for months. The vesicles can appear anywhere on the skin or mucosa, typically appearing on the lips as cold sores, glands, oral mucosa, conjunctiva and cornea, genitalia, anal mucosa and peri-anal tissue.
Herpes symptoms include: inguinal swelling, pain, fever, malaise, headaches, muscle aches, and swollen glands. Some individuals who have the trigeminal nerve compromised with oral herpes, have excruciating facial pain, difficulty swallowing, eating and facial swelling. Individuals with the sacral nerve affected have severe upper leg pain, swelling, and great difficulty walking.
Herpes simplex virus (HSV) infection is recrudescent, residing in the nerve ganglia, and then recurring due to some, as yet unknown, stimulus. Recurrent herpetic infections can be precipitated by almost anything, including: overexposure to sunlight; nutritional deficiencies; stress, menstruation; immunosuppression; certain foods; drugs; febrile illness; etc. Herpes virus can be isolated from cardiac tissue.
HSV 1 and HSV 2 infections pose very serious health threats and can cause causing: blindness; increased cancer risk of the cervix; aseptic meningitis and encephalitis; neonatal deaths; viremia; etc. The devastating effects of this disease go well beyond the medical scope of human suffering. HSV is responsible for serious psychological and emotional distress as well as substantial economic loss to the nation and the world.
Various treatments for herpes have been proposed and have included topical application of such agents as povodone-iodine, idoxuridine, trifluorothymidine, or acyclovir. Such treatments have met with varying degrees of success. Most prior treatments have proven disappointing. Acyclovir, taken orally for systemic treatment of HSV, is somewhat effective. However, acyclovir is only successful in interrupting the replication of the virus. It is not successful in treating an infectious outbreak either systemically or topically. Strains resistant to acyclovir have been reported. Individuals with Auto Immune Deficiency Syndrome (AIDS) are seriously immune-compromised and suffer especially debilitating outbreaks of HSV. Additionally, AIDS individuals may carry acyclovir resistant strains of HSV, which can make acyclovir ineffective for these individuals.
It has been reported that there are currently about 22 million people infected with human immunodeficiency virus (HIV) throughout the world. The largest proportion of new HIV cases has originated in Africa and the Caribbean. The typical progression of HIV infection is divided into different stages: 1) viral transmission; 2) acute retroviral syndrome; 3) seroconversion; 4) a clinical latent period with or without persistent generalized lymphadenopathy (PGL); 5) early symptomatic HIV infection previously known as AIDS-related complex or ARC and more recently referred to as “B symptoms” according to the 1993 CDC classification); 6) acquired immune deficiency syndrome (AIDS) (AIDS indicator condition according to the 1987 CDC criteria and revised 1993 CDC criteria that include a CD4 cell count <200/mm 3 ); and 7) advanced HIV infection characterized by a CD4 cell count <50/mm 3 . CD4 cells are lymphocytes targeted by HIV. In 1993 the CDC changed the definition of AIDS to include all patients with a CD4 count <200/mm 3 ; this definition includes patients in stages 4-7 regardless of symptoms.
The initial acute retroviral syndrome is accompanied by a precipitous decline in CD4 cell counts, high culturable plasma viremia, and high concentrations of HIV RNA in plasma. Clinical recovery occurs and high level HIV RNA plasma viremia is reduced with development of cytotoxic T lymphocyte (CPL) response. The CD4 cell count gradually declines over several years and then shows an accelerated decline at 1.5-2 years before an AIDS-defining diagnosis. HIV RNA concentrations in plasma are relatively stable until the HIV is in a late stage when the CD4 count is <200/mm 3 and the clinical course is characterized by infections, selected tumors, wasting, and neurological complications. Generally, about 10% of patients develop an AIDS-defining diagnosis before the CD4 count decreases to 200/mm 3 . The present median time to an AIDS-defining complication after the CD4 count is 200/mm 3 is 12-18 months. In the absence of therapy directed against HIV or PCP prophylaxis, the average time from viral transmission to an AIDS-defining diagnosis is about 10 years, and survival after an AIDS-defining complication was previously about one year.
The entire sequence of events for an average patient, in the absence of treatment directed against HIV, is approximately ten years from seroconversion to death. The median time from HIV seroconversion to AIDS has been reported to be about 7 years for transfusion recipients, 10 years for hemophiliacs, 10 years for drug users and 8-12 years for gay men. Rates of progression appear similar by sex, race, and risk category if adjusted for quality of care. For patients aged 16-24 years at seroconversion, the median time was 15 years; for those over 35 years at seroconversion, it was 6 years.
HIV infection can be acquired through sexual intercourse, from drug transfusions with contaminated blood, by drug addicts with infected needles, or by prenatal transmission. Symptomatic primary HIV infection, also referred to as an acute retroviral syndrome, has been reported in the preceding risk categories with a frequency of 50-90%. This syndrome has also been noted in seven of eight healthcare workers with HIV transmission following occupational exposure. The time from exposure to onset of symptoms is usually 2-4 weeks, but the incubation may be as long as six weeks. Typical symptoms are: fever, adenopathy, pharyngitis, rash comprising erythematous maculopapular with 5-10 mm lesions on the face and trunk, sometimes extremities including palms and soles or mucocutaneous ulceration on the mouth, esophagus or genitals, myalgias or arthralgias, diarrhea, headache, hepatosplenomegaly, thrush, nausea and vomiting. Neurologic symptoms can include: meningoencephalitis, peripheral neuropathy, facial palsy, Guillain-Barré syndrome, brachial neuritis, radiculopathy, cognitive impairment, and psychosis. The acute illness is generally accompanied by high level HIV viremia with p24 antigenemia, plasma viremia, and high titers of HIV in peripheral blood mononuclear cells.
The cytotoxic T lymphocyte (CTL) response is first and usually precedes detectable humoral response by several weeks. CTL response is accompanied by a 3-5 log decrease in HIV concentration in peripheral blood. The high level of viremia during this acute phase of the illness may be associated with dissemination of the virus to the CNS and lymphatic tissue. Lymph tissue serves as the major reservoir of HIV burden and replication. Infection of non-lymphoid organs with high levels of HIV appears to occur in late-stages of HIV.
The presence of symptoms rather than asymptomatic seroconversion as well as a prolonged illness greater than 14 days appear to correlate with more rapid progression to AIDS. Seroconversion with positive HIV serology generally takes place at 6-12 weeks following transmission such as by transfusion or needles injury to a healthcare worker. The median interval is 63 days. The CTL response is associated with a sharp reduction in quantitive viral load in blood, clinical recovery from the acute retroviral syndrome and return of the CD4 cell count to higher levels that are often in the normal range for most laboratories.
The HIV patient becomes clinically asymptomatic and generally has no findings on physical exam except for Persistent Generalized Lymphadenopathy (PGL) comprising enlarged lymph nodes. Studies of lymph nodes show high concentrations of HIV as extracellular virus trapped on the follicular dendritic cell processes within germinal centers and as intracellular virus predominantly in latent form. The lymph tissue serves as a major reservoir for HIV, the follicular dendritic cells filter and trap free virus and infected CD4 cells, and the viral burden in peripheral blood mononuclear cells is relatively low. With progressive disease, the lymph node configuration is disrupted by HIV.
Virologic studies in patients with asymptomatic HIV infection show high rates of HIV replication with production of an average of 10 9 virions daily. Viral replication is accompanied by massive destruction and the production of 10 9 CD4 cells daily. The turnover of CD4 cells represents 6-7% of the total body CD4 cells so that the entire supply turns over every 15 days. AIDS has been considered a consequence of continuous, high-level replication of HIV-1, leading to virus and immune-mediated termination of CD4 lymphocytes.
Advanced HIV Infection occurs in patients with a CD4 cell count of <50/mm 3 . These patients have limited life expectancy with a median survival of 12-18 months. Virtually all patients who die of HIV-related complications are in this CD4 cell count stratum.
The Food & Drug Administration (FDA) has approved many reverse transcriptase (RT) inhibitors. RT enzymes convert viral RNA into DNA. RT inhibitors can interrupt this process. The RT inhibitor AZT, which is sold under the brand names of Retrovir and zidovudine by Glaxo Wellcome, was approved by the FDA in 1987. The RT inhibitor ddI, which is sold under the brand names of Videx and didanosine by Bristol-Myers Squibb, was approved by the FDA in 1991. The RT inhibitor ddC, which is sold under the brand names of HIVID and dideoxycyytidine by Hoffman-LaRoche, was approved by the FDA in 1992. The RT inhibitor d4T, which is sold under the brand names of Zerit and stavudine by Bristol-Myers Squibb, was approved by the FDA in 1994. The RT inhibitor 3TC, which is sold under the brand names of Epivir and lamivundine by Glaxo Wellcome, was approved by the FDA in 1995. The TR inhibitor Nevirapine, which is sold under the brand name of Viramune by Boehringer Ingelheim, was approved by the FDA in 1996.
The Food & Drug Administration (FDA) has now approved three protease inhibitors for the treatment of human immunodeficiency virus (HIV) infection. Saquinavir sold under the brand name of Invirase by Hoffman-LaRoche Laboratories, was the first protease inhibiting agent to be approved by the FDA. Ritonavir, another protease inhibitor, which is sold under the brand name of Norvir by Abbott Laboratories, received FDA approval in March, 1996 as did Indinavir sold under the brand name of Crixivan by Merck & Co.
Protease inhibitors have a different mechanism of action from that of previously approved anti-HIV drugs, such as the nucleoside analogues AZT and 3TC sold under the brand names of zidovudine and lamivundine by Glaxo Wellcome, ddI and d4T sold under the brand names didanosine and stavudine by Bristol-Myers Squibb, and ddC sold under the brand name of dideoxycytidine by Roche Laboratories. Protease inhibitors block the enzyme which HIV requires for the completion of its replication cycle and formation of viable new viruses. Without the protease enzyme, viral structural proteins cannot be manufactured properly, and faulty, non-infectious virus is formed. The nucleoside analogues block a different enzyme-reverse transcriptase. This action can prevent viral RNA from producing viral DNA which can then incorporate into the DNA of human cells. Combining one or more reverse transcriptase inhibitors with a protease inhibitor, sometimes referred to as a “cocktail,” is claimed to attack HIV replication at two points in the replication cycle. Clinical trials combining saquinavir with AZT, ddC, or both demonstrate a greater decline in the number of HIV particles in the blood, sometimes referred to as viral burden, and a greater increase in CD4 cells (T lymphocytes) than previously observed with reverse transcriptase inhibitors alone. Sometimes, the cocktails have been toxic and ineffective for some patients. Clinical benefit in terms of improved survival or reduced disease progression rate, however, has not yet been fully demonstrated for combination (cocktails) of RT inhibitors and protease inhibitors. Physicians, however, are starting to consider HIV a chronic manageable disease rather than a death sentence.
Saquinavir protease inhibitors have been approved by the FDA for use in combination with reverse transcriptase inhibitors in patients with advanced AIDS. Saquinavir protease inhibitors may be tolerated by some patients without the hematologic or neurologic toxicities encountered with the nucleoside analogues. Certain prescription drugs including rifampin, rifabutin, phenobarbital, dilantin, and dexamethasone, may significantly decrease plasma levels of saquinavir protease inhibitors and should be avoided in patients taking saquinavir. Viral resistance to saquinavir protease inhibitors, as with other anti-HIV drugs has been reported.
Ritonavir and indinavir protease inhibitors appear to be more potent against HIV than the current formulation of saquinavir. Ritonavir protease inhibitors require refrigeration. Ritonavir protease inhibitors are currently used in combination with nucleoside analogues (drugs like AZT) or as monotherapy. An early study treated 32 patients with ritonavir plus AZT plus ddC. After 20 weeks, median CD4 cell counts rose from 83 cells/mm 3 at baseline to 106 cells/mm 3 . Viral load, a measure of the number of viral copies in the blood, decreased by almost 100-fold. Ritonavir is dosed at 600 mg orally twice a day, which can require twelve capsules each day. The drug is available in 100 mg capsules. Side effects are fairly common, including: gastrointestinal symptoms with nausea, vomiting, and diarrhea. Other side effects include numbness and tingling, particularly around the mouth, and liver inflammation comprising a form of hepatitis.
Indinavir protease inhibitors received accelerated FDA approval based on studies demonstrating mean rises in CD4 counts of about 100 cells/mm 3 and drops in viral load of almost 100-fold with a combination of AZT plus 3TC plus indinavir. Indinavir is dosed at 800 mg orally three times per day (2 capsules 3, times daily). In contrast to ritonavir, indinavir can be taken on an empty stomach to improve absorption. Indinavir causes fewer gastrointestinal side effects than ritonavir and seems to be better tolerated overall by some patients. The major side effect of Indinavir protease inhibitors are the development of kidney stones. The drug is partially excreted in the urine and it can crystallize to form stones if adequate hydration is not maintained. Indinavir protease inhibitors can also affect the liver, causing a rise in blood levels of bilirubin, i.e., a bile pigment formed from the breakdown of red blood cells. Indinavir protease inhibitors can also cause drug interactions.
Analysis of resistance to protease inhibitors has not been fully determined. Saquinavir and ritonavir protease inhibitors can currently cost the patient approximately U.S. $600 per month. Indinavir protease inhibitors is priced about 30% below this level. A three-drug combination of AZT plus 3TC plus ritonavir protease inhibitors can cost a patient over U.S $1,000/month. Combinations (cocktails) of RT inhibitors and protease inhibitors can cost as much as $25,000 per year. Although, protease inhibitors may be helpful, the medical community and society have not yet resolved patient cost problems for these expensive drugs.
Staphylococcus is a group of bacteria, also known as Staph or staph (pronounced “staff”), that can cause a multitude of diseases as a result of infection of various tissues of the body. Staphylococcus is a genus of nonmotile gram-positive spherical eubacteria of the family Micrococcaceae that occur singly, in pairs or tetrads, or in irregular clusters and can comprise parasites of skin or parasites of mucous membranes. Staphylococci are the plural of Staphylococcus.
Staph (Staphylococcus) is usually on a person's skin or in their nose or throat. Most of the time, the Staphylococcus bacteria cause no problems or relatively minor skin infections. When the skin is punctured or broken, staph bacteria can enter the wound and cause infections. When staph bacteria enter the bloodstream and spread to other organs, a number of serious infections can occur.
The name Staphylococcus comes from the Greek staphyle meaning a bunch of grapes and kokkos meaning berry, and that is what staph look like under the microscope, like a bunch of grapes or little round berries. Over 30 different types of Staphylococci (staph) can infect humans, but most infections are caused by Staphylococcus aureus.
Staph bacteria cause skin infections, including boils; cellulites, a potentially life-threatening infection that leaves skin inflamed and tender; impetigo, a rash common in young children and infants; and scalded skin syndrome, a serious, blistering condition that mainly affects newborns. Though each condition has specific features, most begin with skin redness, swelling, warmth, tenderness and sometimes fever. Some staph infections are localized; others can cover your entire body.
Staph bacteria can cause illness not only directly by infection such as in the skin, but also indirectly by producing toxins. Staph-related illness can range from mild and requiring no treatment to severe and potentially fatal. However, staph infections can turn deadly if the bacteria burrow deeper into the body, invading the bloodstream, urinary tract, lungs, and/or heart. In the past, most lethal staph infections occurred in people who were hospitalized or had a chronic illness or weakened immune system.
Staph infections can range from minor skin problems to food poisoning, fatal pneumonia, surgical wound infections and endocarditis, a life-threatening inflammation of the heart lining. As a result, signs and symptoms of staph infections vary widely, depending on the location and severity of the infection and on whether the illness results from direct infection with staph bacteria or from toxins the bacteria produce, but most infections are accompanied by fever, chills and sweating.
Staphylococci or staphylococcus (staph) can be found normally in the nose and on the skin many healthy adults. In the majority of cases, the bacteria do not cause disease. However, damage to the skin or other injury may allow the bacteria to overcome the natural protective mechanisms of the body, leading to infection. Anyone can develop a staph infection, although certain groups of people are at greater risk, including: patients in hospitals, newborn infants, breastfeeding women, and people with chronic conditions such as diabetes, cancer, vascular disease, and lung disease. Drug users, people with skin injuries or disorders, intravenous catheters, surgical incisions, and people with a weakened immune system all have an increased risk of developing staph infections.
Staphylococcal disease (staph) of the skin usually results in a localized collection of pus, known as an abscess, boil, or furuncle. The area of the skin affected by staph can become red, swollen, and painful. Staph infections of the skin can progress to impetigo causing a crusting of the skin or cellulites causing inflammation of the connective tissue under the skin and leading to swelling and redness of the area. In some cases, a serious complication known as scalded skin syndrome can develop. In breastfeeding women, Staph can result in mastitis resulting in inflammation of the breast or an abscess of the breast. Staphylococcal breast abscesses can release bacteria into the mother's milk.
Staphylococcal pneumonia (staph) predominantly affects people with underlying lung disease and can lead to abscess formation within the lungs. Infection of the heart valves (endocarditis) can lead to heart failure. Spread of Staphylococci or staphylococcus (staph) to the bones can result in severe inflammation of the bones known as osteomyelitis. Taphylococcal sepsis is a widespread infection of the bloodstream and is a leading cause of shock and circulatory collapse, leading to death, in people with severe burns over large areas of the body.
Toxic shock syndrome is an illness caused by toxins secreted by Staph aureus bacteria growing under conditions in which there is little or no oxygen. Toxic shock syndrome is characterized by the sudden onset of high fever, vomiting, diarrhea, and muscle aches, followed by low blood pressure or hypotension, muscle aches, seizures and headache which can lead to shock and death. There may be a rash resembling sunburn, with peeling of skin. Toxic shock has been linked to the use of certain types of tampons and less often, to skin wounds and surgery.
Staphylococcal (staph) food poisoning is an illness of the bowels that causes nausea, vomiting, diarrhea, and dehydration. It is caused by eating foods contaminated with toxins produced by Staphylococcus aureus . Symptoms usually develop within one to six hours after eating contaminated food and include abdominal cramps, nausea, vomiting and diarrhea. The illness usually lasts for one to three days. Patients with this illness are not contagious, since toxins are not transmitted from one person to another. The illness can be more serious and last longer in children and older adults.
Septic arthritis is often caused by a staph infection. The bacteria usually target the knees, but other joints can be affected, including the ankles, hips, wrists, elbows and shoulders. Staph arthritis can cause swelling and severe pain in the affected joint along with fever and shaking chills.
Community-associated methicillin-resistant staphylococcus aureus (CA-MRSA) can cause skin infections or a deadly pneumonia. Signs and symptoms, which are similar to those of other types of pneumonia, include cough, shortness of breath and chest pain, but the bacteria also cause massive inflammation and destruction of lung tissue.
In cases of minor skin infections, staphylococcal infections are usually diagnosed by their appearance without the need for laboratory testing. More serious staphylococcal infections such as infection of the bloodstream, pneumonia, and endocarditis require culturing of samples of blood or infected fluids. The laboratory establishes the diagnosis and performs special tests to determine which antibiotics are effective against the bacteria.
A serious concern is when staph infections no longer respond to common antibiotics. Although most staph infections can still be successfully treated, methicillin-resistant staphylococcus aureus (MRSA) is a particularly dangerous and drug-resistant form of staph infection called MRSA appeared in hospitals. MRSA infections often begin as a superficial skin problem that can resemble a pimple or spider bite, but which can quickly turn into a deep, painful abscess that requires surgical draining.
Pseudomonas infections are caused by a bacterium Pseudomonas aeruginosa , which is present throughout the world in soil, water, and on the skin of animals and people. Pseudomonas aeruginosa favors moist areas, such as sinks, toilets, pools, and hot tubs, and usually can withstand standard levels of pool chlorination.
Pseudomonas infection can be sudden and severe, or slow in onset and cause little pain. Pseudomonas infections can infect the blood, skin, bones, ears, eyes, urinary tract, heart valves, and lungs. In most cases, however, pseudomonas infections strike only persons who are very ill, usually hospitalized. Pseudomonas infections rarely cause disease in healthy persons, but can infect those who are already sick or who have weakened immune systems. Pseudomonas aeruginosa are opportunistic pathogens or organisms that do not ordinarily cause disease, but multiply freely in persons whose immune systems are weakened by illness or medication. Patients with AIDS have an increased risk of developing serious pseudomonas infections. Hospitalized patients are another high-risk group, because Pseudomonas aeruginosa are often found in hospitals. Pseudomonas infections can be spread within hospitals by health care workers, medical equipment, sinks, disinfectant solutions, and food. People with diabetes are particularly prone to pseudomonas infections. Furthermore, Pseudomonas aeruginosa is the second most common cause of nosocomial pneumonia and the most common cause of intensive care unit (ICU) pneumonia. Risk factors for acquiring a pseudomonas infection also include having a serious illness or undergoing an invasive procedure such as surgery.
Pseudomonas aeruginosa is a common cause of bacterial infections of the blood and is common in patients with blood cancer and patients who have pseudomonas infections elsewhere in the body. Pseudomonas aeruginosa can infect the heart valves of intravenous drug abusers and persons with artificial heart valves. Persons at risk for pseudomonas infections of the bones and joints include diabetics, intravenous drug abusers, and bone surgery patients. Pseudomonas infections of bones and joints result from direct inoculation of the bacteria or the hematogenous spread of the bacteria from other primary sites of infection. Pseudomonas aeruginosa has a particular tropism for fibrocartilagenous joints of the axial skeleton. Pseudomonas aeruginosa causes chronic contiguous osteomyelitis, usually resulting from direct inoculation of bone, and is the most common pathogen implicated in osteochondritis after puncture wounds of the foot. Pseudomonas aeruginosa can cause inflammation of the tissues covering the brain and spinal cord resulting in meningitis and brain abscesses.
Pseudomonas aeruginosa can cause infections in the external ear canal and is sometimes referred to as swimmer's ear. Pseudomonas aeruginosa can cause serious ear infections in elderly patients, and can cause hearing problems, facial paralysis, or even death. Pseudomonas infections of the eye usually follow an injury and can cause ulcers of the cornea that can cause rapid tissue destruction and eventual blindness. The risk factors for pseudomonas eye infections include: wearing soft extended-wear contact lenses; using topical corticosteroid eye medications; being in a coma; having extensive burns; under-going treatment in an ICU; and having a tracheostomy or endotracheal tube.
Even healthy persons can develop a pseudomonas skin rash following exposure to the bacterium in contaminated hot tubs, water parks, whirlpools, or spas. This skin disorder is also known as pseudomonas skin rash or “hot tub” folliculitis. Pseudomonas blood-borne infections are most often seen in IV drug users. Pseudomonas aeruginosa often invades the blood of people with burns and those who have cancer. Pseudomonas aeruginosa can further cause urinary tract infections, usually in people who have had urologic procedures or those who have an obstruction of the urinary tract. Without treatment, an overwhelming infection can lead to dangerously low blood pressure (shock) and death.
Each of the pseudomonas infections described previously has its own set of symptoms. Pseudomonas bacteremia can cause fever, tiredness, muscle pains, joint pains, and chills. Pseudomonas bone infections can cause welling, redness, and pain at the infected site and possibly fever. Pseudomonas meningitis can cause fever, headache, irritability, and clouded consciousness. Pseudomonas ear infections can be associated with pain, ear drainage, facial paralysis, and reduced hearing. Pseudomonas eye infections can cause ulcers that may spread to cover the entire eye, pain, reduced vision, swelling of the eyelids, and pus accumulation within the eye. Pseudomonas pneumonia can cause chills, fever, productive cough, difficult breathing, and blue-tinted skin. Patients with cystic fibrosis with pseudomonas lung infections can experience coughing, decreased appetite, weight loss, tiredness, wheezing, rapid breathing, fever, blue-tinted skin, and abdominal enlargement. Pseudomonas skin infections can cause a range of symptoms from a mild rash to large bleeding ulcers. Pseudomonas folliculitis can include a red itchy rash, headache, dizziness, earache, sore eyes, nose, and throat, breast tenderness, and stomach pain.
Hepatitis is typically caused by hepatitis viruses and can result in inflammation of liver cells and injury to the liver. Hepatitis can also be caused by infections, pregnancy, alcohol, or from herpes simplex virus (HSV), Cytomegalovirus, Epstein-Barr, yellow fever virus, adenoviruses, or Systemic Lupus Erythematosus (SLE). Hepatitis can further be caused from toxins, such as Amanita toxin in mushrooms, carbon tetrachloride, or asafetida. Moreover, hepatitis can also be caused by drugs, such as Paracetamol, amoxycillin, anti tuberculosis medicines, minocycline, methyldopa, nitrofurantoin, isoniazide, or ketoconazole.
Mild hepatitis can heal on its own or can progress to more serious hepatitis resulting in scarring of the liver. Acute hepatitis lasts less than six months. Chronic hepatitis lasts longer than six months. Hepatitis can run a subclinical course in which the affected person may not feel ill. However, when the affected person feels sick and symptomatic, hepatitis can impair functions of the liver that include screening of harmful substances, regulation of blood composition, and production of bile to help digestion.
Clinically, the course of acute hepatitis can vary widely from mild symptoms requiring no treatment to fulminant hepatic failure needing liver transplantation. Acute viral hepatitis is more likely to be asymptomatic in younger people. Initial features of acute viral hepatitis can include nonspecific flu-like symptoms, common to almost all acute viral infections and may include malaise, muscle and joint aches, fever, nausea or vomiting, diarrhea, and headache. More specific symptoms, which can be present in acute hepatitis, are: profound loss of appetite, aversion to smoking among smokers, dark urine, yellowing of the eyes and skin, i.e. jaundice, and abdominal discomfort.
For chronic hepatitis, most patients remain asymptomatic or mildly symptomatic. Some chronic hepatitis patients experience symptoms related to acute hepatitis, as well as abdominal fullness from an enlarged liver or spleen, low grade fever and fluid retention. Jaundice can indicate extensive liver damage. Chronic hepatitis patients can have extensive damage and scarring of liver with cirrhosis leads to weight loss and bruising and bleeding tendencies. Chronic hepatitis patients can also have acne, abnormal menstruation, and lung scarring, as well as inflammation of the thyroid gland and kidneys.
Hepatitis A is an infectious jaundice that is caused by a picornavirus. Hepatitis A is often associated with ingestion of contaminated food or with anal or oral sex. It can cause an acute form of hepatitis and does not have a chronic stage. The patient's immune system makes antibodies against hepatitis A that can provide immunity against future infection. People with hepatitis A are advised to rest, stay hydrated and avoid drinking alcoholic beverages. A vaccine is available that will prevent infection from hepatitis A for life. Hepatitis A can be spread through personal contact, consumption of raw sea food or drinking contaminated water. This occurs primarily in third world countries. The time between the infection and the start of the hepatitis A illness can be from 15 to 45 days. Many sufferers of hepatitis A have relapsing symptoms from 6 months to a year following initial diagnosis of hepatitis A.
Hepatitis B is caused by hepatitis B virus (HBV). Cirrhosis of the liver and liver cancer can result from hepatitis B. Transmission of hepatitis B virus (HBV) can result from exposure to infectious blood or body fluids containing blood. Other types of transmission of hepatitis B virus (HBV) can occur from unprotected sexual contact, blood transfusions, re-use of contaminated needles and syringes, and transmission from mother to child during childbirth. A mother who is positive for the hepatitis B surface antigen has a risk as high as 90% if the mother is also positive for the hepatitis B e antigen. HBV can also be transmitted between people by contact of skin or mucous membrane with secretions or saliva containing HBV.
The primary method of transmission reflects the prevalence of chronic hepatitis B virus (HBV) infection in a given area. In low prevalence areas such as the continental United States and Western Europe, where less than 2% of the population is chronically infected with HBV, HBV is typically acquired from injection drug abuse and unprotected sex are the primary methods. In Eastern Europe, Russia, and Japan, where 2-7% of the population is chronically infected, HBV is predominantly spread among children. In high prevalence areas such as China and South East Asia, transmission of HBV often occurs during childbirth.
Hepatitis B virus (HBV) infection can be prevented by vaccination. Infants born to mothers known to carry hepatitis B can be treated with antibodies to the hepatitis B virus (hepatitis B immune globulin or HBIg). When given with the vaccine within 12 hours of birth, the risk of acquiring hepatitis B is reduced 95%. This treatment allows a mother to safely breastfeed her child. Although pharmaceutical products do not typically clear or resolve a hepatitis B infection, they can stop the hepatitis virus from replicating and prevent liver damage such as cirrhosis and liver cancer.
The hepatitis B virus (HBV) primarily interferes with the functions of the liver by replicating in liver cells, known as hepatocytes. During HBV infection, the host immune response causes both hepatocellular damage and viral clearance. Although the innate immune response does not play a significant role in these processes, the adaptive immune response, particularly virus-specific cytotoxic T lymphocytes (CTLs), can contribute to most of the liver injury associated with HBV infection. By killing infected cells and by producing antiviral cytokines capable of purging HBV from viable hepatocytes, CTLs can eliminate the virus. Although liver damage can be initiated and mediated by the CTLs, antigen-nonspecific inflammatory cells can worsen CTL-induced immunopathology, and platelets activated at the site of infection can cause the accumulation of CTLs into the liver.
Hepatitis C virus (HDV) is a blood-borne, infectious, viral disease that is caused by an ahepatotropic virus, known as Hepatitis C virus (HCV). Hepatitis C infection can cause liver inflammation (hepatitis) that is often asymptomatic, but ensuing chronic hepatitis can result later in cirrhosis causing fibrotic scarring of the liver and liver cancer. Hepatitis C virus (HCV) is spread by blood-to-blood contact with an infected person's blood. The symptoms can be medically managed and some patients can be cleared of the virus by a long course of anti-viral medicines. Although early medical intervention is helpful, people with HCV infection often experience mild symptoms, and consequently do not seek treatment. An estimated 150-200 million people worldwide are infected with HCV. In the U.S., those with a history of intravenous drug use, nasally inhaled drug usage, tattoos, or who have been exposed to blood via unsafe sex or social practices are increased risk for HCV. Hepatitis C virus is the leading cause of liver transplant in the United States.
Hepatitis D virus infection can only occur with a concomitant infection with Hepatitis B virus because the hepatitis D virus uses the Hepatitis B virus surface antigen to form a capsid.
In view of the preceding, it is, therefore, desirable to develop safe and successful method (process) and medicinal composition to treat herpes, pseudomonas, hepatitis, staph (staphylococci) and other infectious diseases.
SUMMARY OF THE INVENTION
An improved method (process) and medicinal composition is provided to treat herpes, pseudomonas, staph (staphylococci), hepatitis, and other infectious diseases. Significantly, the improved method and medicinal composition are safe, economical and effective.
The inventive medicinal composition comprises a quaternary ammonium salt surfactant, a skin protectant and alcohol which provide active ingredients that cooperate with each other in the medicinal composition to alleviate cold sores, rashes or other skin conditions resulting from herpes, pseudomonas, staph, hepatitis, or other infectious diseases. The skin protectant can comprise Allantoin. The alcohol can serve as a pain reliever and can comprise benzyl alcohol and/or isopropyl alcohol.
The quaternary ammonium salt surfactant can comprise one or more of the following: alkyl dimethylbenzylammonium chloride, benzalkonium bromide, benzalkonium chloride, benzalkonium fluoride, alkylbenzyldimethylammonium chloride, alkyldimethylbenzylammonium chloride, n-alkyldimethylbenzylammonium chloride, diisobutylphenoxyethoxethyl dimethylammonium chloride, n-dimethylbenzylammonium chloride, octyldecyldimethylammonium chloride, didecyldimethylammonium chloride, dioctyldimethylammonium chloride, dialkyldimethylammonium chloride, octyldecyldimethylammonium chloride, laurryl dimethylbenzylammonium chloride, o-benzyl-p-chlorophenol, diethyldimethylammonium chloride, doctyldimethylammonium chloride, alkyldimethylbenzylammonium chloride, and alkylbenzyldimethylammonium chloride.
Preferably, the quaternary ammonium salt surfactant comprises benzalkonium halide comprising one or more of the following: benzalkonium chloride, benzalkonium bromide, and/or benzalkonium fluoride. Most preferably, the quaternary ammonium salt surfactant comprises benzalkonium halide and the skin protectant comprises Allantoin. Advantageously, (1) benzalkonium chloride or benzalkonium halide, (2) Allantoin and (3) benzyl alcohol cooperate with each other in the medicinal composition to alleviate cold sores, rashes or other skin conditions resulting from herpes, pseudomonas, staph, hepatitis, or other infectious diseases.
The herpes causing the cold sores, rashes or other skin conditions can comprise: herpes simplex virus 1 (HSV 1), herpes simplex virus 2 (HSV 2), staphylococci (staph), varicella zoster virus (herpes zoster) (shingles), cytomegalovirus, herpetic keratitis, conjunctivitis, human immunodeficiency virus infection (HIV), viral influenza (flu), epstein barr, papilloma virus, viral parainfluenza, adenovirus, viral encephalitis, viral meningitis, arbovirus, arenavirus, picornavirus, coronavirus, syntialvirus, viral infections, roseola infantum, pneumonia, mononucleosis, uveitis, retinitis, human cervical carcinoma, vaginal carcinoma, vulvovaginitis, human herpes IV, Kaposi's sarcoma, cytomegalovirus, or common colds caused by viruses or herpes.
The hepatitis causing the rashes or other skin conditions can comprise hepatitis B virus (HBV) and hepatitis C virus (HCV).
In one preferred embodiment, the medicinal composition comprises by weight: from about 0.01% to about 0.8% by weight aqueous or dry benzalkonium chloride, from about 0.01% to about 99% Allantoin, and from about 0.01% to about 99% benzyl alcohol.
The medicinal composition can also comprise one or more of the following additives or ingredients: water, preferably, sterile (purified) water; methyl cellulose; methyl paraben; potassium sorbate; and/or propyl praben.
The medicinal composition can comprise a formulation without Echinacea or other herbal extracts or herbaceous botanicals. In come circumstances, it may be desirable that the medicinal composition further includes herbal extracts or portions of Echinacea purpurea , such as from about 2% to about 90% by weight of the medicinal composition, preferably, if used, from about 40% to about 60% by weight of the medicinal composition. Significantly, the preferred medical composition and methods for it use preferably excludes undesired Myrtle and/or Centaurea and their extracts, as well as Echinacea angustofolia , aedurid, sodium hydroxide (NaOH), tea tree oil, lapacho extract, licorice root extract, arabinose, betaine, cellulose, copper, fructose, fatty acids, galactose, glucose, iron, potassium, protein, resin, sucrose, and xylose, which can inhibit or retard the unexpected advantageous results of the novel medicinal composition.
A preferred method (process) to treat or alleviate cold sores, rashes or other skin conditions resulting from herpes, pseudomonas, hepatitis, or other infectious diseases, comprises the steps of: substantially inhibiting cold sores, rashes or other skin conditions resulting from herpes, pseudomonas, staph, hepatitis, or other infectious diseases, by applying a medicinal composition on the cold sores, rashes or other skin conditions, or infected regions; and maintaining said composition on the cold sores, rashes, skin conditions, or infected regions until external symptoms and physical manifestations resulting from herpes, pseudomonas, staph, hepatitis, or other infectious diseases substantially disappear about the cold sores, rashes, skin conditions, or infected regions.
The medicinal composition can be applied topically, externally, internally, or orally to a patient, (person, homo sapien or human being) or to an animal, such as a dog, cat, bird, horse, cow, sheep, swine, other farm animal or rodent, to treat or alleviate cold sores, rashes or other skin conditions resulting from herpes, pseudomonas, staph (staphylococci or staphylococcus ), hepatitis, or other infectious diseases.
Desirably, the medicinal composition is applied topically to the cold sores, rashes, skin conditions, or infected regions by: spraying, dabbing, dusting, swabbing, sponging brushing, pouring, dispensing, covering and/or coating. The infected regions can be a patient's lips, mouths, oral mucosa, nasal mucosa, vaginal tissue, labial tissue, anal tissue, periacinal tissue, cutaneous tissue, ocular tissue, conjunctive, and/or eye lids.
In one preferred embodiment, the external symptoms and physical manifestations of cold sores can substantially disappear in about one day or less after the medicinal composition is applied to the cold sores. Desirably, the external symptoms and physical manifestations of vesicular eruption, rash or other skin conditions resulting from herpes simplex virus are resolved in about 19 hours to about 24 hours after the composition is applied to the vesicular eruption, rash or other skin condition.
The method can also comprise: conditioning and treating an active phase herpes lesion on skin or mucosa resulting from herpes of a person (human being) (homo sapien) by sequentially moistening and powdering said active phase herpes lesion. Desirably, moistening comprises wetting the active phase lesion on skin or mucosa with an aqueous solution of the medicinal composition in the absence of and without the following compounds, ingredients or additives: aedurid, sodium hydroxide (NaOH), tea tree oil, lapacho extract, licorice root extract, Myrtle and/or Centaurea and their extracts, arabinose, betaine, cellulose, copper, fructose, fatty acids, galactose, glucose, iron, potassium, protein, resin, sucrose, and xylose.
Advantageously, the inventive medicinal composition, method (process) and treatment yield very attractive, unexpected, surprisingly good and consistent results.
A more detailed explanation of the invention is provided in the following description and appended claims.


DETAILED DESCRIPTION OF THE INVENTION
The following is a detailed description of the preferred embodiments and best modes of practicing the invention.
An improved method (process) and medicinal composition is provided to treat herpes, pseudomonas, staph (staphylococci), hepatitis, and other infectious diseases. The inventive medicinal composition comprises a quaternary ammonium salt surfactant, a skin protectant and alcohol which provide active ingredients and cooperate with each other in the medicinal composition to alleviate cold sores, rashes or other skin conditions resulting from herpes, pseudomonas, staph, hepatitis, or other infectious diseases.
The quaternary ammonium salt surfactant can comprise benzalkonium halide comprising one or more of the following: benzalkonium bromide, benzalkonium fluoride, and/or preferably benzalkonium chloride. The skin protectant can comprise Allantoin (allantoin). The alcohol can serve as a pain reliever and can comprise isopropyl alcohol and/or preferably benzyl alcohol. Advantageously, the benzalkonium chloride, Allantoin and benzyl alcohol cooperate with each other in the medicinal composition to alleviate cold sores, rashes or other skin conditions resulting from herpes, pseudomonas, staph, hepatitis, or other infectious diseases.
The medicinal composition can comprise a formulation without Echinacea or other herbal extracts or herbaceous botanicals. In come circumstances, it may be desirable that the medicinal composition further includes herbal extracts or portions of Echinacea purpurea , such as from about 2% to about 90% by weight of the medicinal composition, preferably, if used, from about 40% to about 60% by weight of the medicinal composition. Significantly, the preferred medical composition and methods for it use preferably excludes undesired Myrtle and/or Centaurea and their extracts, as well as Echinacea angustofolia , aedurid, sodium hydroxide (NaOH), tea tree oil, lapacho extract, licorice root extract, arabinose, betaine, cellulose, copper, fructose, fatty acids, galactose, glucose, iron, potassium, protein, resin, sucrose, and xylose which can inhibit or retard the unexpected advantageous results of the novel medicinal composition.
In one preferred embodiment, the medicinal composition comprises: substantially greater than 0.01% to about 0.8% by weight aqueous or dry benzalkonium chloride, from about 0.01% to about 99% Allantoin, and from about 0.01% to about 99% benzyl alcohol. Preferably, the medicinal composition comprises: substantially greater than 0.02% to 0.3% by weight aqueous or dry benzalkonium chloride, from about 0.05% to about 30% Allantoin; and from about 10% to about 30% benzyl alcohol.
The medicinal composition can also comprise the following additives or ingredients: water, preferably, sterile (purified) water; methyl cellulose; methyl paraben; potassium sorbate; and/or propyl praben. Methyl cellulose is a natural thickener. Methyl paraben and propyl paraben are natural preservative from blueberries. Potassium sorbate is a natural preservative. The ratio of the sterile water to the benzalkonium chloride in the medicinal composition can range from about 30,000:1 to about 250:1, preferably from about 5000:1 to about 750:1. The medicinal composition can comprise a coating, such as a powder, gel, ointment, or paste.
A preferred method (process) to treat or alleviate cold sores, rashes or other skin conditions resulting from herpes, pseudomonas, staph, hepatitis, or other infectious diseases, comprises the steps of: substantially inhibiting cold sores, rashes or other skin conditions resulting from herpes, pseudomonas, hepatitis, or other infectious diseases, by applying a medicinal composition on cold sores, rashes or other skin conditions, or infected regions; and maintaining said composition on the cold sores, rashes, skin conditions, or infected regions until external symptoms and physical manifestations resulting from herpes, pseudomonas, staph, hepatitis, or other infectious diseases substantially disappear about the cold sores, rashes, skin conditions, or infected regions.
In one preferred embodiment, the external symptoms and physical manifestations of cold sores substantially disappear in about one day or less after the medicinal composition is applied to the cold sores. Desirably, the external symptoms and physical manifestations of vesicular eruption, rash or other skin conditions resulting from herpes simplex virus are resolved in about 19 hours to about 24 hours after the composition is applied to the vesicular eruption, rash or other skin condition.
Preferably, an infected area of a person having an infectious outbreak of herpes or other infectious disease can be resolved, within about 1 hour to about 30 hours by topically applying the composition to the infected area of said herpes or said other infectious disease, and maintaining said composition on said infected area for about 1 hours to about 30 hours.
Advantageously, pseudomonas, staph and/or hepatitis are substantially inhibited by applying the medicinal composition on the regions infected by pseudomonas and maintaining the medicinal composition on the regions infected from pseudomonas, staph and/or hepatitis until external symptoms and physical manifestations resulting from pseudomonas, staph and/or hepatitis substantially disappear about the infected regions.
The herpes causing the cold sores, rashes or other skin conditions, can comprise: herpes simplex virus 1 (HSV 1), herpes simplex virus 2 (HSV 2), staphylococci (staph), varicella zoster virus (herpes zoster) (shingles), cytomegalovirus, herpetic keratitis, conjunctivitis, human immunodeficiency virus infection (HIV), viral influenza (flu), epstein barr, papilloma virus, viral parainfluenza, adenovirus, viral encephalitis, viral meningitis, arbovirus, arenavirus, picornavirus, coronavirus, syntialvirus, viral infections, roseola infantum, pneumonia, mononucleosis, uveitis, retinitis, human cervical carcinoma, vaginal carcinoma, vulvovaginitis, human herpes IV, Kaposi's sarcoma, cytomegalovirus, or common colds (caused by viruses or herpes).
The hepatitis causing the rashes or other skin conditions can comprise hepatitis B and hepatitis C.
Benzalkonium Chloride
A surfactant can provide a broad spectrum of antimicrobial action. Surfactants can comprise quaternary ammonium salts containing 6-18 carbon atoms. Preferably, the quaternary ammonium salt surfactant is a mixture of alkyl dimethylbenzylammonium chlorides, which can comprise benzalkonium halide comprising benzalkonium bromide, benzalkonium fluoride and/or most preferably benzalkonium chloride.
The chemical formula of benzalkonium chloride is shown hereinafter. Other types of benzalkonium chloride can be used.









Benzalkonium chloride in aqueous solution is commercially available, among others, under the brand name and trade mark Zephiran® distributed by Sanofi Winthrop Pharmaceuticals (formerly Winthrop Labs).
Benzalkonium chloride can be obtained by extraction, such as a surfactant-mediated solid phase extraction procedure to obtain a concentration of benzalkonium (BA) chloride. For example, dodecyl sulfate attached to a strong anion exchange resin and aqueous samples are passed through a column containing this surfactant-resin material. Benzalkonium chloride can be adsorbed from the aqueous solution onto the sorbent via hydrophobic and electrostatic interactions. Electrostatic attractions occur between the benzalkonium cations and removable dodecyl sulfate anions. Removing this ion pair from the sorbent can result in efficient elutions.
Benzalkonium chloride (alkyl dimethyl benzyl ammonium chloride) is a mixture of alkylbenzyl dimethylammonium chlorides of various alkyl chain lengths. Benzalkonium chloride is a nitrogenous cationic surface-acting agent belonging to the quaternary ammonium group. Benzalkonium chloride has three main categories of use; as a biocide, a cationic surfactant and phase transfer agent in the chemical industry
Benzalkonium chloride is a rapidly acting anti-infective surfactant with a moderately long duration of action. The surfactant is active against bacteria and some viruses, fungi and protozoa. Bacterial spores are considered to be resistant. Solutions of benzalkonium chloride are bacteriostatic or bactericidal according to concentration. The exact mechanism of bacterial action of benzalkonium chloride is unknown but is thought to be due to enzyme inactivation. Activity of benzalkonium chloride generally increases with increasing temperature and pH. Gram-positive bacteria are more susceptible to benzalkonium chloride than gram-negative bacteria. Unfortunately, benzalkonium chloride is inactivated by soaps, anionic detergents, serum, and certain proteins. Benzalkonium chloride has fallen out of favor in some laboratories for the above reasons.
Benzalkonium chloride can be readily soluble in alcohol, and acetone. Dissolution of benzalkonium chloride in water is slow, and aqueous solutions of benzalkonium chloride are preferred, as it is easier to handle. Solutions of benzalkonium chloride can be neutral to slightly alkaline with colors ranging from clear to a pale yellow. Solutions of benzalkonium chloride tend to foam when shaken, have a bitter taste, and a faint almond-like odor in concentrated solutions.
Standard concentrates of benzalkonium chloride can be manufactured as 50% and 80% w/w solutions, and sold under trade names or brand names, such as BC50, BC80, BAC50, BAC80, etc. The 50% solution of benzalkonium chloride is purely aqueous, while more concentrated solutions require rheology modifiers (alcohols, polyethylene glycols, etc.) to minimize increases in viscosity or gel formation under low temperature conditions.
Benzalkonium chloride can be used for disinfectant formulations and for microbial corrosion inhibition in the oilfield sector. Benzalkonium chloride can be considered one of the safest synthetic biocides known and has a long history of efficacious use. Benzalkonium chloride can be useful for antiseptics, hygienic towelettes, and wet wipes.
Alcohol-free benzalkonium chloride solutions can be used for skin disinfection prior to withdrawing blood. Benzalkonium chloride can be used as a preservative and in cosmetics such as eye and nasal drops.
The greatest biocidal activity of benzalkonium chloride is associated with the C12-C14 alkyl derivatives. The mechanism of bactericidal and microbicidal action is thought to be due to disruption of intermolecular interactions. This can cause dissociation of cellular membrane bilayers, which can compromise cellular permeability controls and induces leakage of cellular contents. Other bimolecular complexes of benzalkonium chloride within the bacterial cell can also undergo dissociation.
Enzymes, which finely control a plethora of respiratory and metabolic cellular activities, are particularly susceptible to deactivation by benzalkonium chloride. Furthermore, intermolecular interactions and tertiary structures in such highly specific biochemical systems can be readily disrupted by cationic surfactants, such as benzalkonium chloride.
Benzalkonium chloride solutions can provide rapidly acting biocidal agents with a moderately long duration of action. Benzalkonium chloride is active against bacteria and some viruses, fungi, and protozoa.
Formulations using benzalkonium chloride blended with various quaternary ammonium derivatives can be used to extend the biodcidal spectrum and enhance the efficacy of benzalkonium chloride based disinfection products. This technique has been used to improve virucidal activity of quaternary ammonium-based formulations to healthcare infection hazards such as hepatitis, herpes, HIV, etc. Benzalkonium chloride solutions for hospital use tend to be neutral to alkaline, non-corrosive on metal surfaces, non-staining and safe to use on all washable surfaces.
Allantoin
Allantoin is a skin protectant and is also known as 5-ureidohydantoin or glyoxyldiureide. Allantoin is a chemical compound with a chemical formula (molecular formula) of C 4 H 6 N 4 O 3 and a chemical name of 2,5-Dioxo-4-imidazolidinyl urea. The chemical structure of Allantoin is shown hereinafter:









Allantoin is a white crystalline powder with a molecular weight of 158.1164 g/mol and a melting point of 239° C. Allantoin has solubility in water of 0.5% at 25° C. Allantoin is a diureide of glyoxylic acid. Allantoin is a product of oxidation of uric acid by purine catabolism.
Allantoin is named after the allantois, an amniote embryonic excretory organ in which it concentrates during development in most mammals except higher apes. Allantoin is present in botanical extracts of the comfrey plant. Chemically synthesized, bulk Allantoin is natural-identical, safe, non-toxic, compatible with cosmetic raw materials, and meets CTFA and JSCI requirements.
Significantly, Allantoin is a soothing, anti-irritant, and skin protectant. Allantoin can increase the smoothness of the skin and can promote cell proliferation and wound healing. Allantoin can be useful to help treat skin ulcers, wounds, scalds, burns, sunburns, carbuncles, acne, skin eruptions, fissures, abrasions, impetigo, eczema, and psoriasis. Allantoin can further be useful for the temporary protection of minor cuts and scrapes, as well as to help prevent and temporarily protect chafed, chapped, cracked, wind-burned skin and lips. Allantoin helps relieve dryness and softens cold sores and fever blisters. Allantoin can help treat and prevent diaper rash. Allantoin can be used in medicinal composition, clarifying lotions, various cosmetic lotions and creams, toothpaste, mouthwash, oral hygiene products, shampoos, and lipsticks. Allantoin can also be used in hair and scalp preparations, hair dressing, antiperspirants, deodorants, foot preparations, talcum powder, shaving and after-shave products, baby preparations, eye preparations, rectal preparations, soaps and detergents. The beneficial effects for Allantoin include its use as an active ingredient in over-the-counter cosmetics and can have a moisturizing and keratolytic effect to increase the water content of the extra cellular matrix and enhance the desquamation of upper layers of dead skin cells. Allantoin can form complexes with irritant and sensitizing agents.
Allantoin is a healing, moisturizing, soothing and anti-irritating, keratolytic and non-toxic agent that can useful in dermatological, cosmetic and veterinary preparation. Allantoin is a valuable cell-proliferating healing agent which stimulates healthy tissue formation. Allantoin has a keratolytic and skin softening action.
The amphoteric characteristic of Allantoin permits combination with various chemical substances to form salts and complexes. In combination with various therapeutic substances or compounds, Allantoin synergizes their activity and reduces at their side-effects so as to sensitize irritating or toxic properties. The complex and salts formed with Allantoin have useful dermatotherapeutic properties.
Benzyl Alcohol
Benzyl alcohol is also known as benzal alcohol, benzenecarbinol, benzenemethanol, benzoyl alcohol, hydroxytoluene, phenylmethanol, phenylmethyl alcohol, NCI-C06111, (hydroxymethyl)benzene, and phenylcarbinol. Benzyl alcohol is a chemical compound with a chemical formula (molecular formula) of C 7 H 8 O. The chemical structure of Benzyl alcohol is shown hereinafter:









Benzyl alcohol is a colorless liquid with a melting point of −15° C. and a boiling point of 205° C. Benzyl alcohol has a specific gravity of 1.045, a vapor pressure of 3.7 mm Hg at 77 C, and a flash point of 94° C. Benzyl alcohol can have a sharp burning taste and pleasant aromatic odor. Benzyl alcohol is a natural constituent of a variety of essential oils including jasmine, hyacinth, and ylang-ylang.
Benzyl alcohol is partially soluble in water (4 g/100 mL) and completely miscible in alcohols and ether. Benzyl alcohol can be prepared by the hydrolysis of benzyl chloride using sodium hydroxide:
C 6 H 5 CH 2 Cl+NaOH→C 6 H 5 CH 2 OH+NaCl
Benzyl alcohol can also be prepared via a Grignard reaction by reacting phenylmagnesium bromide (C 6 H 5 MgBr) with formaldehyde, followed by acidification. Like most alcohols, benzyl alcohol reacts with carboxylic acids to form esters.
Benzyl alcohol can exhibit bacteriostatic and antipruritic properties. Benzyl alcohol can be used as a local anesthetic and to reduce pain. Benzyl alcohol is also a useful solvent due to its polarity, low toxicity, and low vapor pressure.
Isopropyl Alcohol
Isopropyl alcohol has a chemical formula (molecular formula) of C 3 H 7 OH) and a structural formula as shown hereinafter.









Isopropyl alcohol is a colorless liquid with a melting point of −89° C. and a boiling point of 82.3° C. Isopropyl alcohol has a density of 0.785 g/cm 3 . Isopropyl alcohol forms an azeotrope with water at 87.4% alcohol. Isopropyl alcohol is soluble or miscible with water, ethyl ether, and ethyl alcohol. Isopropyl alcohol can be produced by combining water and propylene, such as by indirect hydration from the sulfuric acid process or direct hydration.
Isopropyl alcohol is a colorless, flammable chemical compound with a strong odor. Isopropyl alcohol is also known as isopropanol, rubbing alcohol, the abbreviation IPA propan-2-ol, 2-Propanol; sec-Propyl Alcohol; Alcojel; Alcosolve 2; Avantin; Avantine; Combi-Schutz; Dimethylcarbinol; Hartosol; Imsol A; Isohol; Isopropanol; Lutosol; Petrohol; Propol; PRO; Takineocol; 1-Methylethyl Alcohol; iso-C3H70H; 2-Hydroxypropane; Propane, 2-hydroxy-; sec-Propanol; Propan-2-ol; i-Propylalkohol; Alcohol, rubbing; Alcolo; Alcool isopropilico; Alcool isopropylique; Alkolave; Arquad DMCB; iso-Propylalkohol; Isopropyl alcohol; Lavacol; Visco 1152; Alcosolve; ChromaI′; i-Propanol; 2-Propyl alcohol; Spectrar; Sterisol hand disinfectant; UN 1219; (−)-2,3-0-Isopropyl alcohol; Alcohol; Alcowipe; DuPont brand zonyl FSA fluorinated surfactants; DuPont brand zonyl FSJ fluorinated surfactants; DuPont brand zonyl FSN fluorinated surfactants; DuPont brand zonyl FSP fluorinated surfactants; I.P.S.; n-Propan-2-ol; Rubbing alcohol; Sec-propyl; 1-methylethanol; and Propanol-2.
Rubbing alcohol, U.S.P./B.P. is a liquid prepared for topical application. Rubbing alcohol can be prepared from specially denatured alcohol and can contain 68.5%-71.5% volume/volume (vol/vol) (v/v) of absolute (i.e. 100%) alcohol. Individual manufacturers, however, often use their own formulation standards or specifications in which the ethanol content usually ranges from 70-95% v/v.
The British Pharmacopoeia and United States Pharmacopoeia define the term alcohol as a mixture of ethanol (Ethyl Alcohol, C 2 H 5 OH=46.07) and water of fixed proportions. Alcohol, B.P. is 96% ethanol v/v, and Alcohol, U.S.P. is 95% ethanol v/v. Therefore, in Great Britain, the term rubbing alcohol also refers to a mixture using ethyl alcohol.
In the United States, rubbing alcohol, U.S.P. and all preparations coming under the classification of rubbing alcohols must be manufactured in accordance with the requirements of the US Treasury Department, Bureau of Alcohol, Tobacco, and Firearms, using Formula 23-H (8 parts by volume of acetone, 1.5 parts by volume of methyl isobutyl ketone, and 100 parts by volume of ethyl alcohol). Rubbing alcohol contains 68.5-71.5% by volume of absolute ethyl alcohol, the remainder comprising water and the denaturants, with or without color additives, and perfume oils. Rubbing alcohol contains in each 100 mL not less than 355 mg of sucrose octaacetate or not less than 1.40 mg of denatonium benzoate. Rubbing alcohol also exists on pharmacy store shelves without the U.S.P. standard designation. The concentration of the ethyl alcohol component can vary but it is generally printed under the ingredient list of that particular product. A common concentration of ethyl alcohol in rubbing alcohol is 95%.
Isopropyl alcohol can be used as a disinfectant. Isopropyl alcohol or rubbing alcohol is widely used as an antiseptic for sterilizing surfaces or clean minor cuts or abrasions. Rubbing alcohol can be applied externally as a cooling, soothing application for bedridden patients and athletes. Rubbing alcohol can also be used for cleansing the surgeon's hands and instruments and for disinfection of the patient's skin prior to penetration of the skin by a hypodermic needle. As an antiseptic, rubbing alcohol can be good against vegetative bacteria and fair against fungi and viruses. Rubbing alcohol can cool and soothe skin. Isopropyl rubbing alcohol can also be supplied in a 99% concentration, such as for use to harden skin such as in the case of the feet of novice hikers. Sterilizing pads typically contain a 60-70% solution of isopropyl alcohol in water. Isopropyl alcohol can be used as a disinfectant.
Other Embodiments
The improved medicinal composition or medicine, also referred to as Viracea 2 HIV-4, comprises a novel medical composition, formulation, antimicrobial compound and solution. The new antimicrobial medical treatment and microbicidal medicine are successful in treating primarily HIV systemically and can be useful in treating other microbial infections including, but not limited to: varicella zoster virus (herpes zoster) and cytomegalovirus. In some circumstances, it may be desirable to use the novel medicine topically.
While the novel medicine and antimicrobial compound is particularly useful in dramatically inhibiting human immunodeficiency virus infection (HIV), it may be useful in treating other microbial diseases (microbe-causing diseases) such as: Epstein barr, papilloma virus, cellulitis, staphylococci, streptococci, mycobacteria, influenza, parainfluenza, adenoviruses, encephalitis, meningitis, arbovirus, arenavirus, anaerobic bacilli, picornavirus, coronavirus and synsytialvirus, as well as herpes simplex virus, varicella zoster virus and cytomegalovirus.
While the medical treatment and medicine is particularly useful for inhibiting HIV and other infectious diseases in persons (human beings) ( homo sapiens ), they can also be useful for veterinary purposes for treating viral and bacterial infections and infectious diseases in animals, such as: dogs, cats, birds, horses, cows, sheep, swine (pigs and hogs), and other farm animals, as well as rodents and other animals seen in zoos.
Advantageously, the improved medical treatment and medicine of the present invention yielded unexpected, surprisingly good results. This easy to use microbicide solution can provide immediate absorption in parenteral administration. Upon administration, there can be a slight tingling effect. Within minutes of application, a slight medicinal taste in the mouth may be experienced. Initial, in vitro testing of the novel medical treatment and medicine demonstrated extremely surprising inhibitory effects on HIV virus. Desirably, the novel medicine is made from readily available, over the counter (OTC) chemicals or products and provides a safe, comfortable, and economical treatment.
Desirably, the novel medicine (medical composition) includes microbe inhibitors which inhibit, suppress and stop microbial infections from microbe-causing diseases. The microbe inhibitors comprise antimicrobial isolates, botanical extracts or phytochemicals, of at least a portion of one or more of the special plants listed below. The microbe inhibitors can comprise viral inhibitors to inhibit viral diseases, such as: HIV, herpes simplex virus 1 (HSV 1), herpes simplex virus 2 (HSV 2), varicella zoster virus (herpes zoster), cytomegalovirus, epstein barr, papilloma virus, viral influenza, viral parainfluenza, adenovirus, viral encephalitis, viral meningitis, arbovirus, arenavirus, picornavirus, coronavirus, and synstialvirus. The microbe inhibitors can also comprise bacterial inhibitors to inhibit bacterial diseases, such as: cellulitis, staphylococci, streptococci, mycobacterium, bacterial encephalitis, bacterial meningitis, and anaerobic bacilli. In some circumstances, the microbe inhibitors can include fungi inhibitors.
Better results may be obtained if Echinacea and Commiphora (also referred to as Commiphora ) or other plants are not used in the medicine in their raw, untreated and uncut state. For even better results, the medicinal composition can exclude: Myrtle and/or Centaurea and their extracts, as well as Echinacea angustofolia , aedurid, sodium hydroxide (NaOH), tea tree oil, lapacho extract, licorice root extract, arabinose, betaine, cellulose, copper, fructose, fatty acids, galactose, glucose, iron, potassium, protein, resin, sucrose, and xylose.
The improved medical treatment provides a novel method and process for use in treating the above infectious diseases. For some infectious diseases, the microbial inhibitors can be applied and maintained on the microbial infected on the infected area (region or surface) until the external symptoms and physical manifestations of the infection disappear, reside or resolve about the infected area. The medicine can be administered by syringe injection, sublingual intramural, spraying, dabbing, dusting, swabbing, sponging, brushing, pouring, dispensing, covering, or heavily coating the medicine the microbial infected areas, such as: lymph nodes, lymphatic system, T-cells, oral mucosa, nasal mucosa, vagina tissue, labial tissue, rectal tissue, anal tissue, peri-anal tissue, lips, cutaneous tissue, ocular tissue, conjunctiva, and eyelids.
Preferably, the microbial inhibitors or antimicrobial compound is applied systemically with a syringe into the rectal canal or vagina to treat or prevent the sexual transmission of HIV. The microbial inhibitors or antimicrobial compound can be applied in the preceding manner 4-20 times per day for 4 to 18 consecutive days to substantially decrease the viral load of patients infected with HIV, i.e., to decrease the amount of HIV and AIDS virus in the body.
Preferably, the improved medicine, medical composition or microbial compound is a phytochemical concentrate which is combined and simultaneously or concurrently applied with a surfactant, a nutrient, and a carrier, solvent or diluent to provide a microbicide medicinal solution. The nutrient serves as a catalyst, activator, phytochemical initiator, nutritional supplement, and auxiliary carrier. The nutrient can comprise one or more of the following: a water soluble vitamin, a fat soluble vitamin, vitamin A, vitamin B complex, (B vitamin complex), vitamin D, vitamin E, vitamin K, vitamin B1, vitamin B2, vitamin B5, vitamin B6, vitamin B12, vitamin B15, and preferably folacin or folic acid.
To this end, the interesting microbicide solution comprises an antimicrobial detergent surfactant, with botanical extracts. The surfactants preferably are cationic surfactants which can comprise singly or any number of quaternary ammonium chlorides having 6-18 carbons such as alkylbenzyldimethylammonium chloride, mixtures of alkylbenzyldimethylammonium chloride, alkyldimethyl/ethylbenzylammonium chloride, n-alkyldimethylbenzylammonium chloride, diisobutylphenoxyethoxyethyldimethylbenzylammonium chloride, N—(C 12 C 14 C 16 ) dimethylbenzylammonium chloride, benzalkonium chloride, octyldecyldimethyloammonium chloride, didecyldimethylammonium chloride, dioctyldimethylammonium chloride, dialkyldimethylammonium chloride, dialkylmethylbenzylammonium chloride, octyldecyldimethylammonium chloride, dimethylbenzylammonium chloride, laurryldimethylbenzylammonium chloride, o-benzyl-p-chlorophenol, dideryldimethylammonium chloride, doctyldimethylammonium chloride, alkyl(C 14 C 12 C 16 ) dimethylbenzylammonium chloride, and preferably comprises alkylbenzyldimethylammonium chloride most preferably benzalkonium chloride. The range of activity of the cationic surfactant can be 5% to 90% but for best results 8% to 20%. Quaternary ammonium salts are readily available commercially. In some circumstances it may be useful to use other surfactants, such as, but not limited to: DMSO, glycolic acid surfactants, enzyme surfactants, ampholytic surfactants, switterionic surfactants, and nonionic surfactants. The surfactants can comprise detergents, wetting agents, emulsifiers, defoamers, and/or surface tension reducing additives.
Carriers are useful for mixing the constituents, keeping the constituents in solution, and providing an easy method of application to the affected area whether by spray, dropper, or applicator. While an aqueous solution, preferably a sterile aqueous carrier and solvent is preferred for best results, in some circumstances it may be desirable to use other liquid or solid carriers, such as: glycerin, mineral oil, silica, cottonseed oil, coconut oil, vegetable oil, seed oil, fish oil, or animal oil, alcohol, talc, corn meal, beeswax, carnauba wax, beta carotene, garlic oil, camphor oil, soluble vitamins, soluble minerals, rape seed oil, nut oils, olive oil, liposomes, ascorbic acid, evening primrose oil, pycnogenol, grape seed oil, lanolin, Ethocyn, collagen, aloe vera, bee pollen, royal jelly, chondroitin sulfate A, sea vegetables, EDTA, fatty acids, herbs, lecithin, bioflavinoids, grain oils or powders, algae, teas, vinegars, acidophilus, cell salts, ascorbic acids, hydra 5, glandulars, amino acids, psyllium, plant derivatives, or other sterile carriers.
The botanical extracts antimicrobial isolates or phytochemicals contained in this new medicine and medical treatment can be comprised of: myrrha gum resin, sequiterpenses, curzenone, dihydro fuanodien-6-one, 2-methoxylfurandiene, elemol, acetic acid, alpha-amyrone, arabinose, alpha-bisabolene, gamma-bisabolene, cadinene, campesterol, cholesterol, cinnamaldehyde, commiferin, alpha-commiphoric acid, beta-commiphoric acid, gamma-commiphoric acid, commiphorinic acid, m-cresol, cumic alcohol, cuminaldehyde, dipentene, elemol, 3-epi-alpha-amyrin, eugenol, furanodiene, furanodienone, galactose, gum, heerabolene, alpha-heerabomyrrhol, beta-heerabomyrrhol, heeraboresene, limonene, 4-0-methyl-glucuronic acid, n-nonacesane, beta-sitosterol, xylose, caropylenes (carophylenes), lynderstyrene (lindestyrene), arabinose, betaine, copper, echinacen, echinacin B, echinacoside, echinolone, enzymes, fructose, fatty acids, galactose, glucose, glucuronic acid, inulin, inuloid, iron, pentadecadiene, polyacetylene compounds; polysaccharides, such as, but not limited to, arabinogalactan; potassium, protein, resin, rhamnose, sucrose, sulfur, tannins, vitamins a, c, and e, alkylamides, apigenin, arabinogalacta, ascorbic acid, behenic-acid-ethyl-acid, betaine, borneol, bornyl-acetate, caffeic-acid, 2-0-caffeoyl-3-(5-alpha carboxybeta) 3,4 dihydroxyphenyl, 2-0-caffeoyl-3-0 cumaroyltaraic acid, 6-0-caffeoylechinacoside, 2-0-caffeoyl-3-0-feruloyltartaric acid, 2-0-caffeoyltartaric acid, calcium, carbonate, beta carotene, carophyllene, carophyllene-epoxide, chloride, chlorgenic acid, cichoric acid, cichoric-acid-methyl-ester, cobalt, cyanadin-3-0-(beta-d-glycopyranoside), cynadin-3-(6-0-malonyl beta-d-glycopyranoside), cynarin, deca (2e,4e,6e) trienoic acid-isobutylamide, des-rhamnosylverbascoside, 3,5-dicaffeoylquinic acid, 4-5-0 dicaffeoylquinic acid, 2,3-0-diferuloltartaric acid, do-deca-(2e,4e)-dienoic acid-isobutylamide, dodeca-2,4-dien-1-yl isovalerate, dodeca (2e,6z,8e,10e)-tetraenoic acid-isobutylamide, epishobunol, beta-farnesene, 2-0-feruloytartaric acid, germacrene, heptadeca-(8z,11z)-dien-2-one, heteroxylan, humulene 8-12, (e)-10-hydroxy-4,10-dimethyl 4,11-dodecadien-2-one, 13-hydroxyoctadeca-(9z,11e,15z)-trienoic-acid, inulin, iron, isochlorogenic acid, isorhamnetin-3-rutinoside, isotussilagine, kaempferol, kaempferol-3-glucoside, kaempferol-3-nutinoside, limonene, luteolin, luteolin-7-glucoside, magnesium, manganese, 2-methyltetradeca-5,12 diene, 2-methyltetradeca-6,12 dience, methyl-p-hydroxycinnamate, marcene, niacin, palmitic acid, pentadeca-(8z,11z)-dien-2-one, pentadeca-(8z,13z)-dien-11-lyn-2-one, pentadeca-8en-2-one, pentadeca-(8z)-en 2 one, pentadeca-(8z)-en-11,13 dien-2-one, 1-pentadecene, penta-(1,8z)-diene, phosphorous, alpha pinene, beta pinene, polyacetylenes, pontica epoxide, potassium, protein, quercetagetin-7-glucoside, quercetin, quercetin-3-galactoside, quercetin-3-glucoside, quercetin-3-robinoside, quercetin-3-xyloside, quercetin-3-xylosylgalactoside, rhamnoarabinogalactan, riboflavin, rutin, rutoside, selenium, silicate, beta-sitosterol, sitosterol-3-beta o-glucoside, sodium, stigmasterol, sulfate, tartaric acid, tetradeca-(8z)-en-11,13 dien-2-one, thiamin, n-triacontanol, trideca-1-en-3,5,7,9,10-pentayne, tussilagine, vanallin, verbascoside. For better results, the phytochemical concentrates include the above phytochemicals, excluding Arabinose, betaine cellulose, copper, fructose, fatty acids, galactose, glucose, iron, potassium, protein, resin, sucrose, and, xylose.
The botanical extracts, antimicrobial isolates and phytochemicals maybe separated, extracted and isolated from portions of plants, such as: pimpinella anisum, myroxylon, arctostaphylos, carum, capsicum, eugenia mytacea, coriandrum, inula, allium, gentiana, juniperus, calendula, origanum, mentha labiate, commiphora, plantago, rosmarinus, ruta , lamiaceae, meliosa, baptisa, artemisa , sage, mentha, parthenium integrifolium, eucalyptus , asteriacea, and preferably: from the genus Echinacea of the family Astericaea, namely, Echinacea angustofolium , ( Echinacea pallidae ), Echinacea vegetalis, Echinacea atribactilus Echinacea pallidum , and preferably Echinacea purpurea , and its extracts, portions, and cultivars; as well as from the genus Commophora , namely, Commophora myrrha, Commophora molmol, Commophora erythraea , and their cultivars. For best results, the phytochemicals and antimicrobial isolates are extracts from Echinacea purpurea and Commiphora myrrha.
The inventive technology, treatment and medicine yield very attractive, unexpected, surprisingly good and consistent results. Tests show the microbicide solution (medicine) and medical treatment to be extremely useful to: control HIV infection, inhibit attachment of HIV virus to target cells, act as a preventive microbicide, extend the latency periods of HIV and other diseases, and dramatically inhibit HIV and other viruses, while being generally safe to the patient and the environment.
More specifically, a microbicide and treatment are provided to inhibit human immunodeficiency virus, also referred to as human immunodeficient virus or HIV. Desirably, the HIV microbicide and treatment completely inhibits HIV, as well as other infectious microbial diseases, and are safe and non-toxic to humans, animals, and the environment.
The HIV microbicide and medicine can comprise a surfactant and an herbaceous botanical providing a botanical extract, phytochemical, antimicrobial isolate, anti-viral isolate, microbe inhibitor, and viral inhibitor. The preferred microbicide composition can comprise: a surfactant; an aqueous diluent; a nutrient; and the herbaceous botanical of the genus Echinacea (E), of the family Asteracea, species: purpurea, angustofolia, pallidae, vegetalis, atribactilus and the cultivars, as well as the herbaceous botanical of the genus Commiphora species: Commiphora myrrha, Commiphora molmol, Commiphora erythraea , and their cultivars. Preferably, the herbanaceous botanicals are extracts and isolates comprising Commiphora phytochemicals and Echinacea phytochemicals as found in and extracted from Commiphora myrrha and Echinacea purpurea . For best results, the medical treatment and microbicide (medicine) comprises: a cationic surfactant; phytochemicals and/or extracts from Echinacea purpurea and Commiphora myrrha , a sterile aqueous diluent and folacin. The ratio of Commiphora myrrha to Echinacea purpurea preferably ranges from 1:2 to 1:4.
The surfactant provides a certain debridement at the cellular surface level with a broad spectrum of antimicrobial action. Surfactants of this nature can comprise quaternary ammonium salts containing 6-18 carbon atoms. Preferably the quaternary ammonium salt surfactant is a mixture of alkyl dimethylbenzylammonium chlorides, which can be: benzalkonium halide, benzalkonium bromide, benzalthonium chloride and most preferably benzalkonium chloride. The HIV treatment can comprise a 100% active aqueous solution but can also be used in concentrate. The solution can comprise by weight various concentrations of surfactants such as 0.005% to 0.8%, preferably 0.02% to 0.30% and most preferably 0.02% to 0.26%.
The phytochemicals in the botanical Echinacea have demonstrated impressive activity against bacteria, viruses, and some fungi. The exact mechanism is unknown. When the inventive microbicide was tested topically on HIV and HSV 1 & 2, it is effective in treating herpes simplex infectious outbreaks. When tested in vitro, it showed inhibitory activity against HIV-1 and HSV 1 & 2.
The phytochemical concentrate composition comprises the following isolated constituents, botanical extracts, microbial inhibitors, and antimicrobial isolates: polysaccharides, echinacen, echinaceine, echinacoside (caffeic acid ester), echinolone, echinadiol, enzymes, glucuronic acid, inuloid, pentadecadiene, polyacetylene compounds, arabinogalactan, rhamnose, PS I (a 4-0-methylglucoronoarabinoxylan, M r 35 kD) and PS II (an acid rhamnoarabinogalactan, M r 450 kD), cynarin (1,5-di-0-caffeoylquinic acid), chicoric acid (2,3-0-di-caffeoyltartaric acid) and derivatives, alkylamides, keto-alkynes and -alkenes; quinones; oils including: borneol, bornyl acetate; pentadeca-8(z)-en-2one, germacrene D; caryophyllene; caryophyllene epoxide; anthocyanins pyrrolizidine alkaloids; lipophilic amides, isobutylamides; polyacetylenes; myrrha gum resin; curzerenone (furahoeudesmane type); dihydro fuanodien-6-one; 2-methoxyfuranodiene (furanoelemene type); elamol; lyndestyrene (furanogermacrane type); alkylamides, apigenin, arabinogalacta, ascorbic acid, behenic-acid-ethyl-acid, betaine, borneol, bornyl-acetate, caffeic-acid, 2-0-caffeoyl-3-(5-alpha carboxybeta) 3,4 dihydroxyphenyl, 2-0-caffeoyl-3-0 cumaroyltaraic acid, 6-0-caffeoylechinacoside, 2-0-caffeoyl-3-0-feruloyltartaric acid, 2-0-caffeoyltartaric acid, calcium, carbonate, beta carotene, carophyllene, carophyllene-epoxide, chloride, chlorgenic acid, cichoric acid, cichoric-acid-methyl-ester, cobalt, cyanadin-3-0-(beta-d-glycopyranoside), cynadin-3-(6-0-malonyl beta-d-glycopyranoside), cynarin, deca (2e,4e,6e) trienoic acid-isobutylamide, des-rhamnosylverbascoside, 3,5-dicaffeoylquinic acid, 4-5-0 dicaffeoylquinic acid, 2,3-0-diferuloltartaric acid, do-deca-(2e,4e)-dienoic acid-isobutylamide, dodeca-2,4-dien-1-yl isovalerate, dodeca (2e,6z,8e,10e)-tetraenoic acid-isobutylamide, epishobunol, beta-farnesene, 2-0-feruloytartaric acid, germacrene, heptadeca-(8z,11z)-dien-2-one, heteroxylan, humulene 8-12, (e)-10-hydroxy-4,10-dimethyl 4,11-dodecadien-2-one, 13-hydroxyoctadeca-(9z,11e,15z)-trienoic-acid, inulin, iron, isochlorogenic acid, isorhamnetin-3-rutinoside, isotussilagine, kaempferol, kaempferol-3-glucoside, kaempferol-3-nutinoside, limonene, luteolin, luteolin-7-glucoside, magnesium, manganese, 2-methyltetradeca-5,12 diene, 2-methyltetradeca-6,12 dience, methyl-p-hydroxycinnamate, marcene, niacin, palmitic acid, pentadeca-(8z,11z)-dien-2-one, pentadeca-(8z,13z)-dien-11-lyn-2-one, pentadeca-8en-2-one, pentadeca-(8z)-en 2 one, pentadeca-(8z)-en-11,13 dien-2-one, 1-pentadecene, penta-(1,8z)-diene, phosphorous, alpha pinene, beta pinene, polyacetylenes, pontica epoxide, potassium, protein, quercetagetin-7-glucoside, quercetin, quercetin-3-galactoside, quercetin-3-glucoside, quercetin-3-robinoside, quercetin-3-xyloside, quercetin-3-xylosylgalactoside, rhamnoarabinogalactan, riboflavin, rutin, rutoside, selenium, silicate, beta-sitosterol, sitosterol-3-beta o-glucoside, sodium, stigmasterol, sulfate, tartaric acid, tetradeca-(8z)-en-11,13 dien-2-one, thiamin, n-triacontanol, trideca-1-en-3,5,7,9,10-pentayne, tussilagine, vanallin, verbascoside, sequiterpenes; acetic acid, alpha-amyrone, arabinose, alpha-bisabolene, gamma-bisabolene, cadinene, campesterol, cholesterol, cinnamaldehyde, commiferin, alpha-commiphoric acid, beta-commiphoric acid, gamma-commiphoric acid, commiphorinic acid, m-cresol, cumic alcohol, cuminaldehyde, dipentene, elemol, 3-epi-alpha-amyrin, eugenol, furanodiene, furanodienone, galactose, gum, heerabolene, alpha-heerabomyrrhol, beta-heerabomyrrhol, heeraboresene, limonene, 4-0-methyl-glucuronic acid, n-nonacesane, beta-sitosterol, xylose, caropylenes (carophylenes), myrrha gum resin, curzenone, dihydro fuanodien-6-one, and 2-methoxyfurandiene.
For best results, the antimicrobial isolates of the phytochemical concentrate comprise by weight (based upon the total weight of the inventive medical composition): 0.3-9% echinacoside; 0.1-7% PS I (a 4-0-methylglucoronoarabinoxylan, M r 35 kD) and PS II (an acid rhamnoarabinogalactan, M r 450 kD); 0.1-10% cynarin (1,5-di-0-caffeoylquinic acid) and chicoric acid (2,3-0-di-caffeoyltartaric acid) and derivatives; 0.2-4% echinolone; 0.2-8% echinacin B; 0.1-6%; echinaceine; 0.2-7% anthocyanins comprising cyanidin 3-0-β- D -glucopyranoside and 3-0-(6-0-malonyl-β- D -glucopyranoside); 0.01-0.06% pyrrolizidine alkaloids comprising tussilagine and isotussilagine; 0.003-0.009% isomeric dodeca isobutylamides and 2E, 4E,8Z,10E/Z-tetraenoic acid; 0.01-2% caryopylenes; as well as Commiphora myrrh phytochemicals comprising: myrrha gum resin, curzenone, dihydro fuanodien-6-one, 2-methoxyfurandiene, lynderstyrene (lindestrene) sequiterpenes, acetic acid, alpha-amyrone, arabinose, alpha-bisabolene, gamma-bisabolene, cadinene, campesterol, cholesterol, cinnamaldehyde, commiferin, alpha-commiphoric acid, beta-commiphoric acid, gamma-commiphoric acid, commiphorinic acid, m-cresol, cumic alcohol, cuminaldehyde, dipentene, elemol, 3-epi-alpha-amyrin, eugenol, furanodiene, furanodienone, galactose, gum, heerabolene, alpha-heerabomyrrhol, beta-heerabomyrrhol, heeraboresene, limonene, 4-0-methyl-glucuronic acid, n-nonacesane, beta-sitosterol, xylose, caropylenes (carophylenes), and lynderstyrene (lindestyrene).
The phytochemical concentrate can comprise by weight: 2%-90% of the medical composition and solution and preferably comprises not less than 15% of the composition and solution; and for best results, comprises 40%-60% of the medical composition and solution.
The diluent dissolves the benzalkonium chloride (surfactant) and phytochemical concentrates and can act as a carrier in sprays, tubes, and dropper bottles. The preferable diluent is an aqueous diluent and most preferably is a sterile aqueous diluent. The ratio of water in the aqueous solution to benzalkonium chloride can range from 30,000:1 to 250:1 and preferably from 5000:1 to 750:1. The ratio of water to the combined concentrates of benzalkonium chloride and phytochemicals can comprise a range of 2:1 to 100:1 with a preferable range of 4:1 to 40:1, and for best results can comprise a ratio of 6:1 to 20:1.
For best results, the improved microbicidal treatment and medicine (microbicide) for herpes, comprises by weight: 0.02% to 0.3% benzalkonium chloride and to avoid toxicity preferably less than 0.26%; 40% to 60% Echinacea and Commiphora phytochemicals; 0.01% to 25% most preferably 2% to 12% nutrient; and 20% to 60%, most preferably 29.74% to 59.8% sterile water. The medicine (microbicide) desirably comprises a vitamin nutrient which serves as a nutritional carrier and provides a synergistic effect when combined with Commiphora myrrha and Echinacea purpurea . The nutrient can comprise one or more of the following: vitamin A, vitamin B complex, vitamin D, vitamin E, vitamin K, a water soluble vitamin, a fat soluble vitamin, vitamin B1, vitamin B2, vitamin B5, vitamin B6, vitamin B12, vitamin B15, and preferably folacin or folic acid.
While water is the preferred diluent and aqueous carrier, it may be desirable in some circumstances to use other carriers in order to propel the concentrate through a syringe or sprayer, or for greater solubility and efficacy. It may also be desirable in some circumstances to include a viscosity control agent. Furthermore, while it is estimated that the shelf life of the improved medicine is two years, it may be necessary to add an appropriate preservative.
For preferred use, as a microbicide preventative against HIV, the medical solution (medicine) should be applied systemically, vaginally or rectally. The method of application of medicine can be by: syringing, spraying, dabbing, dropper, or other methods. The application or coating of the solution (medicine) should be maintained during coitus. Anionic soaps and anionic detergents, and especially protein content soaps can be contraindicated. Preferably, the area of application should be washed, cleaned and dried prior to application of the medicine. For treatment as an HIV antiviral, the medicine can be applied by syringing the dosage treatment into the rectum or vagina or by other methods.
Phytochemicals
While raw, untreated, unprocessed, non-isolated Echinacea is generally undesirable to treat HIV and herpes intramurally, when, appropriately filtered, intramural administration may be feasible. Significantly, it appears that some, but not all, of the isolated constituents and botanical extracts of Echinacea purpurea and Commiphora Myrrha provide phytochemicals, antimicrobial isolates, botanical extracts and microbe inhibitors which have or exhibit antimicrobial activity that appear to be effective in treating HIV, herpes virus and other infectious diseases.
Significantly, the preferred medical composition and methods for it use preferably excludes undesired Myrtle and/or Centaurea and their extracts, as well as Echinacea angustofolia , aedurid, sodium hydroxide (NaOH), tea tree oil, lapacho extract, licorice root extract, arabinose, betaine, cellulose, copper, fructose, fatty acids, galactose, glucose, iron, potassium, protein, resin, sucrose, and xylose, which can inhibit or retard the unexpected advantageous results of the novel medicinal composition.
As previously stated, the phytochemical concentrate composition comprises the following isolated constituents, botanical extracts, microbial inhibitors, and antimicrobial isolates: polysaccharides, echinacen, echinaceine, echinacoside (caffeic acid ester), echinolone, echinadiol, enzymes, glucuronic acid, inuloid, pentadecadiene, polyacetylene compounds, arabinogalactan, rhamnose, PS I (a 4-0-methylglucoronoarabinoxylan, M r 35 kD) and PS II (an acid rhamnoarabinogalactan, M r 450 kD), cynarin (1,5-di-0-caffeoylquinic acid), acid (2,3-0-di-caffeoyltartaric acid) and derivatives, alklylamides, keto-alkynes and -alkenes; quinones; oils including: borneol, bornyle acetate; pentadeca-8 (z)-en-2one; germacrene D; caryophyllene; caryophyllene epoxide; anthocyanins pyrrolizidine alkaloids; lipophilic amides; isobutylamides; polyacetylenes; myrrha gum resin; curzerenone (furahoeudesmane type); dihydro fuanodien-6-one; 2-methoxyfuranodiene (furanoelemene type); elamol; lyndestrene (furanogermacrane type); alkylamides, apigenin, arabinogalacta, ascorbic acid, behenic-acid-ethyl-acid, betaine, borneol, bornyl-acetate, caffeic-acid, 2-0-caffeoyl-3-(5-alpha carboxybeta) 3,4 dihydroxyphenyl, 2-0-caffeoyl-3-0 cumaroyltaraic acid, 6-0-caffeoylechinacoside, 2-0-caffeoyl-3-0-feruloyltartaric acid, 2-0-caffeoyltartaric acid, calcium, carbonate, beta carotene, carophyllene, carophyllene-epoxide, chloride, chlorgenic acid, cichoric acid, cichoric-acid-methyl-ester, cobalt, cyanadin-3-0-(beta-d-glycopyranoside), cynadin-3-(6-0-malonyl beta-d-glycopyranoside), cynarin, deca (2e,4e,6e) trienoic acid-isobutylamide, des-rhamnosylverbascoside, 3,5-dicaffeoylquinic acid, 4-5-0 dicaffeoylquinic acid, 2,3-0-diferuloltartaric acid, do-deca-(2e,4e)-dienoic acid-isobutylamide, dodeca-2,4-dien-1-yl isovalerate, dodeca (2e,6z,8e,10e)-tetraenoic acid-isobutylamide, epishobunol, beta-farnesene, 2-0-feruloytartaric acid, germacrene, heptadeca-(8z,11z)-dien-2-one, heteroxylan, humulene 8-12, (e)-10-hydroxy-4,10-dimethyl 4,11-dodecadien-2-one, 13-hydroxyoctadeca-(9z,11e,15z)-trienoic-acid, inulin, iron, isochlorogenic acid, isorhamnetin-3-rutinoside, isotussilagine, kaempferol, kaempferol-3-glucoside, kaempferol-3-nutinoside, limonene, luteolin, luteolin-7-glucoside, magnesium, manganese, 2-methyltetradeca-5,12 diene, 2-methyltetradeca-6,12 dience, methyl-p-hydroxycinnamate, marcene, niacin, palmitic acid, pentadeca-(8z,11z)-dien-2-one, pentadeca-(8z,13z)-dien-11-lyn-2-one, pentadeca-8en-2-one, pentadeca-(8z)-en 2 one, pentadeca-(8z)-en-11,13 dien-2-one, 1-pentadecene, penta-(1,8z)-diene, phosphorous, alpha pinene, beta pinene, polyacetylenes, pontica epoxide, potassium, protein, quercetagetin-7-glucoside, quercetin, quercetin-3-galactoside, quercetin-3-glucoside, quercetin-3-robinoside, quercetin-3-xyloside, quercetin-3-xylosylgalactoside, rhamnoarabinogalactan, riboflavin, rutin, rutoside, selenium, silicate, beta-sitosterol, sitosterol-3-beta o-glucoside, sodium, stigmasterol, sulfate, tartaric acid, tetradeca-(8z)-en-11,13 dien-2-one, thiamin, n-triacontanol, trideca-1-en-3,5,7,9,10-pentayne, tussilagine, vanallin, verbascoside sequiterpenes; acetic acid, alpha-amyrone, arabinose, alpha-bisabolene, gamma-bisabolene, cadinene, campesterol, cholesterol, cinnamaldehyde, commiferin, alpha-commiphoric acid, beta-commiphoric acid, gamma-commiphoric acid, commiphorinic acid, m-cresol, cumic alcohol, cuminaldehyde, dipentene, elemol, 3-epi-alpha-amyrin, eugenol, furanodiene, furanodienone, galactose, gum, heerabolene, alpha-heerabomyrrhol, beta-heerabomyrrhol, heeraboresene, limonene, 4-0-methyl-glucuronic acid, n-nonacesane, beta-sitosterol, xylose, caropylenes (carophylenes), lynderstyrene (lindestyrene), caropylenes (carophylenes), myrrha gum resin, curzenone, dihydro frianodine-6-one, 2-methoxyfurandiene, and lynderstyrene (lindestyrene).

Echinacea

Echinacea is a genus of nine species of herbaceous plants in the Family Asteraceae. All are native to eastern and central North America. In is also cultivated in the United Kingdom. The genus name is from the Greek echino, meaning “spiny”, due to the spiny central disk. Echinacea species are perennials which belong to the Aster family. There are 9 different types (species) of Echinacea of the family Astericaea, namely, Echinacea angustifolia ( Echinacea augustofolium )—Narrow-leaf Coneflower; Echinacea atrorubens ( Echinacea atribactilus )—Topeka; Purple Coneflower; Echinacea laevigata ( Echinacea vegetalis )—Smooth Coneflower or Smooth Purple Coneflower; Echinacea pallida ( Echinacea pallidae ) ( Echinacea pallidum ), —Pale Purple Coneflower; Echinacea paradoxa —Yellow Coneflower or Bush's Purple Coneflower; Echinacea purpurea —Purple Coneflower or Eastern Purple Coneflower; Echinacea sanguinea —Sanguine purple coneflower; Echinacea simulata —Wavyleaf Purple Coneflower; Echinacea tennesseensis —Tennessee Coneflower; and cultivars. Of nine Echinacea species, only three are used for medicinal purposes ( Echinacea angustifolia, Echinacea pallida , and Echinacea purpurea ).
Echinacea is also known as an American coneflower, black Susan, black Sampson, combflower, hedgehog, Indian head, Kansas snakeroot, purple coneflower, scurvy root, snakeroot, Niggerhead. Rudbeckia. Brauneria pallida (Nutt.), cock-up-hat, combflower, hedgehog, igelkopf, Indian head, kegelblume, red sunflower, rudbeckia , solhat, and sun hat.
Echinacea plants are herbaceous and drought-tolerant perennials growing to 1 or 2 m in height. Echinacea plants have tall stems, bear single pink or purple flowers and has a central cone that is usually purplish-brown in color. The large cone is actually a seed head with sharp spines that resemble a stiff comb. Echinacea leaves are lanceolate to elliptic, 10-20 cm long and 1.5-10 cm broad. Echinacea bark is thin. Echinacea wood is thick, in alternate porous, yellowish and black transverse wedges, and the rhizome has a circular pith.
Echinacea flowers bloom from early to late summer. Echinacea flowers are a composite inflorescence, with purple (rarely yellow or white) florets arranged in a prominent, somewhat cone-shaped head with the petals of the outer ray florets tend to point downward once the Echinacea flower head opens, thereby forming a cone. Echinacea flowers have a faint aromatic smell, with a sweetish taste, leaving a tingling sensation in the mouth.
The aboveground parts of the plant and roots of Echinacea can be used fresh or dried to make teas, squeezed juice, extracts, or preparations for external use.
The phytochemicals in the botanical Echinacea have demonstrated impressive activity against bacteria, viruses, and some fungi. Echinacea contains active substances that can enhance the activity of the immune system, reduce inflammation, and have hormonal, antiviral, and antioxidant effects. Echinacea can assist in the production of interferon and increases antiviral activity. Echinacea may reduce the severity of symptoms and reduce the duration of illness. Echinacea can improve the migration of white blood cells to attack foreign microorganisms and toxins in the bloodstream. Echinacea can also be an effective therapeutic agent in many infectious conditions.
Echinacea can be used externally or topically, preferably with other phytochemicals or compounds, to help treat various ailments, maladies, symptoms, and conditions, such as: (1) herpes, (2) acne and blemishes, (3) psoriasis, (4) gargles for sore throats, (5) wounds, (6) burns, (7) ulcers, (8) skin inflammations, (9) improve skin tone, (10) dry and cracked skin, (11) upper respiratory problems, (12) boils, (13) psoriasis, (14) eczema, (15) inflammatory skin conditions, (16) relieve pain, (17) insect bites, (18) arthritis (19) lymphatic swelling, (20) staph infections, (21) strep infections, (22) ear infections (also known as otitis media), (23) athlete's foot, sinusitis, and (24) hay fever (also called allergic rhinitis).
Echinacea can be used internally, preferably with other phytochemicals or compounds, to help treat various diseases, ailments, maladies, symptoms, and conditions, such as: (1) coughs, (2) common colds, (3) influenza (flu), (4) stimulate the immune system, (5) bronchitis and respiratory infections, (6) urinary infections, (7) chronic infections, (8) skin diseases, (9) fungal infections, (10) septicemia, (11) slow-healing wounds, (12) chronic fatigue syndrome, (13) venereal diseases, (14) enlarged lymph glands, (15) HIV, (16) hemorrhoids, (17) and a diphtheria, (18) putrid fevers, (19) erysipelas, (20) septicemia, (21) syphilis, (22) vaginal yeast (candida) infections (23) gastroenteritis, (24) weight loss, and (25) stimulate the production of white blood cells. Echinacea can increase bodily resistance to infection Herbalists consider Echinacea one of the best blood purifiers and an effective antibiotic. Echinacea can activate the body's immune system increasing the chances of fighting off any disease.
People are more likely to experience allergic reactions to Echinacea if they are allergic to plants in the daisy family, which includes ragweed, chrysanthemums, marigolds, and daisies. Also, people with asthma or atopy (a genetic tendency toward allergic reactions) may be more likely to have an allergic reaction when taking Echinacea.
The Plains Indians used various species of Echinacea to treat poisonous insect and snake bites, toothaches, sore throat, wounds, as well as mumps, smallpox, and measles. Echinacea angustifolia rhizome was used by North American Plains Indians, perhaps more than most other plants, for various herbal remedies. Echinacea was one of the basic antimicrobial herbs of Eclectic medicine in the mid 1800s through the early 1900s and was used for snakebite and anthrax. Echinacea has become popular in both Europe and America as a herbal medicine.
Echinacea purpurea is a species of a herbaceous plant of the genera Echinacea , which is one of three genera of the family Asteraceae. Echinacea purpurea has a long reported use for its therapeutic and medicinal effects and is commercially available in dry powder form. Some of these reported uses include topical treatment of snake bites, infected wounds, and intra-oral treatment of a sore throat.
The phytochemicals in the botanical Echinacea , and particularly Echinacea purpurea , have demonstrated impressive activity for use in help treating various symptoms, maladies, ailments and diseases. Echinacea purpurea is the preferred type of Echinacea for this application.
The constituents of Echinacea include essential oil, polysaccharides, polyacetylenes, betaine, glycoside, sesquiterpenes and caryophylene. Echinacea also contains copper, iron, tannins, protein, fatty acids and vitamins A, C, and E. The most important immune-stimulating components are the large polysaccharides, such as inulin, that increase the production of T-cells and increase other natural killer cell activity. Echinacea also contains fat-soluble alkylamides and a caffeic acid glycoside called echinacoside that further help contribute to the herb's immune empowering effects.
A phytochemical concentrate composition of Echinacea purpurea comprises the following isolated constituents, botanical extracts, microbial inhibitors, and antimicrobial isolates: polysaccharides, echinacen, echinaceine, echinacoside (caffeic acid ester), echinolone, echinadiol, enzymes, glucuronic acid, inuloid, pentadecadiene, polyacetylene compounds, arabinogalactan, rhamnose, PS I (a 4-0-methylglucoronoarabinoxylan, M r 35 kD) and PS II (an acid rhamnoarabinogalactan, M r 450 kD), cynarin (1,5-di-0-caffeoylquinic acid), chicoric acid (2,3-0-di-caffeoyltartaric acid) and derivatives, alkylamides, keto-alkynes and -alkenes; quinones; oils including: borneol, bornyl acetate; pentadeca-8(z)-en-2one, germacrene D; caryophyllene; caryophyllene epoxide; anthocyanins pyrrolizidine alkaloids; lipophilic amides, isobutylamides; polyacetylenes; myrrha gum resin; curzerenone (furahoeudesmane type); dihydro fuanodien-6-one; 2-methoxyfuranodiene (furanoelemene type); elamol; lyndestyrene (furanogermacrane type); alkylamides, apigenin, arabinogalacta, ascorbic acid, behenic-acid-ethyl-acid, betaine, borneol, bornyl-acetate, caffeic-acid, 2-0-caffeoyl-3-(5-alpha carboxybeta) 3,4 dihydroxyphenyl, 2-0-caffeoyl-3-0 cumaroyltaraic acid, 6-0-caffeoylechinacoside, 2-0-caffeoyl-3-0-feruloyltartaric acid, 2-0-caffeoyltartaric acid, calcium, carbonate, beta carotene, carophyllene, carophyllene-epoxide, chloride, chlorgenic acid, cichoric acid, cichoric-acid-methyl-ester, cobalt, cyanadin-3-0-(beta-d-glycopyranoside), cynadin-3-(6-0-malonyl beta-d-glycopyranoside), cynarin, deca (2e,4e,6e) trienoic acid-isobutylamide, des-rhamnosylverbascoside, 3,5-dicaffeoylquinic acid, 4-5-0 dicaffeoylquinic acid, 2,3-0-diferuloltartaric acid, do-deca-(2e,4e)-dienoic acid-isobutylamide, dodeca-2,4-dien-1-yl isovalerate, dodeca (2e,6z,8e,10e)-tetraenoic acid-isobutylamide, epishobunol, beta-farnesene, 2-0-feruloytartaric acid, germacrene, heptadeca-(8z,11z)-dien-2-one, heteroxylan, humulene 8-12, (e)-10-hydroxy-4,10-dimethyl 4,11-dodecadien-2-one, 13-hydroxyoctadeca-(9z,11e,15z)-trienoic-acid, inulin, iron, isochlorogenic acid, isorhamnetin-3-rutinoside, isotussilagine, kaempferol, kaempferol-3-glucoside, kaempferol-3-nutinoside, limonene, luteolin, luteolin-7-glucoside, magnesium, manganese, 2-methyltetradeca-5,12 diene, 2-methyltetradeca-6,12 dience, methyl-p-hydroxycinnamate, marcene, niacin, palmitic acid, pentadeca-(8z,11z)-dien-2-one, pentadeca-(8z,13z)-dien-11-lyn-2-one, pentadeca-8en-2-one, pentadeca-(8z)-en 2 one, pentadeca-(8z)-en-11,13 dien-2-one, 1-pentadecene, penta-(1,8z)-diene, phosphorous, alpha pinene, beta pinene, polyacetylenes, pontica epoxide, potassium, protein, quercetagetin-7-glucoside, quercetin, quercetin-3-galactoside, quercetin-3-glucoside, quercetin-3-robinoside, quercetin-3-xyloside, quercetin-3-xylosylgalactoside, rhamnoarabinogalactan, riboflavin, rutin, rutoside, selenium, silicate, beta-sitosterol, sitosterol-3-beta o-glucoside, sodium, stigmasterol, sulfate, tartaric acid, tetradeca-(8z)-en-11,13 dien-2-one, thiamin, n-triacontanol, trideca-1-en-3,5,7,9,10-pentayne, tussilagine, vanallin, verbascoside, sequiterpenes; acetic acid, alpha-amyrone, arabinose, alpha-bisabolene, gamma-bisabolene, cadinene, campesterol, cholesterol, cinnamaldehyde, commiferin, alpha-commiphoric acid, beta-commiphoric acid, gamma-commiphoric acid, commiphorinic acid, m-cresol, cumic alcohol, cuminaldehyde, dipentene, elemol, 3-epi-alpha-amyrin, eugenol, furanodiene, furanodienone, galactose, gum, heerabolene, alpha-heerabomyrrhol, beta-heerabomyrrhol, heeraboresene, limonene, 4-0-methyl-glucuronic acid, n-nonacesane, beta-sitosterol, xylose, caropylenes (carophylenes), myrrha gum resin, curzenone, dihydro fuanodien-6-one, and 2-methoxyfurandiene.
Echinacea purpurea is the preferred type of Echinacea for this application. Echinacea purpurea can comprise by weight based upon the total weight of the composition:
from about 0.3% to about 9% by weight echinacoside; from about 0.1% to about 7% by weight PS I (4-O-methyglucoronoarabinoxylan, M r 35 kD) and PS II (acid rhamnoarabinogalactan, M r 450 kD); from about 0.1% to about 10% by weight cynarin (1,5-di-O-caffeoylquinic acid) and chicoric acid (2,3-O-di-caffeoyltartaric acid); from about 0.2% to about 4% by weight echinolone; from about 0.2% to about 8% by weight echinacin B; from about 0.1% to about 6% by weight echinaceine; from about 2% to about 7% by weight anthonocyanins comprising cyanidin 3-O-B-D-glucopyranoside and 3-O-(6-O-malonyl-B-D-glucopyranoside); from about 0.01% to about 0.06% by weight pyrrolizidine alkaloids comprising tussilagine and isotussilagine; from about 0.003% to about 0.009% by weight isomeric dodeca isobutylamides and tetroenoic acid; and from about 0.01% to about 2% by weight carophylenes.
The chemical formula of some of the botanical extracts of Echinacea is shown hereinafter.










Commiphora Myrrha

There are different types of Commiphora (also sometimes referred to as “ Commiphora ”), such as: Commiphora myrrha, Commiphora molmol, Commiphora erythraea , and their cultivars. In the application, Commiphora myrrha , also sometimes referred to as “ myrrha ” is the preferred type of Commiphora.
The chemical formula of some of the botanical extracts of Commiphora myrrha are shown below.









Myrrha is also sometimes referred to as: myrrh, mirre, myrrhis, gummi myrrha, myrrha vera, gum myrrh, Commiphora resin, gruggal gum, gruggal resin, Heerabol myrrh, myrrhe, Manniliche myrrhe, Opopanax , and Hirabol myrrh. Myrrha can comprise gum resin obtained from cuts made in the bark of trees of the genus Commiphora myrrha , i.e. the myrrh tree. Myrrha can also comprise balsamic juices from Balsamodendron myrrha , i.e. a buraceous tree. Myrrha can also be extracted from Osmorhiza or Washingtonia , which is also sometimes referred to a sweet cicely. The myrrh tree is a native in Erythrea, Abyssinia, Somalia, Yemen, Sudan, and elsewhere.
The myrrh-producing Commiphora species are shrubs or small trees with large, sharply pointed thorns on the stem. The unequal ternate leaves are alternate and the small flowers are arranged in terminal panicles. When damaged, the schizogenous resin ducts yield the drug myrrh.
Myrrha is an air-dried oleo-gum resin that exudes from the bark of Commiphora species. The material comprises irregular, rounded grains or lumps of varying sizes with holes and range in color from dark brown and almost black to light or dark orange-brown; some parts may be yellow or colorless to pale yellow. The surface is mostly covered with a grey to yellowish grey powder; the fracture is conchoidal and yields thin, translucent fragments. Myrrha can have a sweet fragrance and a harsh and aromic order. Myrrha can have a bitter and aromatic taste. Myrrha can also be acrid and can stick to the teeth on chewing.
Commiphora molmol and other Commiphora species, insofar as the chemical composition of their gum-resin, are comparable with that of myrrha DAB 10. There is considerable confusion in the literature regarding the sources of myrrh and the identity of the Commiphora species involved. Common (or hirabol) myrrh appears to derive from Commiphora myrrha . Somalian myrrh is said to come from Commiphora molmol . However, the systematic (taxonomic) relationship between Commiphora myrrha and Commiphora molmol is not clear. The source of Abyssinian myrrh is Commiphora madagascariensis or Commiphora abyssinica. Opopanax , which is also referred to as bisabol myrrh or perfumed bdellium is believed to originate from either Commiphora erythraea (Ehrenb) or Opopanax.
The composition of myrrha is very complex and only partially known from 40-60% of myrrha is soluble in ethanol and comprises a resin and an essential oil. Myrrha contains polysaccharides, triterpenoids, triterpene acids and an essential oil containing sesquiterpenes and furano sesquiterpenes. More specifically, Myrrha consists almost entirely of sesquiterpenes. The main components of sesquiterpenes are: furanosesquiterpenes of the germacrane elemane, eudesmane, and guaiane types. In addition, there are sesquiterpene hydrocarbons, and sesquiterpene alcohols, e.g. elemol. Presumably, some of the furanosesquiterpenes are characteristic of pharmaceutical myrrh. Myrrha crude gum or crude mucilage includes 20% proteins and 65% carbohydrates which are made up of galactose, 4-0-methylglucuronic acid, and arabinose. Commiphora myrrhaphyto chemicals comprise: acetic acid, alpha-amyrone, arabinose, alpha-bisabolene, gamma-bisabolene, cadinene, campesterol, cholesterol, cinnamaldehyde, commiferin, alpha-commiphoric acid, beta-commiphoric acid, gamma-commiphoric acid, commiphorinic acid, m-cresol, cumic alcohol, cuminaldehyde, dipentene, elemol, 3-epi-alpha-amyrin, eugenol, furanodiene, furanodienone, galactose, gum, heerabolene, alpha-heerabomyrrhol, beta-heerabomyrrhol, heeraboresene, limonene, 4-0-methyl-glucuronic acid, n-nonacesane, beta-sitosterol, xylose, caropylenes (carophylenes), myrrha gum resin, curzenone, dihydro fuanodien-6-one, 2-methoxyfurandiene, and lynderstyrene (lindestyrene).
Macro and microscopically, myrrha can appear as a brownish yellow powder characterized by yellowish splinters or spherical grains of various sizes, along with fine granular material which swells in water. In chloral-hydrate mounts, there are only a few fragments of tissue from the plant source: reddish brown fragments of cork, individual and groups of polyhedral to oblong stone cells, partly with greatly thickened, pitted, and lignified walls and brownish contents; fragments of thin-walled parenchyma and sclerenchymatous fibres, and irregular prismatic to polyhedral crystals of calcium oxalate.
Myrrha should be protected from light and moisture in well-closed containers. It is best with a desiccant, since the carbohydrate part of the drug readily absorbs water. Preferably, myrrha should not be stored in powdered form.
Myrrha has many medicinal benefits. Myrrha is a pungent, stimulating, astringent, carminative, astringent and aromatic herb. Myrrha also has anti-catarrhal, anti-inflammatory, antimicrobial, antiseptic, astringent, balsamic, carminative, cicatrisant, emmenagogue, expectorant, fungicidal, sedative, digestive, stomachic, tonic, and vulnerary properties.
Significantly, Myrrha can be used externally as a topical preparation to help treat infected wounds, minor skin inflammations, as well as inflammation of the throat, gums and mouth, including mouth ulcers, gingivitis, stomatitis and sinusitis. Myrrha can also help treat sores, such as: eczemas, skin ulcers and bed sores.
Myrrha can be helpful to relieve spasms, promote healing, fight inflammation, and reduce digestive discomfort. Myrrha also enhances cell regeneration.
Myrrh can be used internally to help treat stomach and chest problems, bronchial infection, and menstrual problems.
Myrtle
The Myrtle ( Myrtus ) is a genus of one or two species of flowering plants in the family Myrtaceae, native to southern Europe and northern Africa. Myrtle are evergreen shrubs or small trees, growing to 5 m tall. The leaves of Myrtle are entire, 3-5 cm long, with a pleasantly fragrant essential oil. The star-like flowers of Myrtle have five petals and sepals, and an amazingly large number of stamens. Petals are usually white, with globose blue-black berries containing several seeds.
The Common Myrtle Myrtus communis , is widespread in the Mediterranean region and is also by far the most commonly cultivated. The other species, Saharan Myrtle M. nivellei , is restricted to the Tassili n'Ajjer mountains in southern Algeria and the Tibesti Mountains in Chad, where it occurs in small areas of sparse relict woodland near the centre of the Sahara Desert; it is listed as an endangered species.
Preferably, the medical composition excludes undesired Myrtle and its extracts, which can inhibit or retard the unexpected advantageous results of the novel medicinal composition.

Centaurea

Centaurea (Cen-tau-ré-a) is a genus of about 350-500 species of herbaceous thistle-like flowering plants in the family Asteraceae, and is widely found in the United States and Canada. Common names for different species of Centaurea include star-thistle, cornflower, knapweed and bluet. Some species of Centaurea are cultivated as ornamental plants in gardens.
Species of the genus Centaurea include: Centaurea adpressa, Centaurea aggregata, Centaurea akamantis, Centaurea alpestris, Centaurea alpina, Centaurea americana ( Plectocephalus americanus ] )—American Star-thistle, Centaurea appendicigera —endemic in Turkey, Centaurea argentea, Centaurea aspera —Rough Star-thistle, Centaurea atropurpurea, Centaurea babylonica, Centaurea behen, Centaurea bella, Centaurea bovina, Centaurea bulbosa, Centaurea cachinalensis, Centaurea calcitrapa —Purple Star-thistle or Caltrop—Europe, Centaurea calcitrapoides, Centaurea chilensis, Centaurea cineraria —Dusty Miller, Centaurea clementei, Centaurea cyanus —Cornflower, Centaurea dealbata, Centaurea debeauxii, Centaurea depressa, Centaurea diffusa —Diffuse Knapweed, Centaurea diluta, Centaurea dschungarica, Centaurea eriophora, Centaurea floccosa, Centaurea gayana, Centaurea glastifolia, Centaurea grinensis, Centaurea gymnocarpa, Centaurea horrida, Centaurea hypoleuca, Centaurea iberica —A spiny Mediterranean species thought to be the “thistle” mentioned in Genesis, Centaurea imperialis, Centaurea jacea, Centaurea kasakorum, Centaurea kopetaghensis, Centaurea kotschyana, Centaurea leucophylla, Centaurea macrocephala, Centaurea maculosa —Spotted Knapweed; eastern Europe; introduced in North America, now an invasive weed which releases a toxin that reduces growth of forage species, Centaurea marschalliana, Centaurea melitensis, Centaurea moschata —Sweet Sultan, Centaurea monocephala, Centaurea montana —Perennial Cornflower, Centaurea nira —Black Knapweed or Common Knapweed, Centaurea nigrescens —Tyrol Knapweed; southern and eastern Europe, Centaurea orientalis, Centaurea ovina, Centaurea paniculata, Centaurea phrygia, Centaurea pindicola, Centaurea polypodiifolia, Centaurea pulcherrima, Centaurea ragusina, Centaurea rothrockii ( Plectocephalus rothrockii ), Centaurea ruthenica, Centaurea rutifolia, Centaurea sadleriana —Pannonian Knapweed, Centaurea scabiosa —Greater Knapweed, Centaurea seridis, Centaurea sibirica, Centaurea simplicicaulis, Centaurea solstitialis —Yellow Star-thistle; Europe, Centaurea squarrosa, Centaurea stenolepis, Centaurea stoebe, Centaurea sulphurea, Centaurea transalpina, Centaurea tchihatcheffii —, Mogan Lake, Ankara, Turkey, Centaurea trichocephala, Centaurea triniifolia, Centaurea triumfettii, Centaurea uniflora , and Centaurea virgata.
Preferably, the medical composition excludes undesired Centaurea and its extracts, which can inhibit or retard the unexpected advantageous results of the novel medicinal composition.
Folic Acid
The preferred nutrient is folic acid for best results. Folic acid, also referred to as folacin, pteroylglutamic acid, foldine, folaemin, foliamin, folicet, folipac, follettes, folsan, folvite, incafolic, millafol or cytofol, is a yellow, crystalline, water-soluble vitamin of the B complex group essential for cell growth and reproduction. Folic acid functions as a coenzyme with vitamins B 12 and vitamin C in the breakdown and utilization of proteins and in the formation of nucleic acids and heme in hemoglobin. Folic acid also increases the appetite and stimulates the production of hydrochloric acid in the digestive tract. Folic acid is stored in the liver and may be synthesized by the bacterial flora of the gastrointestinal tract. Deficiency of folic acid can result in poor growth, graying hair, glossitis, stomatitis, gastrointestinal lesions, and diarrhea, and it may lead to megaloblastic anemia. Deficiency is caused by inadequate dietary intake of the vitamin, malabsorption, or metabolic abnormalities. Need for folic acid is increased as in pregnancy, infancy, and stress. Folic acid is both heat and light labile, and considerable loss of the vitamin occurs when it has been stored for a long period. Folic acid is nontoxic and is effective in treating specific deficiency states. The chemical formula of folic acid is shown below.









The chemical structure of folic acid is shown hereinafter:









Pteroylglutamic Acid
Folic Acid
The folic acid molecule contains glutamic acid, p-aminobenzoic acid, and a pterin; the combination of the pterin and p-aminobenzoic acid is termed pterocid acid. The structure shown is the pteroylglutamic acid of liver. The folic acid produced by bacteria contains three glutamic acid residues combined in γ-glutamyl linkage. Many animal tissues contain pteroylheptaglutamic acid, the glutamic acid residues again being in γ-glutamyl linkage. Synthetic pteroylpolyglutamic acids, in which the glutamic acid molecules are linked in a-glutamyl bonds, are active in bacterial growth assays; pteroyl-γ-glutamic acids are effective both in bacteria and in the treatment of macrocytic anemia in man. An enzyme in animal tissues hydrolyzes the naturally occurring pteroylpolglutamate compounds to pteroylmonglutamic acid and free glutamic acid.
Another structural formula of pteroylglutamic acid (PteGlu 1 ) is shown below.

























Position
Radical
Congener











N 5
—CH 3
CH 3 H 4 PteGlu
Methyltetrahydrofolate


N 5
—CHO
5-CHOH 4 PteGlu
Folinic acid (Citrovorum Factor)


N 10
—CHO
10-CHOH 4 PteGlu
10-Formyltetrahydrofolate


N 5-10
—CH—
5,10-CHH 4 PteGlu
5,10-Methenyltetrahydrofolate


N 5-10
—CH 2 —
5,10-CH 2 H 4 PteGlu
5,10-Methylenetetrahydrofolate


N 5
—CHNH
CHNHH 4 PteGlu
Formiminotetrahydrofolate


N 10
—CH 2 OH
CH 2 OHH 4 PteGlu
Hydroxymethyltetrahydrofolate



The structures and nomenclature of pteroylglutamic acid (folic acid).
































Major portions of the folic acid molecule include a pteridine ring linked by a methylene bridge to paraminobenzoic acid, which is joined by an amide linkage to glutamic acid. While pteroylglutamic acid is the common pharmaceutical form of folic acid, it is neither the principal folate congener in food nor the active coenzyme for intracellular metabolism. Following absorption, PteGlu 1 is rapidly reduced at the 5, 6, 7, and 8 positions to tetrahydrofolic acid (H 4 PteGlu 1 ), which then acts as an acceptor of a number of one-carbon units. These are attached at either the 5 or the 10 position of the pteridine ring or bridge these atoms to form a new five-membered ring.
Vitamin B 12 and folic acid are dietary essentials for man. A deficiency of either vitamin results in defective synthesis of DNA in any cell that attempts chromosomal replication and division. Since tissues with the greatest rate of cell turnover show the most dramatic changes, the hematopoietic system is especially sensitive to deficiencies of these vitamins. Clinically, the earliest sign of deficiency is a megaloblastic anemia, where the derangement in DNA synthesis results in a characteristic morphological abnormality of the precursor cells in the bone marrow. Abnormal macrocytic red blood cells are the product, and the patient becomes severely anemic.
Methylcobalamin supports the methionine synthetase reaction, which is essential for normal metabolism of folate. Methyl groups contributed by methyltetrahydrofolate (CH 3 H 4 PteGlu 1 ) are used to form methylcobalamin, which then acts as a methyl group donor for the conversion of homocysteine to methionine. This folate-cobalamin interaction is pivotal for normal synthesis of purines and pyrimidines and, therefore, of DNA. The methionine synthetase reaction is largely responsible for the control of the recycling of folate cofactors; the maintenance of intracellular concentrations of folylpolyglutamates; and, through the synthesis of methionine and its product, S-adenosylmethionine, the maintenance of a number of methyylation reaction. Since methyltetrahydrofolate is the principal folate congener supplied to cells, the transfer of the methyl group to cobalamin is essential for the adequate supply of tetrahydrofolate (H 4 PteGlu 1 ), the substrate for a number of metabolic steps. Tetrahydrofolate is a precursor for the formation of intracellular folylpolyglutamates; it also acts as the acceptor of a one-carbon unit in the conversion of serine to glycine, with the resultant formulation of 5,10 methylenetetrahydrofolate (5,10-CH 2 H 4 PteGlu). The latter derivative donates the methylene group to deoxyuridylate for the synthesis of thymidylate—an extremely important reaction in DNA synthesis. In the process, the 5,10-CH 2 H 4 PteGl is converted to dihydrofolate (H 2 PteGlu). The cycle is then completed by the reduction of the H 2 PteGlu to H 4 PteGlu by dihydrofolate reductase, the step that is blocked by folate antagonists such as methotrexate. Other pathways also lead to the synthesis of 5,10 methylenetetrahydrofolate.
Table A
Biosynthesis of Folic Acid




The biosynthesis of folic acid is shown below. The symbol ppp represents triphosphate.









TABLE A



Biosynthesis of Folic Acid

The biosynthesis of folic acid is shown below. The symbol ppp represents triphosphate






























Folate can be transported to tissues as CH 3 H 4 PteG 1 . The liver actively reduces and methylates PteGlu 1 (and H 2 or H 4 PteGlu 1 ) and then transports the CH 3 H 4 PteGlu 1 into bile for reabsorption by the gut and subsequent delivery to tissues, CH 3 H 4 PteGlu acts as a methyl donor for the formation of methylcobalamin and as a source of H 4 PteGlu and other folate congeners, as described previously. Folate is stored within cells as polyglutamates.
Surfactants
While benzalkonium chloride is the preferred surfactant for best results, in some circumstances it may be desirable to use other quanternary ammonium surfactants or other surfactants.
The quaternary ammonium compound can be dicocodimonium chloride, which is also known as dicoco alkyldimethyl, chlorides or dicoco dimethyl ammonium chloride or Di-C8-18-alkyldimethyl, chlorides. This can be used in combination with isopropanol, such as 20-30% isopropanol. The preferred source of quaternary compound comprises: 70-80% quaternary ammonium compound and less than 0.03% methyl chloride, has a specific gravity of about 0.87 at 115 degrees F., a vapor pressure of 33 mm/Hg at 68 degrees F., an initial boiling point of 180 degrees F. at 760 mm/Hg, and a volatility of 20-30%, and is produced under the brand name CarSpray 300 by Witco Corporation, Dublin, Ohio, USA. The quaternary compound can provide disinfecting qualities and serves as a fungicide to treat fungus and yeast infections.
Other quaternary ammonium compounds may be useful, such as produced under the brand name Jet Quat 2C-75 by Jetco Chemicals, Inc. of Corsicana, Tex., USA, or produced under the brand names Carspray 400 and Carnauba Spray 200 by Witco Corporation, Dublin, Ohio, USA, or containing 9% denatured ethyl alcohol such as sold under the brand name BTC 2125M by Stephan Company, Northfield, Ill., USA, or the following MAQUAT products comprising n-alkyl dimethyl benzyl ammonium chloride produced by Mason Chemical Company, Arlington Heights, Ill., USA. LC-12S (67% C12, 25% C14, 7% C16, 1% C18), MC 1416 (5% C12, 60% C14, 30% C16, 5% C18), MC1412 (40% C12, 50% C14, 10% C16), SC-18 stearyl paste or flake (5% C16, 95% C18), TC-76 or MQ-2525 (5% C12, 60% C14, 30% C16, and 5% C18) and MC6025-50% (25% C12, 60% C14 and 15% C16). Jet Quat 2C-75 comprises: 50-75% dicoco dimethyl quaternary ammonium chloride, 20-50% isopropyl alcohol, has a specific gravity of 0.88 and a boiling point of 180 degrees F. CarSpray 400 comprises: 55-65% quaternary ammonium compounds, 20-30% amines, C14-18 & C16-18 unsaturated, alkyl, ethoxylated, 10-20% isopropanol, and less than 0.03% methyl chloride, and has a specific gravity of approximate 0.88 at 75 degrees, F, a vapor pressure of 33 mm/Hg at 68 degrees F., an initial boiling point of 180 degrees F. at 760 mm/Hg, and a volatility of 10-20%. Carnauba Spray 200 comprises: 50-60% quaternary ammonium compounds, 10-20% isopropanol, 15-25% water, 1-10% alkoylated carnauba wax, and less than 0.03% methyl chloride, and has a specific gravity of about 0.90 at 80 degrees F., a vapor pressure of 33 mm/Hg at 68 degrees F., an initial boiling point of 180 degrees F. at 760 mm/Hg, and a volatility of 20-40%.
Nonionic surfactants are surface-active compounds which do not ionize in water solution. Often times these possess hydrophilic characteristics by virtue of the presence therein of an oxygenated chain (e.g., a poly-oxyethylene chain), the lyophilic portion of the molecule being derived from fatty acids, phenols, alcohols, amides or amines. Exemplary compounds are the poly-(ethylene oxide) condensates of alkyl phenols, e.g. the condensation product formed from one mole of nonyl phenol and ten moles of ethylene oxide, and the condensation products of aliphatic alcohols and ethylene oxide, e.g. the condensation product formed from 1 mole of tridecanol and 12 moles of ethylene oxide.
The nonionic surfactants can comprise phenol ethoxylates comprising a condensate product of ethylene oxide and an alkyl phenol or an aliphatic alcohol. The nonionic surfactants preferably comprise nonophenol ethoxylate such as T-DET, and/or octaphenol ethoxylate. The nonionic surfactants are reaction products of ethylene oxide and nonolphenol and/or octalphenol. The ratio of the phenol to the ethylene oxide can range from 2:20 to 4:16 and preferably is about 8:12.
Nonionic synthetic surfactants can comprise nonionic detergents. Nonionic synthetic surfactants can also be formed by condensing ethylene oxide with a hydrophobic base formed by the condensation of propylene oxide with propylene glycol. The hydrophobic portion of the molecule which, of course, exhibits water insolubility has a molecular weight of from about 1200 to 2500. The addition of polyoxyethylene radicals to this hydrophobic portion tends to increase the water solubility of the molecule as a whole and the liquid character of the product can be retained up to the point where polyoxyethylene content is about 50% of the total weight of the condensation product. Other nonionic synthetic surfactants can include: the polyethylene oxide condensates of alkylphenols, e.g. the condensation products of alkylphenols or dialkylphenols wherein the alkyl group contains from about 6 to 12 carbon atoms in either a straight chain or branched chain configuration, with ethylene oxide. The ethylene oxide can be present in amounts equal to 8 to 25 moles of ethylene oxide per mole of alkylphenol. The alkyl substituent in such compounds can be derived from polymerized propylene, diisobutylene, n-octene, or n-nonene.
Nonionic surfactants can also be produced from the condensation of ethylene oxide with the reaction product of propylene oxide and ethylenediamine, e.g. compounds containing from about 40% to about 80% polyoxyethylene by weight and having a molecular weight of from about 5,000 to about 11,000 resulting from the reaction of ethylene oxide groups with a hydrophobic base comprising the reaction product of ethylenediamine and excess propylene oxide; the base having a molecular weight on the order of 2,500 to 3,000.
Other nonionic surfactants include the condensation product of aliphatic alcohols having from 8 to 18 carbon atoms, in either straight chain or branched chain configuration, with ethylene oxide, e.g. a coconut alcohol ethylene oxide condensation having from 10 to 30 moles of ethylene oxide per mole of coconut alcohol, and the coconut alcohol fraction having from 10 to 14 carbon atoms.
Further nonionic surfactants include long chain tertiary amine oxides corresponding to the following general formula: R 1 R 3 R 2 N→O wherein R1 is an alkyl radical of from about 8 to 18 carbon atoms, and R 2 and R 3 are each methyl or ethyl radicals. The arrow in the formula is a conventional representation of a semi-polar bond. Examples of amine oxides suitable for use include: dimethyldodecylamine oxide, dimethyloctylamine oxide, dimethyldecylamine oxide, dimethyltetradecylamine oxide, and dimethylhexadecylamine oxide.
Other nonionic surfactants can include: long chain tertiary phosphine oxides corresponding to the following general formula RR′R″P→O wherein R is an alkyl, alkenyl or monohydroxyalkyl radical ranging from 10 to 18 carbon atoms in chain length and R′ and R″ are each alkyl or monohydroxyalkyl groups containing from 1 to 3 carbon atoms. The arrow in the formula is a conventional representation of a semi-polar bond. Examples of suitable phosphine oxides are: dimethyldodecylphosphine oxide, dimethyltetradecylphosphine oxide, ethylmethyltetradecylphosphine oxide, cetyldimethylphosphine oxide, dimethylstearylphosphine oxide, cetylethylpropylphosphine oxide, diethyldodecylphosphine oxide, diethyltetradecylphosphine oxide, dipropyldodecylphosphine oxide, bis-(2-hydroxymethyl)dodecylphosphine oxide, bis-(2-hydroxyethyl)dodecylphosphine oxide, (2-hydroxy propyl)methyltetradecylphosphine oxide, dimethyloleylphosphine oxide, and dimethyl-(2-hydroxydodecyl)phosphine oxide.
In some circumstances it may be useful to use other surfactants such as: another cationic surfactant, an ampholytic surfactant or a zwitterionic surfactant.
The cationic surfactants can include cationic detergents. The cationic surfactants comprise compounds which ionize in an aqueous medium to give cations containing the lyophilic group. Typical of these compounds are the quaternary ammonium salts which contain an alkyl group of about 12 to about 18 carbon atoms, such as lauryl benzyl dimethyl ammonium chloride.
Ampholytic surfactants are compounds having both anionic and cationic groups in the same molecule. Exemplary of such compounds are derivatives of aliphatic amines which contain a long chain of about 8 to about 18 carbon atoms and an anionic water solubilizing group, e.g., carboxysulfo, sulfo or sulfato. Examples of ampholytic detergents are: sodium-3-dodecylaminopropane sulfonate, sodium-N-methyl taurate, and related substances such as higher alkyl disubstituted amino acids, betaines, thetines, sulfated long chain olefinic amines, and sulfated imidazoline derivatives.
Zwitterionic surfactants can include synthetic detergents. Zwitterionic surfactants are generally derivatives of aliphatic quaternary ammonium compounds in which the aliphatic radical can be a straight chain or branched and wherein one of the aliphatic substituents contains from about 8 to 18 carbon atoms and one contains an anionic water solubilizing group, e.g., carboxy, sulfo, or sulfato. Examples of compounds falling within this definition are: 3-(N,N-dimethyl-N-hexadecyl ammonio)-propane-1-sulfonate and 3-(N,N-dimethyl-N-hexadecyl ammonio)-2-hydroxy propane-1-sulfonate.
Clinical Pharmacology
When the Echinacea and Commiphora phytochemicals (antimicrobial isolates, botanical extracts and microbe inhibitors) were mixed, combined and applied with: a surfactant, preferably benzalkonium chloride; a nutrient carrier, preferably folic acid; and a sterile aqueous carrier; the results were unexpected and surprisingly good in resolving (treating) HIV, and other infectious diseases and the effectiveness of the medicine (microbicide) dramatically increased. Significantly, when tested in vitro, the unique compound demonstrated unexpectedly and surprisingly good antiviral activity against HIV including inhibition of the attachment of HIV to target cells. When the synergistic medicine was tested topically in vivo, herpes simplex infections were immediately arrested. When the synergistic medicine was tested in vitro, the benzalkonium chloride surfactant was substantially less toxic and within a safe level and there was a higher level of inhibitory activity against HIV and HSV 1 & 2. The synergism interaction and mixing of the Echinacea and Commiphora phytochemicals, folic acid, and surfactant were demonstrated and observed by viewing the rapid solubility of the components when mixed and the slight adhesive quality created by the properties in solution. Furthermore, the chemical properties of the Echinacea and Commiphora phytochemicals, surfactant nutrient carrier (nutrient), and aqueous carrier enhanced stabilization and increased reactivity which is useful in treating infectious diseases.
The medicine can be used in varying dilutions on: oral and nasal mucosa; vaginal tissue; labial tissue; anal and peri-anal tissue; penile tissue; cutaneous tissue; open subcutaneous tissue; and in higher dilutions on ocular infections and preferably rectal or vaginal administration. By varying the concentrations, the medicine may possibly be administered parenterally. The medicine may be contraindicated in vaginal or anal passages; in pack dressing; in the ear canal; occlusive dressings; casts or ingestion and such use may produce irritation or chemical burns. It may not be advisable to use the medicine to treat anaerobic fungal infections, since some fungi may be resistant.
Examples 1-7
In Vivo Testing
In an initial, topical application, in-vivo study that was undertaken to evaluate the effects of the medical treatment and medicine of the present invention upon seven human test subjects who had been tested positive for HSV 1 or 2. The subjects were treated topically with the medicine comprising benzalkonium chloride surfactant in an aqueous solution (at a ratio of 1:750) in combination with the herbaceous botanical Echinacea purpurea in powdered form containing the previously listed phytochemicals. Application of the composition was made by a two-step procedure by first wetting the affected area or vesicle with the benzalkonium chloride surfactant in an aqueous solution by spraying, dabbing, or using a dropper; then applying a coating of the powdered phytochemicals over the wetted area by either swab or manually sprinkling the powder onto the infected area. An important aspect in this treatment was maintaining complete coverage of the affected area for the duration of the outbreak. Therefore, the area of outbreak was kept covered with the medical composition by reapplying as needed. Of the seven subjects, six were female, and one was male. At the beginning of this study, the age of the male was 38, the female subjects were ages 8, 27, 30, 32, 38, and 39. There were twelve infectious outbreaks over approximately six weeks. Nine of the outbreaks were HSV 2, genital herpes, and three were HSV1, cold sores. The 8 year old and the 27 year old females exhibited the HSV 1 (cold sores). The 30 year old, 38 year old and the 39 year old females exhibited the HSV 2 (genital herpes). The 38 year old also had a HSV 1 cold sore. The male exhibited HSV 2 (genital herpes). All subjects tested had a well established history of the disease and could identify the standard course of their disease. To obtain objective data, none of the test subjects knew anything about the test treatment or any action of the medicine. On repeat tests, the subjects were told that there may be placebos mixed in the samples of formula.
In seven cases, the antimicrobial compound (medicine) was applied directly on tissue at the prodrome stage. In five cases, the antimicrobial compound was applied directly on erupted vesicles. The antimicrobial compound was reapplied as necessary to maintain coverage.
Observations: With each application of the medicine, each individual (test subject) reported a tingling sensation for a few seconds. They also reported that there was a substantial degree of adherence of the medicine (antimicrobial) compound to the vesicle(s) or affected area. The adherence of the composition to the epithelial tissue remained to a degree even after showering or water rinsing the area.
Results: The results of the testing of the 7 subjects with the medical treatment and medicine were unexpectedly surprisingly good and very consistent. In each case, the subject happily reported that once the composition (medicine) was applied to the affected area, the pain completely stopped within 10 to 20 minutes when nothing in the past had ever eased pain before. In the seven cases, where the compound (medicine) was applied at the prodrome stage, the subjects reported that the pain stopped, all symptoms that would have previously escalated to full outbreak ceased and the outbreak never again occurred. All external symptoms and physical manifestations of herpes disappeared within a few hours after the medicine was applied. In the five cases, where the compound (medicine) was applied to erupted vesicles, the subjects reported that the pain stopped in minutes and the burning, itching and irritation resolved in two to four hours and the vesicles dried up and were gone in twenty-one hours. In all cases, the other more extreme, debilitating symptoms of: fever, malaise, inguinal swelling, weeping sores and painful urination stopped, once the medicine was applied.
In follow-up, where subjects had been given a supply of the composition (medicine) to test on future outbreaks, it was reported that if the initial signs of an outbreak exhibited, signaling the prodrome stage of an outbreak, the compound (medicine) was immediately applied by the subjects as per instructions and the outbreak was fully arrested and resolved. Significantly, it was also reported by subjects who were accustomed to experiencing several outbreaks annually, that they had remarkably longer latency periods. In a three year follow-up with one individual who had reported severe outbreaks monthly for four years prior to use of this medicine, she now reports that she has not had an outbreak in over a year since using this medicine.
Additional Observations: One human male subject reported that after the initial application during the prodrome phase of an outbreak, he showered and forgot to reapply the composition (medicine) for a period of approximately 30 hours. Consequently, several vesicles erupted and began to coalesce. The subject proceeded to reapply the composition (medicine) and thereafter kept the area well coated with the composition. Subsequently, the outbreak resolved in 21 hours in the same manner as described with the other human subjects.
Another observation indicated that the composition (medicine) may be weakened or less effective in the presence of certain proteins or soaps. One human female subject, may have been overly zealous in cleansing the affected area prior to application of the composition (medicine). This occurred during a third outbreak after having success with the composition (medicine) on the two prior outbreaks. In this instance, when the composition (medicine) was applied, there was no familiar tingling sensation and no relief from symptoms. Approximately 24 hours elapsed before she sought any advice and the outbreak had escalated to the full vesicular eruption stage with all the foregoing symptoms of the disease. She was instructed to thoroughly rinse any soap residue from the area, dry the area and reapply the composition (medicine). After following the instructions, she reported that the outbreak has been fully resolved, as it had in the two prior outbreaks, by applying the medical composition.
Examples 8-13
Dermatological and Veterinary Testing
Animal testing to determine any possible dermatological allergic reaction induced by the medical composition (medicine) was undertaken. Six animal subjects were used. The animals included 3 female rabbits (ages unknown); 2 dogs (1 female 2 year old, and 1 male 9 year old); one, 3 year old neutered male cat. In these animal tests, the above composition (medicine) was applied, in the previously stated method, to the inside of the outer ear of each animal. In all instances, the area being treated was kept coated with the compound for twenty-four hours, matching the time human subjects had used. The testing performed on the six animal subjects indicated that there were no signs of dermatological irritation or allergic reaction.
Example 14
The above medical compound containing viral inhibitors was also tested on a papilloma virus caused wart on the muzzle of a two year old gelded thoroughbred horse. Papilloma virus warts are difficult to treat. The wart measured 25 mm in diameter. The antimicrobial compound (medicine) was applied twice daily. The wart was then measured at each application.
Results: Quite unexpectedly, the wart decreased dramatically in size by approximately 3 mm per day while the medicine was applied to the wart and on the fifth day fell off completely. It was observed that, at first the surface layers of the wart began to degrade, exposing large erythematous papules. Then interestingly, the warts did not just diminish in size by flaking or peeling, they diminished at the point of attachment on the subject's epidermis and fell off still somewhat intact with no sequela scarring.
In an ongoing, long term in vivo study of this invention, which began with the first seven subjects in April of 1989 and has now spanned 7 years, approximately 100 infectious outbreaks have been treated with the medicine in different concentrations, as described previously. In all cases the surprisingly good results were the same: 1. Pain disappears in minutes; 2. No outbreak occurs when the composition is applied at the prodrome stage; 3. The outbreak resolves in twenty-one hours when applied at the vesicular stage.
In Vitro Testing
Laboratory testing was undertaken at the University Of Chicago, Clinical Microbiology Laboratories to determine inhibitory activity in vitro of the medical treatment and composition (medicine). The laboratory testing was conducted by the Associate Director, PhD, and Associate Professor of Pathology. The in vitro testing of the medical composition, referred to as the “Drug” below, yielded surprisingly good results. It was determined that the medical treatment and composition had unexpectedly, surprising excellent inhibitory activity on HSV 1 and HSV 2. It was stated by the pathologist, that he had tested “hundreds” of other compounds and had never seen anything as good as what this compound did.
The following are the tests of the medicine that were conducted and results that were obtained at The University of Chicago. For ease of interpreting some of the scientific data and test results, the following definitions apply:
“MEM” pertains to Minimal Essential Medium. This is the culture medium used in laboratories for growing the cells upon which tests were run. “Fibroblast” is a mesenchyme human cell (a cell found in connective tissue, blood, bone, lymphatics, and cartilage). “IC 50 ” pertains to the Inhibitory Concentrate. For this testing a 50% endpoint was selected, as is typical. The number following indicates the greatest dilution below 50%. Therefore it is the definition of the endpoint. If an area under a dilution is left blank, it indicates that there may have been toxicity at that dilution, the test may not have been worth reading, or no interpretable data is available. If an area under dilution is marked with a hyphen (-), it indicates that there are no plaques and there is a successful inhibition of herpes (HSV).

Examples 15-17
In these in vitro tests, the following drugs (medicine) was used:
Drug #1.=Benzalkonium chloride surfactant in an aqueous solution at a ratio of 1:750. The surfactant in the aqueous solution was filtered before use and diluted in an equal volume of 2×MEM to give a 1:1500 dilution in 1×MEM.
Drug #2 =Echinacea powder (photochemicals) in an aqueous solution. This preparation was extracted by warm infusion in sterile water. The extracted phytochemicals was centrifuged and filtered before use. The filtered phytochemicals were diluted in an equal volume of 2×MEM to give the undiluted preparation in 1×MEM.
Drug #3 =Echinacea powder (phytochemicals) were extracted and combined with benzalkonium chloride surfactant by a cold infusion process. The combined preparation was centrifuged and filtered before use and diluted in an equal volume of 2×MEM to give the undiluted preparation in 1×MEM.
1. Three 24-compartment plates were inoculated with fibroblasts. Three different extractions (for comparison) in five concentrations of the composition were used to screen for antiviral activity in concentrations of: undiluted, 1:2, 1:4, 1:8, and 1:16 in 1×MEM. There were four control compartments on each plate containing MEM without drug.
2. The growth media was removed from the compartments and 200 ul of HSV-1 was added to each compartment of the upper half of each plate. HSV-1 was diluted 1:5000 (2.0 ul of stock HSV-1 in 10 mL of MEM). The virus titer was 3×10 6 per mL. Also, 200 ul of HSV-2 was added to each compartment of the lower half of each plate. HSV-2 was diluted 1:2,000 (5.0 ul of stock HSV-2 in 10 mL of MEM). The virus titer was 6×10 5 per mL.
3. The plates were incubated at 37° C. for two hours.
4. The inoculum was removed and one mL of the MEM containing Drugs #1-3 were added to the four compartments. The concentration of the drug compared to the MEM is indicated below.










TABLE 1



Concentration
Undiluted
1:2
1:4
1:8
1:16












Drug (ul)
4000
2000
1000
500
250

MEM (ul)
—
2000
3000
3500
3750






















5. Results: HSV-1, liquid overlay, Drug added immediately after virus absorption.
Plate 1, Drug #1 contaminated with bacteria! No growth, maybe debris. Plate 2, Drug #2 contaminated with bacteria! No growth, maybe debris. Plate 3, Drug #3 The results are indicated in Tables 2 and 3 below.






TABLE 2



Drug #3 HSV 1 Test Results






Concentration











undiluted
1:2
1:4
1:8
1:16













plaques
54
toxic
toxic
—
6*
12**


plaques
42
toxic
toxic
—
4*
16**

Average
48



5
14
IC 50 > 1:16






























TABLE 3 Drug #3 HSV 2 Test Results Concentration undiluted 1:2 1:4 1:8 1:16 plaques 46 toxic toxic — 22* 32** plaques 49 toxic toxic — 21* 28** Average 48 22 30 IC 50 = 1:8 *slight toxicity. **very small plaques Comments: Testing with the medicine (Drug #3) provided
excellent results. The cells look fine with no contamination. At the lower dilutions, the preparation may be toxic to some of the cells. This preparation was unexpectedly successful in its inhibitory activity.

Examples 18-20
Three 24-compartment plates were inoculated with fibroblasts and the following drugs.
Test Drug #1A=Benzalkonium chloride surfactant in an aqueous solution. The benzalkonium chloride surfactant was prepared by making a 1:375 dilution in water (32 ul in 12.0 mL of sterile water). This was filtered before use. This was diluted in an equal volume of 2×MEM to give 1:750 dilution in 1×MEM. The dilution was done to maintain the ratio.
Test Drug #2A= Echinacea purpurea powder (phytochemicals) in an aqueous solution. This preparation was a 50 mg/mL solution (300 mg in 6.0 mL of water) of Echinacea purpurea powder in sterile water. The mixture was vortexed and refrigerated for four hours. The Echinacea powder preparation was centrifuged at 3500 rpm for 15 minutes at 10° C. and filtered before use and then diluted in an equal volume of 2×MEM to give the undiluted preparation in 1×MEM.
Test Drug #3A= Echinacea purpurea powder (phytochemicals) dissolved in benzalkonium chloride surfactant. This preparation was a 50 mg/mL solution (300 mg in 6.0 mL of benzalkonium chloride, 1:375). The mixture was vortexed and refrigerated for four hours. The phytochemical and surfactant mixture was centrifuged at 3500 rpm for 15 minutes at 10° C. and filtered before use, and then diluted in an equal volume of 2×MEM to give the undiluted preparation in 1×MEM.
1. Three plates were used to screen the three drug preparations. The concentrations needed to screen for antiviral activity were 1:2, 1:4, 1:8, and 1:16 in 1×MEM. There were four control compartments on each plate containing MEM without drug.
2. The growth media was removed from the compartments and 200 ul of HSV-1 was added to each compartment of the upper half of each plate. HSV-1 was diluted 1:5000 (2.0 ul of stock HSV-1 in 10 mL of MEM). The virus titer was 3×10 6 per mL.
3. The plates were incubated at 37° C. for four hours.
4. The inoculum was removed and one mL of the MEM containing drugs #1A-3A were added to the four compartments.







TABLE 4





Concentration









Undiluted
1:2
1:4
1:8
1:16













Drug (ul)
4000
2000
1000
500
250


MEM (ul)
—
2000
3000
3500
3750


























5. Results: HSV-1, liquid overlay, composition added immediately after virus absorption.





TABLE 5



Drug #1A - HSV 1 Test Results





Concentration









1:2
1:4
1:8
1:16
1:32














plaques
70
toxic
toxic
toxic
toxic
toxic


plaques
68


plaques
58


plaques
74


Average
70


IC 50





Comments: These compartments have a fine precipitate over the cells. Benzalkonium chloride probably precipitates with the protein in the medium.




































TABLE 6



Drug #2A - HSV 1 Test Results





Concentration









1:2
1:4
1:8
1:16
1:32













plaques
72
—
—
—

9*
12*

plaques
74
—
—
—

7
8

plaques
79
—
—
—

4
12

plaques
71
—
—
—

7
11

Average
70


IC 50 > 1:32



Comments: Although there were some plaques, they were very small.




































TABLE 7



Drug #3A - HSV 1 Test Results





Concentration









1:2
1:4
1:8
1:16
1:32












plaques
72
toxic
toxic
toxic
toxic
—*

plaques
68



—

plaques
67



—

plaques
70



—

Average
70


IC 50 > 1:32



Comments: Although there was some toxicity, this drug was very successful in inhibiting the virus, there did not appear to be any plaques.































Examples 21-24
Four 24-compartment plates were inoculated with fibroblasts.
Test Drug #1B=Benzalkonium chloride surfactant in an aqueous diluent. The benzalkonium chloride was prepared by making a 1:1000 dilution in water (10 ul in 10.0 mL of sterile water). This was filtered before use and diluted in an equal volume of 2×MEM to give 1:2000 dilution in 1×MEM. (500 ul drug plus 500 ul of 2×MEM).
Test Drug #2B= Echinacea purpurea powder (phytochemicals) in an aqueous solution. This preparation was a 50 mg/mL solution (250 mg in 5.0 mL of water) of Echinacea purpurea powder in sterile water. The mixture was vortexed and refrigerated for four hours. This Echinacea powdered preparation was centrifuged at 3500 rpm for 15 minutes at 10° C. and filtered before use, and diluted in an equal volume of 2×MEM to give the undiluted preparation in 1×MEM. (500 ul drug plus 500 ul of 2×MEM).
Test Drug #3B= Echinacea purpurea powder (phytochemicals) dissolved in benzalkonium chloride surfactant. This preparation was a 50 mg/mL solution (250 mg in 5.0 mL of benzalkonium chloride, 1:1000). The mixture was vortexed and refrigerated for four hours. The Echinacea phytochemicals and surfactants were centrifuged at 3500 rpm for 15 minutes at 10° C. and filtered before use, and then diluted in an equal volume of 2×MEM to give the preparation in 1×MEM (500 ul drug plus 500 ul of 2×MEM).
Test Drug #4B= Echinacea purpurea powder (phytochemicals) in an aqueous solution (diluent) and then mixed with benzalkonium chloride surfactant at a ratio of 1:1000. This preparation was a 50 mg/mL solution (250 mg in 5.0 mL in 5.0 mL of water) of Echinacea purpurea powder in sterile water. The mixture was vortexed and refrigerated for four hours. The aqueous phytochemicals were centrifuged at 3500 rpm for 15 minutes at 10° C. and filtered before use. This preparation was diluted in an equal volume of benzalkonium chloride at a ratio of 1:1000, to get the Echinacea -benzalkonium chloride mixture. This mixture was diluted with equal volume of 2×MEM to give the 1:4 preparation in 1×MEM (500 ul drug #1 and 250 ul drug #2 plus 500 ul of 2×MEM).
1. Four plates were used to screen the four drug preparations. The concentrations needed to screen for antiviral activity were 1:20, 1:40, 1:80, and 1:160 and 1:320 in 1×MEM. There were four control compartments on each plate containing MEM without drug.
2. The growth media was removed from the compartments and 200 ul of HSV-1 was added to each compartment of the upper two rows of each plate. HSV-1 was diluted 1:5000 (2.0 ul of stock HSV-1 in 10 mL of MEM). The virus titer was 3×10 6 per mL. Also, 200 ul of HSV-2 was added to each compartment of the lower half of each plate. HSV-2 was diluted 1:2,000 (5.0 ul of stock HSV-2 in 10 mL of MEM). The virus titer was 6×10 5 per mL.
3. The plates were incubated at 37° C. for four hours.
4. The inoculum was removed and one mL of the MEM containing drugs #1-4 was added to the four compartments.







TABLE 8





Concentrate









1:20
1:40
1:80
1:160
1:320













Drug (ul)
400
200
100
50
25


MEM (ul)
3600
3800
3900
3950
3975


























5. Results: HSV-1, liquid overlay, drugs added immediately after virus absorption.





TABLE 9



Drug #1B - HSV 1 Test Results





Concentration









1:20
1:40
1:80
1:160
1:320














plaques
37
toxic
toxic
toxic
toxic
15?*


plaques
45




18?*


Average
41



IC 50





Comments: Slightly toxic, test was difficult to read.


HSV-2, liquid overlay, drugs added immediately after virus absorption.



































TABLE 10



Drug #1B - HSV 2 Test Results






Concentration











1:20
1:40
1:80
1:160
1:320














plaques
38

toxic
toxic
toxic
toxic
21


plaques
42





17

Average
40





19
IC 50 > 1:320



Comments: Test was too toxic to give a good reading.


































TABLE 11



Drug #2B - HSV 1 Test Results






Concentration











1:20
1:40
1:80
1:160
1:320













plaques
39
2*
8*
23*
24
44


plaques
40
3
18
11
28
38

Average
40
3
13
17
26

IC 50 > 1:80



Comments: Small plaques.


































TABLE 12



Drug #2B - HSV 2 Test Results






Concentration











1:20
1:40
1:80
1:160
1:320












plaques
48
21
33



plaques
52
22
38

Average
50
21.5
35.5

IC 50 > 1:20



































TABLE 13



Drug #3B - HSV 1 Test Results






Concentration











1:20
1:40
1:80
1:160
1:320













plaques
44
1*
17
31
37



plaques
46
—
16
28
27

Average
45
—
17
30
32

IC 50 > 1:40



Comments: Although there was some toxicity, drug very successful there did not appear to be any plaques.


































TABLE 14



Drug #3B - HSV 2 Test Results






Concentration











1:20
1:40
1:80
1:160
1:320













few cells

11*
27
30
35



plaques
44
10
32

Average
44
11
29.5



IC 50 > 1:20



Comments: A difficult test to get a really good reading. However the drug has successful inhibitory activity.


































TABLE 15



Drug #4B - HSV 1 Test Results






Concentration











1:40
1:80
1:160
1:320
1:640













plaques
47
toxic
toxic
toxic
33



plaques
48


28

Average
48


30


IC 50 > 1:320



Comments: Too toxic at the higher levels. Nonetheless, there was inhibitory activity at 1:320


































TABLE 16



Drug #4B - HSV 2 Test Results






Concentration











1:40
1:80
1:160
1:320
1:640













plaques
38
toxic
toxic
toxic
2*
16


plaques
40



4
20

Average
39



3
18
IC 50 > 1:640



Comments: Toxicity probably due to the benzalkonium chloride. The drug at the 1:320 dilution showed very strong inhibitory activity.





























The in vitro tests of Examples 21-24 used raw materials which were not refined. Nevertheless, the tests demonstrate surprisingly good viral inhibitory activity and a probable synergy between the constituents.
In the preceding in vitro tests where Drugs #3, 3A and 3B, were Echinacea purpurea phytochemicals extracted and combined with benzalkonium chloride surfactant, the resultant medicine, demonstrated the greater antiviral activity, and most remarkably demonstrated a synergy between the components: Echinacea purpurea and benzalkonium chloride. This can possibly be explained by a shared stability and enhanced reactivity between the two components. The benzalkonium chloride in the synergistic mixture exhibited a lesser degree of toxicity and the synergistic combination (medicine) exhibited a greater degree of antiviral activity, particularly with HSV-2.
HIV Tests
Viracea-1 and Viracea-2 were tested for evaluation of anti-HIV activity in acute infection model assays. Additional assays were performed to evaluate the range and mechanism of action of the two compounds.
Compounds Viracea-1 and Viracea-2 were supplied as solutions. Formulation included filtering of the solution and centrifugation. The high test concentration used in each assay varied from a 1:5 dilution to a 1:100 dilution in tissue culture medium. Each compound was stored at 70° C. prior to use. In these tests, the following drugs (composition) were used.
Viracea 1=
Viracea 2=
Propagation and Quantitation of Cell Lines and Virus Stocks
Cells utilized in the compound screening assays were designated as the CEM-SS cell line. These cells are highly susceptible to infection with HIV, rapidly form multinucleated syncytia, and are eventually killed by HIV. These cells are easily maintained (2-7×10 3 cells per ml) in RPMI 1640 tissue culture medium supplemented with 10% fetal bovine serum, glutamine, and antibiotics. The cells are passaged twice weekly at 1:20 dilution. Passage number is logged each week and the cells are discarded after twenty weeks of passage and fresh CEM-SS cells are thawed and utilized in the assay. Stocks of CEM-SS cells have been frozen in liquid nitrogen in 1 ml NUNC vials in 90% fetal calf serum and 10% dimethyl sulfoxide (DMSO). Following thawing, CEM-SS cells are routinely ready to be utilized in the primary screen assay after two weeks in culture. Prior to replacing a late passage cell line, the new CEM-SS cells are tested in the screening assay protocol utilizing the current stock of infectious virus and AZT. If the infectivity of the virus is significantly different on the new cells or if AZT appears less active than expected the new cells will not be entered into the screening program. Mycoplasma testing is routinely performed on all cell lines (see above).
Virus pools are prepared and titrated in CEM-SS cells, placed in 5 ml aliquots, and frozen at −135° C. After thawing, unused virus is discarded to avoid changes in infectious titer. Optimization assays have documented a one-log reduction in virus titer upon the first freeze-thaw cycle, and less drastic titer reduction with subsequent rounds of freeze-thaw. Virus pools are prepared by the acute infection of 5×10 5 CEM-SS cells with HIV in a volume of 200 μl at a multiplicity of infection determined to give complete cell killing at day 7 post-infection (approximately 0.05 for the III B isolate of HIV-1 and 0.01 for the RF isolate of HIV-1). Infection is allowed to proceed for one hour at 37° C. and then the cells are transferred to a T25 flask and the volume is increased to 2 ml. On day 1 post-infection the volume is brought to 5 ml and on day 2 the volume is increased to 10 ml. Beginning on day 4, the cells are pelleted, the supernatant is saved and the cells are resuspended in a fresh 10 ml aliquot of tissue culture medium. Complete medium changes on a daily basis, rather than allowing growth of the cells in the medium for longer periods of time, allows the virus inoculum utilized in the primary screen to remain relatively undepleted of nutrients when it is used to infect cells. The staining reaction utilized (XTT) requires that the glucose concentration remain high. Wells depleted of glucose by cell growth will not permit metabolic conversation of the tetrazolium dye to the formazan product.
Cell-free supernatants from the acutely infected cells are saved on day 4, day 5, day 6, and day 7. An aliquot of supernatant is saved separately on each day for use in titer determination. Titer determinations include reverse transcriptase activity assay, endpoint titration or plaque assay (CEM-SS) quantitation of infectious particles, and quantitation of cell killing kinetics. It has been determined that peak levels of infectious virus are produced in the acutely infected cultures as the viability of the cells falls through the 50% level. Since the primary screening assay quantifies the protective effects of a compound by its ability to inhibit HIV-induced cytopathic effects, the quantity of virus required to kill CEM-SS cells in 6 days is routinely utilized to determine the amount of virus required per well in the primary screening assay. Each of the daily pools is titrated in the primary screening XTT assay protocol by performing two-fold dilutions of the virus beginning at a high test concentration of 50 μl of virus per well. The tetrazolium dye XTT staining method is utilized to determine the exact amount of virus required to kill all the CEM-SS cells in each well and this minimum amount of virus is utilized for performance of all primary testing. Identical methods are utilized to prepare all virus isolates utilized in the laboratory, including laboratory derived strains of HIV-1, HIV-2 and STV. Clinical isolates utilized are passaged in fresh human cells and the methods for the growth of these cells and the production of virus pools is described below.
Microtiter Antiviral XTT Assay
Cell Preparation
CEM-SS cells or other established human cell line used in these experiments were passaged in T-150 flasks for use in the assay. On the day preceding the assay, the cells were split 1:2 to assure they would be in an exponential growth phase at time of infection. On the day of assay the cells were washed twice with tissue culture medium and resuspended in fresh tissue culture medium. Total cell and viability counting was performed using a hemacytometer and trypan blue dye exclusion. Cell viability was greater than 95% for the cells to be utilized in the assay. The cells were pelleted and resuspended at 2.5×10 4 cells per ml in tissue culture medium. Cells were added to the drug-containing plates in a volume of 50 μl.
Virus Preparation
A pretitered aliquot of virus was removed from the freezer (−80° C.) and allowed to thaw slowly to room temperature in a biological safety cabinet. The virus was resuspended and diluted into tissue culture medium such that the amount of virus added to each wall in a volume of 50 μl will be the amount determined to give complete cell killing at 6 days post-infection. In general the virus pools produced with the IIIB isolate of HIV required the addition of 5 μl of virus per well. Pools of RF virus were five to ten fold more potent, requiring 0.5-1 μl per well. TCID 50 calculation by endpoint titration in CEM-SS cells indicated that the multiplicity of infection of these assays ranged from 0.005-2.5.
Plate Format
The format of the test plate has been standardized and contained cell control wells (cells only), virus control wells (cells plus virus), drug toxicity control wells (cells plus drug only), drug colorimetric control wells (drug only) as well as experimental wells (drug plus cells plus virus).
Examples 25-48
XTT Staining of Screening Plates
After 6 days of incubation at 37° C. in a 5% CO 2 incubator the test plates were analyzed by staining with the tetrazolium dye XTT. XTT-tetrazolium is metabolized by the mitochondrial enzymes of metabolically active cells to a soluble formazan product, allowing the rapid quantitative analysis of the inhibition of HIV-induced cell killing by anti-HIV test substances. On day 6 post-infection plates were removed from the incubator and observed. The use of round bottom microtiter plates allows rapid macroscopic analysis of the activity of a given test compound by the evaluation of pellet size. The results of the macroscopic observations were confirmed and enhanced by further microscopic analysis. XTT solution was prepared only as a stock of 1 mg/ml in PBS. Phenazine methosulfate (PMS) solution was prepared at 15 mg/ml in PBS and stored in the dark at −20° C., XTT/PMS stock was prepared immediately before use by diluting the PMS 1:100 into PBS and adding 40 ml per ml of XTT solution. Fifty microliters of XTT/PMS was added to each well of the plate and the plate was reincubated for 4 hours at 37° C. Adhesive plate sealers were used in place of the lids, the sealed plate was inverted several times to mix the soluble formazan product and the plate was read spectrophotometrically at 450 nm with a Molecular Devices Vmax plate reader. Using a % CPE Reduction, % Cell Viability, IC 25, 50 & 95 . TC 25, 50 & 95 and other indices were calculated.





TABLE 17



IN VITRO ANTIVIRAL RESULTS

XTT ASSAY

FOR VIRACEA 1














1
2
3
4
5
6
7
8
9
10
11
12










Reagent

Plastic



Background

Background














0.169
0.160
0.160
0.159
0.154
0.167
0.066
0.063
0.058
0.061
0.063
0.067














Experimental


Experimental



Tox
cc/vc
High Conc.
Tox
Tox
Low Conc.
cc/vc
Tox
















1.498
1.461
0.196
0.378
0.278
1.466
1.474
0.204
0.211
0.208
1.517
1.511

1.392
1.461
0.192
0.196
0.293
1.414
1.479
0.205
0.247
0.185
1.496
1.497

1.333
1.426
0.318
1.410
1.372
1.356
1.482
0.242
0.182
0.215
1.478
1.519

1.208
0.219
1.134
1.181
1.110
1.206
1.487
0.219
0.208
0.215
0.189
1.512

1.032
0.193
0.940
0.828
0.968
0.944
1.480
0.192
0.207
0.254
0.309
1.506

0.656
0.222
0.596
0.582
0.544
0.572
1.464
0.206
0.254
0.186
0.184
1.468







Colorimetric Background-High
Colormetric Background-Low


Concentrations
Concentrations
















0.289
0.182
0.168
0.171
0.166
0.167
0.163
0.173
0.172
0.166
0.164
0.180































































TABLE 18



VIRACEA 1








STRN
RF














Reagent
0.62
Drug Viracea 1
25%
50%
95%

Virus Control
0.058
TC
1:66
1:18.5
1:10

Cell Control
1.312
IC
1:625
1:400

Differential
1.254
Antiviral Index (AJ)
9.47
21.6
































TABLE 19



VIRACEA 1









Antiviral
Cytotoxicity



Drug Viracea 1
Test Values
Test Values











Row on

Mean
% Red in
Mean
% Cell
Colorimetric


Plate
Conc. (um)
O.D.
Viral CPE
O.D
Viability
Control














Based on
Low
B
0.00003
−.030
0%
1.313
100%
0.018

values of

C
0.0001
−.010
0%
1.324
100%
0.003

columns

D
0.00032
−.011
0%
1.334
100%
0.005

7 through 12

E
0.001
−.015
0%
1.328
100%
0.010

(right side

F
0.0032
−.013
0%
1.321
100%
0.011

of plate)

G
0.01
−.006
0%
1.303
99%
0.002

Based on

B
0.032
0.059
5%
1.315
100%
0.006

values of

C
0.1
0.003
0%
1.237
94%
0.005

columns

D
0.32
0.804
64%
1.173
89%
0.010

1 through 6

E
1
0.915
73%
1.039
79%
0.007

(left side

F
3.2
0.673
54%
0.807
62%
0.020

of plate)
high
G
10
0.228
18%
0.326
25%
0.127














































TABLE 20



IN VITRO ANTIVIRAL RESULTS

XTT ASSAY

FOR VIRACEA 2














1
2
3
4
5
6
7
8
9
10
11
12










Reagent

Plastic



Background

Background














0.169
0.163
0.164
0.166
0.160
0.170
0.074
0.072
0.067
0.067
0.067
0.068














Experimental


Experimental



Tox
cc/vc
High Conc.
Tox
Tox
Low Conc.
cc/vc
Tox
















1.468
1.421
0.461
0.257
1.170
1.467
1.501
0.207
0.222
0.214
1.506
1.503

1.321
1.397
1.316
0.209
0.191
1.340
1.494
0.200
0.202
0.204
1.446
1.487

0.906
1.345
0.249
0.764
0.836
0.953
1.485
0.227
0.179
0.179
1.453
1.500

0.219
0.256
1.190
0.207
0.210
0.234
1.491
0.204
0.190
0.228
0.192
1.506

0.168
0.190
0.161
0.161
0.148
0.157
1.503
0.237
0.195
0.202
0.186
1.501

0.242
0.223
0.238
0.239
0.230
0.242
1.495
0.201
0.204
0.227
0.189
1.503






Colormetric Background-High
Colorimetric Background-Low

Concentrations
Concentrations
















0.258
0.172
0.159
0.165
0.163
0.165
0.165
0.166
0.166
0.171
0.159
0.169































































TABLE 21



VIRACEA 2








STRN
RF







Regent
0.165
Drug Viracea 2
25%
50%
95%

Virus Control
0.041
TC
1:450
:250
1:100

Cell Control
1.263
IC
1:900

Differential
1.222
Antiviral Index (AI)
2.02





























TABLE 22



VIRACEA 2









Antiviral
Cytotoxicity



Drug Viracea 2
Test Values
Test Values











Row on

Mean
% Red in
Mean
% Cell
Colorimetric


Plate
Conc. (um)
O.D.
Viral CPE
O.D
Visibility
Control














Based on
Low
B
0.00003
0.004
0%
1.335
100%
0.004

values of

C
0.0001
0.002
0%
1.331
100%
−.006

columns

D
0.00032
−.017
0%
1.321
100%
0.006

7 through 12

E
0.001
0.000
0%
1.332
100%
0.001

(right side

F
0.0032
0.004
0%
1.336
100%
0.001

of plate)

G
0.01
0.005
0%
1.334
100%
0.000

Based on

B
0.032
0.090
7%
1.302
100%
0.000

values of

C
0.1
0.368
30%
1.167
92%
−.002

columns

D
0.32
0.410
34%
1.764
61%
0.000

1 through 6

E
1
0.002
0%
1.067
5%
−.006

(left side

F
3.2
−.056
0%
−.010
0%
0.007

of plate)
High
G
10
−.063
0%
−.016
0%
0.093









































Examples 49-54
Reverse Transcriptase Activity Assay
A microtiter based reverse transcriptase (RT) reaction was utilized. Tritiated thymidine triphosphate (NEN) (TTP) was resuspended in distilled H 2 0 at 5 Ci/ml. Poly rA and oligo dT were prepared as a stock solution which was kept at −20° C. The RT reaction buffer was prepared fresh on a daily basis and consists of 125 μl 1MEGTA, 125 μl dH 2 O, 125 μl Triton X-100, 50 μl 1M Tris (pH 7.4), 50 μl 1MDTT, and 40 μl 1MMgCl 2 . These three solutions were mixed together in a ratio of 1 parts TTP, 2.5 parts poly rA:oligo dT, 2.5 parts reaction, the reaction buffer and 4 parts distilled water. Ten microliters of this reaction mixture was placed in a round bottom microtiter plate and 15 μl of virus containing supernatant was added and mixed. The plate was incubated at 37° C. and incubated for 60 minutes. Following reaction, the reaction volume was spotted onto filter mats, washed 6 times for 5 minutes each in a 5% sodium phosphate buffer, 2 times for 1 minute each in distilled water, 2 times for 1 minute each in 70% ethanol, and then dried. The dried filter mat was placed in a plastic sample bag. Betaplate scintillation fluid was added and the bag was heat sealed. Incorporated radioactivity was quantitated utilizing a Wallac Microbeta scintillation counter.





TABLE 23



VIRACEA-1: PBMC/ROJO

REVERSE TRANSCRIPTASE ACTIVITY





Conc.














1:0
1:100,000
1:32,000
1:10,000
1:3200
1:1000
1:320
1:100
1:32
1:10
















Sample 1
28139
31490
35838
42526
39967
38024
20042
12715
415
1742

Sample 2
24587
35989
35757
32780
34191
25895
16677
7587
12495
12513

Sample 3
32527
34334
34782
31899
43755
34038
28838
10896
4251
7551

Sample 4
28418
33938
35459
35768
39304
32652
21852
10399
5720
7269

% VC
100.0
119.4
124.8
125.9
138.3
114.9
76.9
36.6
20.1
25.6






































TABLE 24



VIRACEA-1: PBMC/ROJO

TOXICITY VALUES





Conc.














1:0
1:100,000
1:32,000
1:10,000
1:3200
1:1000
1:320
1:100
1:32
1:10
















Sample 1
2.029
2.167
2.200
2.137
1.975
2.025
0.966
0.764
0.840
1.033

Sample 2
2.120
2.234
2.169
2.203
2.263
1.895
1.009
0.696
0.916
1.058

Sample 3
1.879
2.176
2.160
2.053
2.038
1.847
0.916
0.734
0.768
1.128

Sample 4
2.009
2.192
2.176
2.131
2.092
1.922
0.964
0.731
0.841
1.073

% VC
100.0
109.1
108.3
106.1
104.1
96.7
48.0
38.4
41.9
53.4






































TABLE 25



VIRACEA-2: PBMC/ROJO

Reverse Transcriptase Activity





Conc.














1:0
1:100,000
1:32,000
1:10,000
1:3200
1:1000
1:320
1:100
1:32
1:10
















Sample 1
28139
31734
36488
34880
31240
2287
7436
463
96
38

Sample 2
24587
27559
33120
23103
33408
20550
9478
265
103
81

Sample 3
32527
24114
23828
28137
23174
25825
11132
309
77
55

Sample 4
28418
27802
31145
26677
29274
16221
9349
346
92
58

% VC
100.0
97.8
109.6
100.9
103.0
57.1
32.9
1.2
0.3
0.2






































TABLE 26



VIRACEA-2: PBMC/ROJO

TOXICITY VALUES





Conc.














1:0
1:100,000
1:32,000
1:10,000
1:3200
1:1000
1:320
1:100
1:32
1:10
















Sample 1
2.029
1.547
1.460
1.488
1.345
1.354
0.860
0.546
0.429
0.611

Sample 2
2.120
1.503
1.548
1.622
1.902
1.489
0.971
0.529
0.434
0.627

Sample 3
1.879
1.364
1.463
1.720
1.649
1.223
0.772
0.451
0.433
0.633

Sample 4
2.009
1.471
1.490
1.610
1.632
1.355
0.868
0.509
0.432
0.624

% VC
100.0
73.2
74.2
80.1
81.2
67.5
43.2
25.3
21.5
31.0

































ELISA
ELISA kits were purchased from Coulter. The assay is performed according to the manufacturer's recommendations. Prior to ELISA analysis, the reverse transcriptase activity assays were routinely performed and the values were used for incorporated radioactivity in the RT activity assay to determine the dilution of samples required for the ELISA. Control curves were generated in each assay to accurately quantititate the amount of capsid protein in each sample. Data was obtained by spectrophotometric analysis at 450 nm using a Molecular Devices Vmax plate reader. P24 concentrations were calculated from the optical density values by use of the Molecular Devices software package Soft Max.
Infectious Particles
Infectious virus particles were quantitated utilizing the CEM-SS plaque assay and the Quantitative infectivity assay for HIV-1 and HIV-2. Flat bottom 96-well microtiter plates were coated with 50 μl of poly-L-lysine at 50 μg/ml for 2 hours at 37° C. The wells were then washed with PBS and 2.5×10 5 CEM-SS cells were placed in the microtiter well where they became fixed to the bottom of the plate. Enough cells were added to form a monolayer of CEM-SS cells in each well. Virus containing supernatant was added from each well of the XTT phase, including virus and cell controls and each serial dilution of the test substance. The number of syncytia were quantitated in the flat bottom 96-well microtiter plate with an Olympus CK2 inverted microscope at 4 days following infection. Each syncytium resulted from a single infectious HIV virion.
Anti-HIV Activity in Fresh Human Cells: Assay in Fresh Human T-Lymphocytes
Fresh human peripheral blood lymphocytes (PBL) were isolated from voluntary Red Cross donors, seronegative for HIV and HBV. Leukophoresed blood is diluted 1:1 with Dulbecco's phosphate buffered saline (PBS), layered over 14 mL of Ficoll-Hypaque density gradient in a 50 mL centrifuge tube. Tubes were then centrifuged for 30 minutes at 600×.g. Banded PBLs were gently aspirated from the resulting interface and subsequently washed 2× with PBS by low speed centrifugation. After final wash, cells were enumerated by trypan blue exclusion and re-suspended at 1×10 7 /mL in RPMI 1640 with 15% Fetal Bovine Serum (FBS), 2 mM L-glutamine, 4 μg/mL PHA-P and allowed to incubate for 48-72 hours at 37° C. After incubation, PBLs were centrifuged and reset in RPMI 1640 with 15% FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 10 μg/mL gentamicin, and 20 U/mL recombinant human IL-2. PBLs were maintained in this medium at a concentration of 1-2×10E6/mL with bi-weekly medium changes, until use in the assay protocol.
For the PBL assay, PHA-P stimulated cells from at least two normal donors were pooled, set in fresh medium at 2×10E6/mL and plated in the interior wells of a 96 well round bottom microplate at 50 μL/well. Test drug dilutions were prepared at a 2× concentration in microtiter tubes and 100 μL of each concentration is placed in appropriate wells in a standard format. 50 μL of a predetermined dilution of virus stock was placed in each test well. Wells with cells and virus alone were used for virus control. Separate plates were identically set without virus for drug cytotoxicity studies using an XTT assay system.
In the standard PBL assay (MOI: 0.2), the assay was ended on day 7 following collection of cell free supernatant samples for reverse transcriptase activity assay. In the low MOI PBL assay (MOI: 0.02), supernatant samples were collected on day 6, day 11, and day 14 post-infection and analyzed for RI activity. Tritiated thymidine triphosphate (NEN) (TTP) was resuspended in distilled H 2 O at 5 Ci/ml. Poly rA and oligo dT were prepared as a stock solution which was kept at −20° C. The RT reaction buffer was prepared fresh on a daily basis and consists of 125 μl 1MEGTA, 125 μl dH 2 O, 110 μl 10% SDS, 50 μl 1M Tris (pH 7.4), 50 μl 1M DTT, and 40 μl 1M MgCl 2 . These three solutions were mixed together in a ratio of 2 parts TTP, 1 part poly rA:oligo dT, and 1 part reaction buffer. Ten microliters of this reaction mixture was placed in a round bottom microtiter plate and 15 μl of virus containing supernatant was added and mixed. The plate was incubated at 37° C. in a water bath with a solid support to prevent submersion of the plate and incubated for 60 minutes. Following reaction, the reaction volume was spotted onto pieces of DE81 paper, washed 5 times for 5 minutes each in a 5% sodium phosphate buffer, 2 times for 1 minute each in distilled water, 2 times for 1 minute each in 70% ethanol, and then dried. Opti-Fluor O was added to each sample and incorporated radioactivity was quantitated utilizing a Wallac 1450 Microbetaplus liquid scintillation counter.
Tritiated thymidine incorporation was measured in parallel cultures at day 7. Each well was pulsed with 1 μCi of tritiated thymidine and the cells were harvested 18 hours later with a Skatron cell harvester onto glass fiber filter papers. The filters were dried, placed in a scintillation vial with 1 ml of scintillation cocktail and incorporated radioactivity was quantitated on a Packard Tri-Carbh 1900 TR liquid scintillation counter.
Examples 55-78
Anti-HIV Activity in Fresh Human Cells; Assay in Fresh Human Monocyte Macrophages
For isolation of a adherent cells, 3×10 6 non-PHA stimulated peripheral blood cells were resuspended in Hanks buffered saline with calcium and magnesium supplemented with 10% human AB serum. The cells were placed in a 24-well microtiter plate at 37° C. for 2 hours. Non-adherent cells were removed by vigorously washing six times. The adherent cells were cultured for 7 days in RPMI 1640 tissue culture medium with 15% fetal bovine serum. The cultures were carefully monitored for confluency during this incubation period. Infection of the cells was performed with the monocytotropic HIV-1 strains BaL or ADA and the matched pair of AZT-sensitive and AZT-resistant virus isolates. Each of these virus isolates was obtained from the NLAID AIDS Research and Reference Reagent Program. High titer pools of each of these viruses have been harvested from infected cultures of peripheral blood adherent cells and frozen in 1.0 ml aliquots at −80° C. Monocyte-macrophage monolayers were infected at an MOI of 0.1. Compounds to be evaluated in the monolayers were infected at an MOI of 0.1. Compounds to be evaluated in the monocyte-macrophage assay were added to the monolayers shortly before infection in order to maximize the potential for identifying active compounds.
At two days post-infection, the medium was decanted and the cultures washed twice with complete medium in order to remove excess virus. Fresh medium alone or medium containing the appropriate concentration of drugs was added and incubation continued for an additional 5 days. XTT-tetrazolium or trypan blue exclusion assays for cell viability and HIV p24 ELISA assays for production of p24 core antigen were performed on Day 7 post-infection. ELISA kits were purchased from Coulter. Control curves were generated in each assay to accurately quantitate the amount of capsid protein in each sample. Data was obtained by spectrophotometric analysis at 450 nm using a Molecular Devices Vmax plate reader. P24 concentrations were calculated from the optical density values by use of the Molecular Device software package Soft Max.





TABLE 27



MACROPHAGE ASSAY FOR VIRACE 1

pg/mL P-24 Activity







AZT Control

Viracea #1

μM
vs. ADA
Dilution
vs. ADA












4
20.94
27.07
57.73
1:100
110.1
46.0
78.9

1.28
3.66
11.46
35.99
1:312
145.2
87.3
143.0

0.410
25.96
20.94
27.07
1:976
505.4
126.9
590.1

0.131
28.19
28.19
57.17
1:3051
811.9
98.4
652.5

0.042
34.87
79.47
105.70
1:9536
129.6
1055.0
1106.0

0.013
149.10
279.60
217.70
1:29802
1058.0
1098.0
1266.0

0.004
470.80
660.90
912.30
1:93132
1185.0
1067.0
1195.0

0.0014
919.00
1150.00
678.70
1:291038
1043.0
754.0
1287.0

0.0004
1005.00
1252.00
954.10
1:909494
1053.0
1035.0
712.7







































TABLE 28



MACROPHAGE ASSAY FOR VIRACEA 2

pg/mL P-24 Activity







AZT Control

Viracea #2

μM
vs. ADA
Dilution
vs. ADA












4
8.65
8.65
17.45
1:100
42.19
22.95
34.49

1.28
9.20
6.45
25.15
1:312
4.25
15.25
41.09

0.410
13.60
10.00
16.35
1:976
14.70
17.45
39.44

0.131
53.74
13.60
62.54
1:3051
63.64
26.25
48.79

0.042
82.89
72.44
96.63
1:9536
48.79
570.60
180.80

0.013
175.80
168.70
316.00
1:29802
278.60
243.50
450.80

0.004
914.90
891.20
499.20
1:93132
305.60
599.80
435.90

0.0014
821.90
594.80
983.10
1:291038
548.10
947.90
913.20

0.0004
1097.00
1160.00
1098.00
1:909494
814.80
790.60
820.80







































TABLE 29



MACROPHAGE ASSAY FOR VIRACEA 1

pg/mL P-24 Activity







AZT Control

Viracea #1

μM
vs. XTT
Dilution
vs. XTT












4
1.947
1.750
2.022
1:100
1.936
1.754
2.089

1.28
2.244
2.021
2.097
1:312
1.835
1.850
1.931

0.410
2.205
2.107
2.144
1:976
2.039
2.007
1.992

0.131
2.067
2.223
2.191
1:3051
2.040
1.710
1.903

0.042
2.357
2.175
2.339
1:9536
2.156
2.057
2.156

0.013
2.506
2.204
2.160
1:29802
2.073
1.573
1.858

0.004
2.372
2.325
2.191
1:93132
2.225
1.978
2.433

0.0014
2.558
2.091
1.884
1:291038
2.037
1.559
2.169

0.0004
2.037
2.389
2.166
1:909494
2.405
2.198
2.275







































TABLE 30



MACROPHAGE ASSAY FOR VIRACEA 2

Toxicity Studies Absorbance







AZT Control

Viracea #1

μM
vs. ADA
Dilution
vs. ADA












4
1.140
0.981
1.427
1:100
1.271
1.244
1.289

1.28
1.692
1.318
0.985
1:312
1.081
1.154
1.393

0.410
1.505
1.258
1.522
1:976
1.073
1.183
1.536

0.131
1.427
1.347
1.043
1:3051
1.482
1.032
1.518

0.042
1.534
1.725
1.720
1:9536
1.031
1.330
1.053

0.013
1.818
1.526
1.363
1:29802
1.344
1.449
1.497

0.004
1.578
1.112
1.034
1:93132
1.024
1.554
1.446

0.0014
1.386
1.350
1.133
1:291038
1.692
1.112
1.411

0.0004
1.451
1.081
1.342
1:909494
1.182
1.163
1.373







































TABLE 31



ANTI-HIV MACROPHASE ASSAY (P24)

For VIRACEA #2-4

P24 Activity (pg/mL)





Dilution














0
1:909494
1:291038
1:93132
1:29802
1:9536
1:3051
1:976
1:312
1:100
















Sample 1
1366.8
1347
524.1
634.4
457.5
349.9
193.5
138
120.9
46.96

Sample 2
1366.8
1151
693.8
782.2
321.5
228
271.4
190.2
4.718
96.46

Sample 3
1366.8
1000
877.9
642.9
507
382.2
136.1
202.1
171.7
92.5

Average
1366.8
1166.0
695.6
686.5
428.7
320.0
200.3
176.8
99.1
78.5

% VC
100.0
85.3
51.1
50.2
31.4
23.4
14.7
12.9
7.3
5.8







































TABLE 32



VIRACEA #2-4

XTT Toxicity Value (Absorbance)













Dilution
0
1:909494
1:291038
1:93132
1:29802
1:9536
1:3051
1:976
1:312
1:100















Sample 1
3.293
3.85
3.606
3.787
3.693
3.657
2.927
3.134
3.131
3.393

Sample 2
3.293
3.005
3.662
3.542
3.685
3.828
3.408
2.833
3.074
3.263

Sample 3
3.293
3.457
3.648
2.59
2.808
2.558
2.735
2.932
2.892
3.345

Average
3.293
3.437
3.639
3.306
3.395
3.348
3.023
2.966
3.032
3.334

% CC
100.0
104.4
110.5
100.4
103.1
101.7
91.8
90.1
92.1
101.2


































TABLE 33



ANTI-HIV MACROPHASE ASSAY (P24) For VIRACEA #2-5

P24 Activity (pg/mL)













Dilution
0
1:909494
1:291038
1:93132
1:29802
1:9536
1:3051
1:976
1:312
1:100















Sample 1
1298.2
1350
793.9
1001
515.9
274
196.3
65.8
16.28
3.904

Sample 2
1298.2
1350
858.6
851
780.4
393.3
102.9
110.2
38.79
16.28

Sample 3
1298.2
1454
1262
801.2
837.8
396.1
222.2
113.1
42.73
15.72

Average
1298.2
1384.7
971.5
884.4
711.4
354.5
173.8
96.4
32.6
12.0

% VC
100.0
106.7
74.8
68.1
54.8
27.3
13.4
7.4
2.5
0.9


































TABLE 34



VIRACEA #2-5

XTT Toxicity Value (Absorbance)













Dilution
0
1:909494
1:291038
1:93132
1:29802
1:9536
1:3051
1:976
1:312
1:100















Sample 1
3.139
3.459
3.568
3.567
3.634
3.562
3.134
3.311
3.171
2.974

Sample 2
3.139
3.018
3.295
3.505
3.533
3.359
2.833
3.313
3.133
2.909

Sample 3
3.139
3.21
3.261
3.263
3.297
3.051
2.932
2.829
3.151
3.35

Average
3.139
3.228
3.375
3.445
3.488
3.312
2.966
3.151
3.152
3.078

% CC
100.0
102.9
107.5
109.7
111.1
105.5
91.8
100.4
100.4
98.0


































TABLE 35



IN VITRO ANTI-HIV MACROPHASE ASSAY For VIRACEA 1

P24 (pg/mL)













Dilution
0
1:909494
1:291038
1:93132
1:29802
1:9536
1:3051
1:976
1:312
1:100















Sample 1
1171.0
712.7
1287.0
1196.0
1266.0
1106.0
652.5
590.1
143.0
78.9

Sample 2
1171.0
1035.0
754.0
1067.0
1098.0
1055.0
98.4
126.9
87.3
46.0

Sample 3
1171.0
1053.0
1043.0
1185.0
1058.0
129.6
811.9
505.4
145.2
110.1

Average
1171.0
933.6
1028.0
1149.0
1140.7
763.5
520.9
407.5
125.2
78.3

% VC
100.0
79.7
87.8
98.1
97.4
65.2
44.5
34.8
10.7
6.7


































TABLE 36



VIRACEA 1

XTT Toxicity Value (Absorbance)













Dilution
0
1:909494
1:291038
1:93132
1:29802
1:9536
1:3051
1:976
1:312
1:100















Sample 1
2.275
2.275
2.169
2.433
1.856
2.156
1.903
1.992
1.931
2.089

Sample 2
2.275
2.198
1.559
1.978
1.573
2.057
1.710
2.007
1.850
1.754

Sample 3
2.275
2.405
2.037
2.225
2.073
2.156
2.040
2.089
1.835
1.936

Average
2.275
2.293
1.922
2.212
1.835
2.123
1.884
2.013
1.872
1.926

% CC
100.0
100.8
84.5
97.2
80.6
93.3
82.8
88.5
82.3
84.7


































TABLE 37



IN VITRO ANTI-HIV MACROPHASE ASSAY For VIRACEA 2

P24 (pg/mL)













Dilution
0
1:909494
1:291038
1:93132
1:29802
1:9536
1:3051
1:976
1:312
1:100















Sample 1
1045.9
820.80
913.20
435.90
450.80
180.80
48.78
39.44
41.09
34.49

Sample 2
1046.8
790.60
947.90
599.80
243.50
570.60
26.25
17.45
15.25
22.95

Sample 3
1045.8
814.80
548.10
305.60
276.60
48.79
63.64
14.70
4.25
42.19

Average
1045.8
808.7
803.1
447.1
324.3
266.7
46.2
23.9
20.2
33.2

% VC
100.0
77.3
76.8
42.8
31.0
26.6
4.4
2.3
1.9
3.2


































TABLE 38



VIRACEA 2

XTT Toxicity Value (Absorbance)













Dilution
0
1:909494
1:291038
1:93132
1:29802
1:9535
1:3051
1:976
1:312
1:100















Sample 1
1.439
1.373
1.411
1.446
1.497
1.053
1.518
1.536
1.393
1.289

Sample 2
1.439
1.163
1.112
1.554
1.494
1.330
1.032
1.183
1.154
1.244

Sample 3
1.439
1.182
1.692
1.024
1.334
1.031
1.482
1.073
1.081
1.271

Average
1.439
1.239
1.405
1.341
1.442
1.138
1.344
1.264
1.209
1.268

% CC
100.0
86.1
97.6
93.2
100.2
78.1
93.4
87.8
84.0
88.1





























Examples 79-90
Binding and Fusion Inhibition Assays
These assays utilized HeLa-CD4-LTR-β-galactosidase cells which employ a tat protein-induced transactivation of the β-galactosidase gene driven by the HIV-1 long terminal repeat (LTR) promoter. The assay was used to quantitate both the binding of infectious virons to cells and cell-cell fusion events. Infected cells form syncytia which can be easily counted microscopically after incubation with X-gal. The HIV binding inhibition assay involved plating 1×10 4 HeLa-CD4-LTR-B-galactosidase cells in 200 μl in flat bottom, 96-well microtiter plates. The cells were incubated overnight, medium was removed and replaced with 100 μl of various concentrations of ISIS 5320 or control compound. One hour later 100 μl of virus-containing medium was added to each well. Cells were incubated for an additional hour and the monolayer was washed extensively to remove unbound virus and extracellular compound. At 48 hours, the cells were fixed and stained with X-gal. Blue multinuclear cells were than counted under an inverted microscope. The cell-cell fusion inhibition assay was also performed in flat bottom, 96, well microtiter plates. HeLa-CD4-LTR-β-galactosidase cells (5×10 3 ) were added to each well and incubated with test compound for 1 hours prior to the additional of 5×10 3 HL2/3 cells (28). Cells were incubated for an additional 48 hours and fixed and stained with X-gal. Blue syncytia were counted microscopically. Staining of the cells was performed by fixing the cells with a solution of 1% formaldehyde and 0.2% glutaraldehyde and staining the fixed cells with 4 μM potassium ferrocyanida, 4 μM potassium ferricyanide, 2 μM MgCl 2 and 0.4% X-gal in PBS. Trans-activation of β-galactosidase expression was also monitored by ELISA Cell extracts were prepared by freeze-thaw and assayed for β-galactosidase activity according to the manufacturer's recommendations. The results of the ELISA were quantitated spectrophotometrically at 405 nm using a Molecular Devices Vmax microtiter plate reader.





TABLE 39



Beta-gal Fusion Assay: Viracea #1/SK1

Number of Blue Cells per Well










Dilution
0
1:3200
1:1000
1:320
1:100
1:32
1:10












Sample 1
49.0
10.0
11.0
19.0
4.0
6.0
3.0

Sample 2
32.0
15.0
10.0
4.0
5.0
5.0
8.0

Sample 3
59.0
15.0
10.0
5.0
4.0
4.0
4.0

Mean
46.7
13.3
10.3
9.3
4.3
5.0
5.0

% VC
100.0
28.6
22.1
20.0
9.3
10.7
10.7

STD DEV
29.3
6.2
1.2
18.0
1.2
2.1
5.



































TABLE 40



Beta-gal Fusion Assay: Viracea #1/SK1

PERCENT TOXICITY










Dilution
0
1:3200
1:1000
1:320
1:100
1:32
1:10












Sample 1
1.596
1.574
1.931
1.925
1.34
1.576
1.63

Sample 2
1.578
1.692
1.734
1.728
2.152
1.633
1.711

Sample 3
1.66
1.38
1.811
1.646
1.647
1.308
1.545

Mean
1.612
1.649
1.825
1.768
1.946
1.672
1.629

% Viability
100.0
96.1
113.2
109.6
120.7
103.07
101.0


































TABLE 41



Beta-gal Fusion Assay: Viracea #2/SK1

Number of Blue Cells per Well










Dilution
0
1:3200
1:1000
1:320
1:100
1:32
1:10












Sample 1
49.0
26.0
16.0
17.0
10.0
2.0
1.0

Sample 2
32.0
18.0
16.0
11.0
3.0
2.0
0.0

Sample 3
59.0
19.0
20.0
14.0
5.0
3.0
1.0

Mean
46.7
21.0
17.3
14.0
6.0
2.3
0.7

% VC
100.0
45.0
37.1
30.0
12.8
5.0
1.4

STD DEV
29.3
9.3
4.9
6.4
7.7
1.2
1.2



































TABLE 42



Beta-gal Fusion Assay: Viracea #2/SK2

PERCENT TOXICITY










Dilution
0
1:3200
1:1000
1:320
1:100
1:32
1:10












Sample 1
1.441
1.59
1.965
1.972
1.799
1.932
0.829

Sample 2
1.5
1.543
1.83
1.835
1.897
1.386
0.882

Sample 3
1.425
1.536
1.839
1.867
2.036
1.615
0.758

Mean
1.455
1.558
1.875
1.891
1.911
1.644
0.823

% Viability
100.0
106.9
129.0
130.0
131.3
113.0
56.6


































TABLE 43



Beta-gal Fusion Assay: Viracea #1

Number of Blue Cells per Well










Conc.
0
1:3200
1:1000
1:320
1:100
1:32
1:10












Sample 1
38.0
37.0
47.0
37.0
42.0
55.0
18.0

Sample 2
48.0
34.0
75.0
37.0
37.0
50.0
14.0

Sample 3
32.0
41.0
48.0
52.0
57.0
64.0
9.0

Mean
39.3
37.3
56.7
42.0
45.3
56.3
13.7

% VC
100.0
94.9
144.1
106.8
115.3
143.2
34.7

STD DEV
20.5
8.9
40.4
22.0
26.5
18.0
11.5



































TABLE 44



Beta-gal Fusion Assay: Viracea #1

PERCENT TOXICITY










Conc.
0
1:3200
1:1000
1:32
1:100
1:32
1:10












Sample 1
1.425
1.951
1.981
1.815
1.796
1.639
1.644

Sample 2
1.5
1.971
1.983
1.826
1.833
1.845
1.547

Sample 3
1.441
1.913
1.942
1.835
1.823
1.932
1.644

Mean
1.455
1.945
1.969
1.825
1.817
1.872
1.612

% Viability
100.0
133.6
135.3
125.4
124.9
126.6
110.7


































TABLE 45



Beta-gal Fusion Assay: Viracea #2

Number of Blue Cells per Well










Conc.
0
1:3200
1:1000
1:320
1:100
1:32
1:10












Sample 1
38.0
64.0
50.0
56.0
40.0
50.0
0.0

Sample 2
48.0
56.0
77.0
54.0
53.0
54.0
0.0

Sample 3
32.0
44.0
46.0
42.0
48.0
47.0
0.0

Mean
39.3
54.7
57.7
50.7
47.0
50.3
0.0

% VC
100.0
139.0
146.6
128.8
119.5
128.0
0.0

STD DEV
20.5
25.6
42.9
19.3
16.7
8.9
0.0



































TABLE 46



Viracea #2

PERCENT TOXICITY










Conc.
0
1:3200
1:1000
1:320
1:100
1:32
1:10












Sample 1
1.425
1.998
1.87
1.85
1.592
0.956
0.174

Sample 2
1.5
1.911
1.959
1.904
1.645
0.988
0.174

Sample 3
1.441
1.976
1.902
1.939
1.623
0.965
0.182

Mean
1.456
1.962
1.914
1.898
1.620
0.970
0.177

% Viability
100.0
134.8
131.5
130.4
111.3
56.6
12.1





























Topical Microbicide Assay
MEI 180 cervical epithelial cells were plated in the interior walls of a 96-well flat bottom microtiter plate at a density of 5×10 cells per well and incubated overnight. Chronically infected H9 cells wee treated with 200 μg/ml mitomycin C in complete medium for one hour, washed extensively and resuspended at 4×10 5 per ml. The concentration of mitomycin C used resulted in the killing of the chronically infected cells within 48 hours of treatment, allowing sufficient time for cell-cell transmission of virus to the ME-180 cells while assuring that the virus endpoint quantification would not include a contribution from the chronically infected cells. Antiviral compounds and chronically infected cells (2×10 4 ) were added to each well containing ME180 cells and incubated for 6 hours. Following co-cultivation the monolayer was washed extensively and fresh medium added. Medium was removed and fresh medium added at 24 and 48 hours post-infection to remove dead lymphocytes. On day 6 post-infection, supernatant samples were removed and analyzed for virus content by p24 ELISA.
CD4 Expression Assays
Quantitation of the effect of Viracea on CD4 expression was performed using standard flow cytometric techniques. Cells were treated with Viracea for one hour at 37° C. in tissue culture medium. Briefly, 10 6 CEM-SS cells were incubated with or without compound for 60 minutes at room temperature. Anti-CD4 monoclonal antibody (20 μl, 3 μg/ml) (Becton-Dickinson, San Jose, Calif.) was added, and cells were incubated at 4° C. from 40 min. Cells were then washed twice with PBS, resuspended in 1° C. paraformaldehyde, and analyzed using a Becton-Dickinson FACSort flow cytometer.
Macromolecular Synthesis
CEM-SS cells were cultured in triplicate in the presence or absence of compound 24 hours at 37° C. in a humidified CO 2 incubator. At 24 hours, 1 μCi of [methyl- 3 H]-thymidine, [5- 3 H]-uridine, or [3, 4, 5- 3 H]-leucine was added to the culture and incubation was continued for an additional 8 hours. The cells were transferred to glass fiber filter papers by use of Skatron cell harvester. The glass fibers were washed with distilled water, placed in a scintillation vial and the quantity of incorporated radioactivity quantitated with a Packard Tri-Carb scintillation counter.
HIV Test Results
Viracea-1 and Viracea-2 were evaluated in the microtiter anti-HIV assay which quantifies the ability of a test compound to inhibit HIV replication and HIV-induced cell destruction. The two compounds were determined to be active against the RF strain of HIV-1 in CEM-SS cells. Viracea-1 inhibited HIV-induced cytopathic effects (IC 30 ) at 1:400 dilution, while Viracea-2 exhibited an IC 25 at a 1:900 dilution and did not reach a 50% inhibitory value. Both Viracea-1 and Viracea-2 exhibited toxicity (TC 30 ) to the CEM-SS cells at dilutions of approximately 1:20 and 1:250, respectively. The positive control compound, ddC, exhibited the expected level of activity against the RF virus.
Viracea-1 and Viracea-2 were evaluated for activity in fresh human PBMCs infected with the clinical HIV isolate ROJO. This low passage isolate has been defined as a drug sensitive (AZT, ddC, nevirapine) syncytium-inducing virus isolate. Neither Viracea-1 nor Viracea-2 inhibited the replication of this isolate at nontoxic concentrations. Further evaluation of the compounds in PMBCs infected with ROJO were performed using IL2 stimulation of the PBMCs rather than PHA blastogenesis. Again, no activity was detected below concentrations which inhibited the growth of the PBMCs. AZT exhibited the expected level of activity in these assays.
Viracea-1 and Viracea-2 were evaluated in fresh human monocyte-macrophages infected with the low passage clinical isolate ADA. In these assays, both compounds exhibited high levels of activity with Viracea-2 being clearly superior. The 50% effective concentration of Viracea-1 and Viracea-2 was 1:4000 and 1:10000, respectively. Toxicity was not detected to the monocyte-macrophage monolayer by morphological examination or by XTT-Tetrazolium staining. AZT exhibited the expected level of activity in these assays.
Viracea-1 and Viracea-2 were found to inhibit the attachment of infectious virus to the CD4-expressing HeLa-CD4-LTR-β-galactosidase cells. Inhibition of binding of virus to the target cells was detected at dilutions of approximately 1:1000 to 1:3200 for both compounds. Neither compound had any antiviral effect on the fusion of the envelope-expressing HL2/3 cells with the HeLa-CD4-LTR-β-galactosidase cells. Toxicity was noted for both compounds in the fusion assay where compound was present for the full duration of the assay as well as with Viracea-2 in the binding assay where compound was only present for 2 hours. Chicago Sky Blue, a sulfonated dye, exhibited the expected level of activity in each of these assays.
Viracea-2 prevented the transmission of virus from chronically infected lymphocytes to the ME 180 cervical epithelial cell line at a dilution of approximately 1:500 (IC 30 ). Toxicity was not detected in this assay to the ME180 cells. In this assay, the drug was present during the time of infection only (4 hours). Dextran sulfate (positive control, sulfated polysaccharide) and dextran (negative control) exhibited the expected level of activity in these assays.
Viracea-2 had no effect on the expression of CD4 on the cell surface.
Inhibition of the incorporation of thymidine (DNA), uridine (RNA) or leucine (protein) into high molecular weight macromolecules was observed at dilutions greater than 1:320. The inhibition of macromolecule synthesis paralleled the toxicity of the compounds in CEM-SS cells.
Summary of HIV Test Results
Viracea-1 and Viracea-2 inhibit HIV infection in established T-cells with a narrow therapeutic index. Viracea-1 and Viracea-2 potentially inhibit HIV replication in monocyte-macrophages. Viracea-1 and Viracea-2 inhibit the attachment of virus to target cells but do not prevent the fusion of infected and uninfected cells. Viracea-2 inhibits the transmission of virus in a topical microbicide assay and may be useful in the prevention of sexual transmission of HIV. Viracea-2 has no effect on cell surface CD4 expression.
Prevention and Treatment
The antimicrobial compound provides an antimicrobicide and medicine which can (1) help prevent the sexual transmission of HIV; (2) control viral load of HIV and other viruses; (3) eradicate HIV; (4) extend the latency periods of autoimmunedificiency syndrome (AIDS) in patients who have contracted HIV; (5) decrease pain and suffering of HIV patients; (6) lower the infectious spread of HIV; and (7) provide better and more successful treatment of patients with HIV. The medical treatment can also resolve the physical symptoms of an infectious outbreak of HIV, herpes simplex virus 1 or 2 (HSV 1 or HSV 2) or other infectious microbial diseases. The preceding can be accomplished by systemically applying or injecting the above described preferred antimicrobial compound (medicine) with a syringe into the rectal canal (rectum, rectal tissue, anus or anal tissue) or the vagina (vaginal tissue) of a patient infected with HIV or other infectious microbial disease for 8-12 times per day, preferably 10 times a day at intervals of every two hours, for a period of 10-18 consecutive days, preferably 14 consecutive days (two weeks) for best results. The dosage, concentration, and amount of the antimicrobial compound (medicine) can be varied depending on the severity and extent of the disease as well as the age, sex, weight, race and health of the patient. Desirably, the infected area is rinsed (washed) and dried to remove any soap or residue on the infected area before the antimicrobial compound (medicine) is applied. For treatment herpes simplex virus 1 or 2, the antimicrobial compound can be applied on the infected area, such as for 19-24 hours. Preferably, vesicular eruption of herpes virus is resolved in 19-24 hours and herpes lesions are consequently healed.
Among the many advantages of the medical treatment and medicine (compositions) of the invention are:
1. Outstanding performance in rapidly resolving cold sores, rashes or other skin conditions resulting from herpes, pseudomonas, staph, hepatitis, and other infectious diseases. 2. Superior results in relieving pain from cold sores, rashes or other skin conditions resulting from herpes, pseudomonas, staph, hepatitis, and other infectious diseases. 3. Superb treatment resulting from herpes, pseudomonas, staph, hepatitis, and other infectious diseases. 4. Beneficial to resolve herpes simplex virus 1 (HSV 1), herpes simplex virus 2 (HSV 2), staphylococci (staph), varicella zoster virus (herpes zoster) (shingles), cytomegalovirus, herpetic keratitis, conjunctivitis, human immunodeficiency virus infection (HIV), viral influenza (flu), epstein barr, papilloma virus, viral parainfluenza, adenovirus, viral encephalitis, viral meningitis, arbovirus, arenavirus, picornavirus, coronavirus, syntialvirus, viral infections, roseola infantum, pneumonia, mononucleosis, uveitis, retinitis, human cervical carcinoma, vaginal carcinoma, vulvovaginitis, human herpes IV, Kaposi's sarcoma, cytomegalovirus, and common colds resulting from viral infections. 5. Reduces many of the serious emotional and mental anguish resulting from herpes, pseudomonas, staph, hepatitis, and other infectious diseases. 6. Decreases worldwide economic loss from herpes, pseudomonas, staph, hepatitis, and other infectious diseases. 7. Excellent medicinal preparation for newborns, children, adults and animals. 8. Pleasant, attractive and easy to use.

9. Safe.
10. Economical. 11. Dependable. 12. Effective.

Although embodiments of the invention and examples have been shown and described, it is to be understood that various modifications, substitutions, and additions, as well as other uses, and rearrangements of compounds, ingredients, parts, components, process steps, methods, procedures, and treatment, can be made by those skilled in the art without departing from the novel spirit and scope of this invention.