Immune Technologies, LLC Product Description

I.          Executive Summary

Immune Technologies^TM, “LLC” (“the Company” and “IT”) is a Florida company that provides treatment technologies for viral, fungal, bacterial and protozoal diseases and health conditions, particularly HIV/AIDS and Rheumatoid Arthritis, based on a patented technology invented by its medical director, Stephen Herman, M.D.

The Company’s lead therapeutics are Alphamir ^TM, as a prescription-only medicine to combat HIV/AIDS, and Arthromir^TM for Rheumatoid Arthritis.  Both of these inventions utilize Trioxolane derivatives that, upon introduction to the body’s aqueous ambience, reduce and produce high-energy oxygen metabolites within the blood stream as well as activate certain receptor cells in the oropharyngeal cavity.  These receptor cells cause the body to produce CD4 cells that are the precursor cells to the evolvement of lymphocytes, and thereby modulate the natural immune process in the body.

On May 24^th, 2001, the Company received provisional two-year registrations for use of Alphamir^TM as a prescription-only medicine to combat HIV/AIDS and for Arthromir^TM for Rheumatoid Arthritis.  The two-year registrations are renewed in five-year intervals.  These approvals came after completing clinical trials at the Kenya Medical Research Institute.  Immune Technologies has provided updated clinical data to the Pharmacy Poisons Board at the one-year anniversary in May 2002.  These approvals allow the resultant medicines to be used as ethical drugs in 21 sub-Saharan African countries that are members of the East African Medical Conference.

AIDS IS OUR FOCUS:  In Africa, the number of people suffering from HIV/AIDS has reached upwards of 20,000,000 in 2002 and 42,000,000 worldwide.  These figures are expected to increase to nearly 100,000,000 by 2010.  With HIV/AIDS reaching pandemic magnitudes, the Company is focusing its product launch initiatives on HIV/AIDS in Africa and then to other countries, Europe and the United States. 

HOW WE ARE UNIQUE:  Our approach to combat disease is unique from the common practice of using antimicrobial agents that can be toxic; rather, it utilizes the basic science within the white blood cells, called ‘the respiratory burst.’  The respiratory burst is the process by which the body’s immune system produces bio-oxidants to destroy abnormal cells and invading microorganisms.  Trioxolane emulates this process by producing oxidative metabolites that are anti-microbial, immuno-stimulatory and non-toxic.  This approach reinforces the natural immune system in combating harmful agents and regulates the production of serotonin, prostaglandin and inflammatory interleukins.

SAFETY PROFILE:  AlphamirTM is a safe and non-toxic substance.  The safety profile was performed over a period of 6 years between 1988 and 1994 in four laboratories located in the United States, Cuba, and Kenya.   Three of the facilities were medical academic institutions (University of California at Irvine, Kenya Medical Research Institute, and University of Cuba, LaHabana) and the fourth was a professional contract research organization located in California.  Nineteen different safety and toxicity studies were performed including:  minimum inhibition concentration assays, minimum bacterial concentration assays, acute systemic toxicity, dermal toxicity, oral toxicity, ocular irritation, primary skin irritation, vaginal and rectal irritation, AMES mutagenesis assays, micronuclei in mouse bone marrow, and other topical studies were performed in order to substantiate the effectiveness, safety, and tolerability of AlphamirTM.  Of these nineteen studies, a total of 312 mice, 65 rats, and 70 rabbits were used in order to substantiate the safety of the product.  

IT CLINICAL TRIALS:  AlphamirTM and ArthromirTM’s clinical investigator is Davy Kiprotich Koech BSc, MS, PhD, Cbiol, MIBiol, FinstPM, SS, CuD (Hon.), OGW.  For the past 14 years, Dr. Davy Koech has served as Chief Research Officer and Director of the Kenya Medial Research Institute (KEMRI).  He is the author of 161 refereed journal publications in the fields of Leishnamiasis, Schistosomiasis, Malaria, AIDS, and numerous other diseases. Research physicians, under Dr. Koech’s direction, conducted anecdotal, Phase I, and Phase II HIV/AIDS and Rheumatoid Arthritis studies which were completed at KEMRI on a total of 176 patients.  From 1992-1993 a Phase I double blind clinical trial with 30 patients; 3 study arms with 10 patients each – Oropharyngeal, Suppository, and Control was performed for AIDS.  The immunological results were:  CD4+ lymphocyte levels in groups treated with AlphamirTM increased 100+% versus 0% change for the control group.  From 1999 - 2001 the AIDS Phase II Open label clinical trial with randomized dose ranging for efficacy, safety and tolerability of AlphamirTM was performed with 43 patients which consisted of 3 study arms at buccal dose levels of Group 1 - 100 mg, Group 2 - 200 mg, and Group 3 - 400 mg.  Results from   Group 1 showed consistent and steady rise in weight; 50% of patients showed significant rise in CD4+ count with 33% drop in viral load.  The successful results led both drugs to provisional fast tract registrations as a prescription medicine for ethical use with continued research efforts.  Follow-up continues with monthly viral loads and no adverse effects encountered.  Funds are needed to begin Phase III clinical trials administering Alphamir as monotherapy and in combination therapy with other drugs/therapies.

IT STRATEGY:  The Company will simultaneously focus its attention on significant distribution of Alphamir ^TM for HIV/AIDS and Arthromir ^TM for Rheumatoid Arthritis in Africa at very low costs while it looks to promote its products with a pharmaceutical partner in developed markets.  IT suggests that the only viable and effective approach to contain HIV/AIDS must include a comprehensive program to:

1)      administer a low-cost self-testing procedure which we will co-market;

2)      treat infected HIV/AIDS population with Alphamir; and

3)      administer a vaginal gel for the prevention of transmission which we are developing.       

The demand for the use of this treatment strategy is great enough to support incrementally larger requirements of funding.  At this time, we are actively pursuing funds for Phase III clinical trials, to execute this treatment strategy, and manufacture and distribute the products.  It should be noted that the best scientific evidence supports the fact that viral strains cannot develop resistance to Alphamir ^TM and this represents a major breakthrough in the ability to effectively control the AIDS pandemic.

PROBLEM AND SOLUTION:  IT is taking an integrated approach with a cost effective treatment program.  Estimates to treat the current 42 million plus infected people range from approximately $316 to $3,325 per year using presently available Highly Active Antiretroviral Treatment (HAART).  In addition, the cost of the medical infrastructure for distribution, monitoring and training necessary for use of these treatment programs is prohibitive throughout Africa.  IT is launching Alphamir ^TM with a cost of $205 per year per patient for an orally prescribed medication not requiring water to swallow.  Efforts are being made to bring those costs down substantially for Third World countries.  The dosage of 100 mg, 4 times a day (10 drops/25 mg under the tongue 4 times) is expected to be available in tablet form that melts in the mouth without water.

The Company’s proprietary technology is unique and is closely guarded.  The technology is protected by existing and future pending patents.  The process and use of Trioxolane is patented in the U.S.A., Mexico, and Canada.  Its use will offer the production of ethical drugs that will be further tested in clinical trials for the treatment of viral, bacterial, and fungal diseases. Subsequent patents are to be issued and will cover our invention internationally.

II.        Market Review

IT’s registrations are valid in twenty-one African countries that are members of the East African Medical Conference to fight the symptoms of HIV/AIDS and Rheumatoid Arthritis.  In Africa, the number of people suffering from HIV/AIDS has reached upwards of 20,000,000 in 2002 and 42,000,000 worldwide.  These figures are expected to increase to nearly 100,000,000 by 2010.  In 2001, five million people were newly infected, three million died of AIDS (8,000 people a day), leaving 13.2 million children orphaned and a death total of more than 21,800,000 people thus far. 

The Company plans to commence its efforts to obtain approval for the use of its products as ethical drugs in Europe, Asia, Latin America, and South America.  Simultaneously, Immune Technologies will begin IND discussions with the US Federal Drug Administration (FDA).  In the United States, it is reported that a new drug costs upward of $800,000,000 and 10 years to receive US FDA approval.  In comparison, to date, $10,000,000 and 14 years of research have gone into developing Alphamir ^TM.  The Company believes that once the efficacy of Alphamir ^TM is demonstrated aboard, combined with its track record in 600+ patients as safe and non-toxic, US FDA approval should be less expensive and fast tracked. 

III.       Competition

Presently there are numerous costly alternatives to Alphamir™ for treatment of HIV/AIDS in Africa.  Estimates to treat the current 20 million plus infected people range from approximately $316 to $3,325 per year using presently available Highly Active Antiretroviral Treatment (HAART).  In addition, the cost of the medical infrastructure for distribution, monitoring and training necessary for use of these treatment programs is prohibitive throughout Africa.

Our data suggests that Alphamir™ is equally or more effective than current treatments.  Clinical trials with Alphamir™ have shown no toxic effects and require minimal special training and monitoring.  We are confident that clinical trials of Alphamir™ will demonstrate that it can be used as an effective vaginal gel to prevent transmission of HIV.  This will be the only truly effective means of curbing the AIDS pandemic.  No other current technology has this potential.  In addition, the cost of our product is lower than the currently available treatments on the market.  Immune Technologies is also cost competitive when compared with the generic treatments known today.  Alphamir™ is 1/5^th the cost of the lowest offer from proprietary companies.

According to Kenyan government figures, only 2,500 out of 200,000 AIDS patients in Kenya are currently receiving antiretrovirals, (ARVs).  The drugs are only available in high-cost private hospitals such as the Aga Khan, the Nairobi, the Pandya Memorial Hospital, the private wing of the public Kenyatta National Hospital, and a few church-run hospitals."  While generic AIDS drugs are now allowed into Kenya under compulsory licensing provisions in the Industrial Property Act of July 2001, which came into force in May this year, one major problem is that a majority of the patients say that the drugs are not available.  Some patients are switching to other drugs and then developing resistance.  "We are going to end up with serious resistance and two years from today we might have no ARVs that we can use," Dr. John Wesongo of Nairobi's Mbagathi Hospital. 

Nicholas Otieno, who has been living with HIV/AIDS since 1992, speaks of the irregular supplies of ARVs, which can lead to HIV-resistance to the treatments.  "Since I started taking antiretroviral drugs last July, I have twice been unable to get my regular supply of Zerit.  Once I could get Epivir as a substitute, but that costs 4000 Kenya shillings (US$50) per month instead of the usual KES 440 (US$5) I pay for Zerit.  The other time I went without a substitute for two weeks."  Liza Kimbo of the Kenya Coalition for Access to Essential Medicines says the problem of drug shortages started last year when the five major drug companies lowered the prices of their AIDS drugs for Africa by between 50 -80%....it's the big five.  It's the companies that have provided these drugs at the beneficial prices are the ones that are then affected with the shortages.  Inter Press Service–April 23, 2002

Kenya has said yes to generic ARVs, but failed to win funds from the Global Fund for HIV/AIDS, TB & Malaria.  Upon approval, the government planned for 300,000 people in Kenya to receive generic drugs IF they received the funding.  Nairobi hopes that the generics will undercut the prices of branded ARVs, which despite manufacturers 'reductions' still cost US $850 a year for the cheapest triple therapy.  That is less than a tenth of the price in the developed world, but still too much for the 10 million people in Kenya who live on less than US $1 a day. 

Generic triple therapies including AZT, 3TC, and nevirapine are now down to prices of around US $295 a year but still dear in the African context.  Ellen Hoen, coordinator of the globalization section of the Medicines Sans Frontiers (MSF) campaign for access to essential medicines, knows of other courses costing only US $209.  It's still not low enough 'but the prices could go down further', she says.

The Ministry has already announced its intention to make a reapplication to the Global Fund when its board sits again in September.

IV.       Alphamir™ – Technology Overview

Immune Technologies, “LLC”, has developed an entirely new medicinal agent, Trioxolane, designated Alphamir™.  This agent was developed in response to the increasingly urgent need to treat the worldwide threat of viral epidemics, as well as to the increasing occurrence of antibiotic-resistant organisms.  We believe Alphamir™ is a major breakthrough in therapeutic medicine and its broad efficacy brings a new understanding of the human immune system and our natural disease fighting mechanisms.

The most broadly successful therapy for human disease is provided by our own immune system.  This includes all infectious, neoplastic, and autoimmune diseases.  An intact and functioning immune system maintains a disease-free state, while a suppressed or dysfunctional immune system leaves us susceptible to a wide spectrum of maladies, almost without restriction. 

The process by which Alphamir™ acts as a microbicidal agent is best explained through the understanding of the Respiratory Burst; the natural way our bodies fight microbes and tumor cells.  White blood cells produce high-energy bio-oxidants, to kill viruses, fungi, and bacteria and to modulate immune function.  Through significant in-vitro, pre-clinical and clinical research studies, Immune Technologies has demonstrated that Alphamir™ augments this natural process.

a.                  Biochemistry of Alphamir™

Phagocytes employ, as antimicrobial agents, a number of compounds generated by partial reduction of oxygen.  Oxygen is initially reduced to superoxide (O₂^-) by a membrane-associated flavoprotein.  This process occurs in a respiratory burst via glucose oxidation in the hexose monophosphate shunt.  Oxidized NADPH participates as follows:

(O₂ + NADPH yields 2O₂^- + NADP^- + H^-) subsequently by dismutation (superoxide dismutase) 2O₂^- + 2H⁺₂ yields O₂ + H₂O₂.  Present theory suggests that microbicidal action by phagocytes is mediated by myeloperoxidase that catalyzes the conversion of H₂O₂ and Cl^- to hypochlorous acid (HOCl).  In support of the effectiveness of this mechanism it is interesting to note that 2X10^-7 M of HOCl generated by 10⁶ neutrophils will destroy 15x10⁷ e.coli in milliseconds.  In short, neutrophils purposely generate large quantities of reactive oxidants for microbicidal purposes.  Interestingly, HOCl is the sole active ingredient in bleach.  HOCl quickly reacts with primary or secondary amines to form an additional family of microbicidal agents called chloramines. 

The term “respiratory burst” refers to a coordinated series of metabolic events that take place when phagocytes are exposed to appropriate stimuli.  This group of events underlies all oxygen-dependent killings by phagocytes. 

b.         The Basic Science

Alphamir^TM incorporates Trioxolane derivates that, upon introduction to the body’s aqueous ambience, reduce and produce high-energy oxygen metabolites within the blood stream as well as activate certain receptor cells in the pharyngeal cavity.  These receptor cells cause the body to produce cells that are the precursor cells to the evolvement of lymphocytes, and thereby modulate the natural immune process of the human body.

More specifically, Alphamir^TM acts in vivo through the production of free radicals.  Neutrophils and other phagocyte leukocytes can utilize these free radicals to produce multiple pathogenic toxins thus materially increasing the efficiency of the immune system to kill invading microorganisms.  In addition, these free radicals exert a strong direct microbiocidal effect even in the absence of leukocytes.  Free radicals also exert both an immunomodulatory and immunostimulatory effect by chemotactically signaling the immune system that a pathogen/leukocyte interaction is occurring.  Little is known as to how Alphamir^TM works on a molecular level.  Effects can be divided into long-term and short-term actions.  Short-term effects include such things as lysis of pathogenic organisms, abnormal cells, and toxins.  Alphamir^TM may have activity by destroying or blocking the activity of inflammatory agents such as prostaglandins. 

Long-term effects are mediated via immune system modulation and stimulation as well as enzyme system modulation.  It has been shown that uptake of these radicals in infected cells is preferential in a ratio of 4 to 1 with uninfected cells.  It appears that Alphamir^TM also plays a role in cell membrane stabilization.

c.                   Safety & Toxicity Studies  (Note:  A more robust explanation of the Safety & Toxicity Studies can be viewed in the Appendix A)

Alphamir^TM is a safe and non-toxic substance.  The safety profile was performed over a period of 6 years between 1988 and 1994 in four laboratories located in the United States, Cuba, and Kenya.  Three of the facilities were medical academic institutions (University of California Irvine, Kenya Medical Research Institute, and University of Cuba, La Habana ) and the fourth was a professional contract research organization located in California.  19 different safety and toxicity studies were performed including: Minimum Inhibition Concentration assays, Minimum Bacterial Concentration assays, acute systemic toxicity, dermal toxicity, oral toxicity, ocular irritation, primary skin irritation, vaginal and rectal irritation, AMES Mutagenesis assays, Micronuclei in Mouse Bone Marrow, and other topical studies were performed in order to substantiate the effectiveness, safety, and tolerability of Alphamir^TM.  Of these 19 studies, a total of 312 mice, 65 rats, and 70 rabbits were used in order to substantiate the safety of the product.  The following includes a summary of important results:

·        Alphamir^TM was considered non-irritant to ocular tissue in the rabbit. 

·        The product would be considered dermally non-toxic to the rabbit; the LD50 was greater than 2.3 ml/kg of body weight of a 1% (w/v) solution of the test article in the solvent. 

·        The product would be considered a slight irritant to the skin.

·        The oral, single dose, LD50 of Alphamir^TM was determined to be greater than 5,010 mg/kg body weight in the rat.

·        The results show that the rats can tolerate daily I.P. of Alphamir^TM at a dose of 214 mg/kg that is almost 25 times higher than that proposed for AIDS patients (8.7 mg/kg).  

·        Female rats given Alphamir^TM I.P. doses up to 63 mg (586 mg/kg) in 0.5 cc tolerate a higher dose level [63 mg (586 mg/kg)] than male rats.  The Alphamir^TM level tolerated by rats is at least 380 mg/kg. 

·        The result was an LD50 of Alphamir^TM in mice has been calculated to be 700 mg/kg.  The minimum tolerable dose is 600 mg/kg.

·        Medium lethal dose was not obtained because only one male rat died in 4 hours after a single oral dose larger than 5 g/kg.  Substances with a LD50 larger than 5 g/kg orally administered to rodents are considered as non-toxic or moderately toxic substances.  Thus, this pharmaceutical preparation of Alphamir^TM is practically non-toxic when orally administered.

·        Alphamir^TM does not show a mutagenic effect at any of the studied concentration for all strains included in AMES Test.  Because of the phenotypic characteristics of Salmonella stocks selected in the AMES Test, Alphamir^TM does not present a mutagenic effect of the kind of mutations due to a frame shift running nor to changes of base pairs. 

Toxic concentration values found in some strains do not correspond to the reported inhibitory concentrations for Alphamir^TM in several pathogenic bacteria.

·        No toxicity sign was observed during a period of 15 days in mice of both sexes, after being topically administered with a larger dose of 5000 mg/kg.  There was no sign of dermic irritation during the time of observation.  Alphamir^TM does not show any toxicity sign due to its topical administration in single doses to mice of both sexes.

d.                  The Clinical Investigator 

Davy Kiprotich Koech  BSc, MS, PhD, Cbiol, MIBiol, FinstPM, SS, CuD (Hon.), OGW:  Since 1989, Dr. Davy Koech has served as Chief Research Officer and Director of the Kenya Medical Research Institute.  He is the author of 161 refereed journal publications in the fields of Leishmaniasis, Schistosomiasis, Malaria, AIDS, and numerous other diseases.  He has performed research under numerous grants and programs with the World Health Organization’s Immunology Research and Training Centre, served as a Fulbright-Hays Scholar/Pharmacologist at Duquesne University, Montefiore Hospital (Department of Neurosurgery), and Harvard University Medical School (Departments of Medicine and Biological Chemistry).  Since 1988 Dr. Koech has served as a member of the World Health Organization’s Regional Panel of Experts on AIDS and as a member of the International Council for Infectious Diseases.  He was honored to receive the Presidential Award (State Investiture) of the Decoration of the Silver Star of Kenya (S.S.) in 1984 and the Presidential Award (State Investiture) of the Decoration of the Order of the Grand Warrior of Kenya in 1989 both in recognition of distinguished service rendered to his nation.  Dr. Koech is a member of Who’s Who in Science and a member of the International Who’s Who in Medicine.  Dr. Koech was also honored to receive the Medal of Honor commemorating distinguished lifelong achievements from the American Biographical Institute in 1987.

Dr. Koech received his BSc (Chemistry & Zoology) from the University of Nairobi in 1974, his MS (Pharmacology – Majored in Clinical Pharmacology) from Duquesne University in Pittsburgh, PA in 1977, and his Ph.D. (Medical Pathology, Immunology) from the University of Nairobi.  Dr. Koech is a member of the East African Society of Parasitologists, British Society for Immunology, British Transplantation Society, Institute of Biology (U.K.), Institute of Professional Managers and Administrators (Fellow), Britain, International Society of Infectious Diseases, Association of Physicians of East and Central Africa, and Kenya Association of Clinical Pathologists. 

e.        Phase I Clinical Trial – HIV/AIDS     Efficacy of Trioxolane Alphamir^TM In the Clinical Management of HIV Seropositive and Symptomatic Individuals – 1992-1993 

After sufficient safety and toxicity studies were performed, a Phase I study protocol was developed and deployed at the Kenya Medical Research Institute by physicians under the direction of its Chief Research Officer, Dr. Davy Koech.  This Phase I double blind 12 week clinical trial in consenting individuals infected with HIV-1 using Alphamir^TM was performed to evaluate the clinical efficacy and safety of Alphamir^TM in the management of symptoms associated with HIV infections and to assess the changes in immunological profiles in patients receiving the drug. 

Patients of either sex were included in the study who had symptoms of ARC or AIDS with laboratory confirmation of HIV seropositivity by both ELISA and Western Blot or the equivalent tests, were between the ages of 12-60 years old, had a Karnofsky performance score (KPS) of between 40-90, and gave informed consent to participate.  Patients under the age of 12, pregnant woman and lactating mothers, patients on specific anti-HIV medication such as AZT and interferon’s, those on systemic or local steroids, and patients with renal impairment were excluded from the study. 

The study divided the 30 patients into three groups of 10 patients each selected as described by the criteria above.  The Pharyngeal and Suppository Groups each received 125mg daily while the control received a placebo.  The results of the trial demonstrate a significant decline in clinical complaints with a substantial increase in CD4+ lymphocyte levels as compared to the controls where HIV related clinical complaints and CD4+ lymphocyte levels remained consistent or marginally depressed.  There were no adverse side effects reported.  Please see results below:

KPS = Karnofsky performance score

f.          Phase II Clinical Trial – HIV/AIDS 1999-2001       

Dose Range Determination of Trioxolane (Alphamir^TM) for Efficacy, Safety and Tolerance in Individuals with HIV/AIDS – Performed at Kenya Medical Research Institute.  (Note:  for a more complete description with data, please see Appendix B)

The most recent study was an open label, randomized dose ranging clinical trial for efficacy, safety and tolerability of Trioxolane (Alphamir^TM) in asymptomatic HIV-1 individuals with CD4 counts of 100-500 cells/ul of whole blood.  In this study, 48 patients with proven HIV-1 status were recruited. 

The study consisted of three arms at buccal dose levels of 100mg (Group 1), 200 mg (Group 2), and 400 mg (Group 3) daily for a period of 4 months.  The patients CD4+ T cell counts were divided into low and medium, 100-250 cell/ul and 251-500 cell/ul of whole blood respectively before random allocation to the 3-treatment dose levels.  Efficacy was determined largely on the basis of changes in the levels of CD4+ T cells as well as those of viral loads.  Toxicity was assessed by monitoring liver, renal, and bone marrow functions.  Follow-up was done every two weeks.  At each follow-up, all toxicity and efficacy assessments were done.  Because of costs and the known slow pace of change, samples for the analysis of viral loads were taken every month, but those of two monthly intervals were analyzed.  CD4+ T cell levels were determined every two weeks.

A total of 48 individuals were recruited into the formal study.  Six of them could not complete the study for reasons not related to the medication.  Three were due to relocation of their work place following posting by their employers; two were due to the frequent travels in and out of the country that could not offer sufficient time for regular follow-up and visits.  One simply indicated that he was too busy to adhere to follow-up timetable.  However, they still insisted that they wanted to continue with the medication.  They were then given medication, but were withdrawn from the study.  A total of 43 out of 48 subjects had complete examinations (clinical) and full laboratory results for various measurements completed and assessed at baseline and at each of the eight follow-up visits up to 16 weeks.  There were 17 patients in Group 1 of whom 14 completed the study, 16 in Group 2 of whom 15 completed and 15 in Group 3 of whom 14 completed the study.

CD4+ T cell levels showed better increase over the base line in those on daily doses of 100 mg.  The levels tended to plateau during the third month of treatment at an average of 30% increase over the baseline.  Some had increases of more than 60 percent over the baseline.  On the average, 50 percent of the patients on 100 mg had significant increase in CD4+ T cells while it was 47 percent in those on 200 mg daily dose and 14 percent in those on 400 mg daily dose.   A reduction of 50 percent or more of the baseline in viral load was considered significant while an increase of 100 percent or more was considered significant in the other direction.

Alphamir^TM at 100 mg/day given in 4 divided doses had a good effect on the viral load reduction of 66.7% at 2 months.  This degree in viral load reduction dropped by 33.3% by 16 weeks.  Similarly, the observed significant reduction in log viral load dropped by over 50% at 16 weeks.  At 200 mg, given in 4 divided doses, Alphamir^TM showed a reduction in viral load of 46% at 2 months.  This degree of reduction dropped by 29% by 16 weeks.  At 400 mg similarly administered, the viral load drop was only 30% at 2 months, but this drop was insignificant. 

At 16 weeks the number of patients with this minimal drop in viral loads rose to 64%.  But only one person showed any significant drop in the log viral load drop at both 4 weeks and 16 weeks follow-ups.

18 out of 43 registered a significant reduction in their viral load.  Ten of them were on 100 mg daily dose while 5 were on 200 mg daily dose and the remaining 3 were on 400 mg daily dose.  None of those on 100 mg daily dose registered any significant increase in their viral load.

The drug, Alphamir^TM when given in four equal doses at a daily dose of 100 mg, is able to significantly increase the levels of CD4⁺ T cells.  From the foregoing, the data suggest that the lower dose appears to cause a better drop in viral loads than the higher dose. 

However, even this lower dose showed a better effect at 2 months than at 16 weeks.  This suggests that a much lower dose than 100 mg per day might be what is required for a possible sustained viral load reduction.  The drug is able to cause significant viral load reduction.  The 100 mg daily dose was found to be better than 200 mg or 400 mg daily dose.  Further follow-up led to the conclusion that a daily dose of 100 mg gave better results and hence, all follow-up as well as subsequent patients were put on 100 mg daily dose beginning August 2000.  Those with previous weight loss gained an average of 1.2k (2.6 lb) by the fourth week and this has been maintained.  Because of the known slow pace of change, the viral loads are being done every month.  Additional data have so far not been analyzed but no adverse effects have been encountered.  The results obtained so far justify the need for facilitating wider use of this drug in the clinical management of HIV/AIDS. 

g.         Future Development

It is expected that in the coming year at least three other diseases’ clinical trials will be undertaken and completed.  The Company intends to proceed with clinical trials of Prophamir™, the use of Trioxolane as a prophylactic for women against sexually transmitted diseases including HIV/AIDS. 

The Company expects that the results of the studies on HIV/AIDS and the resultant approval by the Kenyan authorities will embrace Alphamir™ as the drug of choice for Africa.  Two factors will influence that decision:  the efficacy of Trioxolane in treatment of the symptoms of HIV and the product’s low cost.  Whereas the present cost of drugs to maintain an HIV patient in the United States is some $3,000 to $10,000 annually, the generic version cost $ 316 annually from HAART. It will be possible with appropriate funding to achieve better results, with minimal side effects, and reduce the cost to approximately $205 per patient per year or less in Africa.  In addition, the use of Alphamir™ requires minimal patient monitoring and training. 

In the near future, the Company will commence with the requirements to obtain approvals in the U.S., Europe, Asia and South America.  At this time, it is not clear how long it will take to fulfill those requirements; however, it is anticipated that the results achieved in Africa will highly impact that time span.  Funding is being sought now to increase the number of patients for Phase III of the clinical trials.

h.         Treatment Strategy

We are taking an integrated approach with a cost effective treatment program, as the spread of HIV/AIDS will soon reach catastrophic proportions.  Our non-toxic, efficacious, low cost treatment is part of our treatment strategy to reduce the proliferation of HIV/AIDS.

Immune Technologies recommends that the only viable and effective approach to contain HIV/AIDS must include a comprehensive program to:

 1) administer a low cost self-testing procedure which we will co-market;

 2) treat the infected HIV/AIDS population with Alphamir; and

 3) administer a vaginal gel for the prevention of transmission which we are developing.

  The demand for the use of this treatment strategy is great enough to support incrementally larger requirements of funding.  It should be noted that the best scientific evidence supports the fact that viral strains cannot develop resistance to Alphamir™ and this represents a major ingredient in the ability to effectively control the AIDS pandemic.

i.          Product Launch

The Federal governments of the registered African countries traditionally control the purchase of drugs and its dissemination to its citizens.  Immune Technologies, “LLC” is working on a product launch strategy at this time. 

It is expected that this plan will incorporate a local liaison person who will represent the Company to the health authorities.  While each of the governments will deal with the Company directly, our liaison will maintain our relationship with the authorities.  Through this plan, the Company will also offer a distribution plan that will incorporate wholesalers, pharmacies, NGOs, and other local and regional groups.   

The Kenya Medical Research Institute (KEMRI), Nairobi, Kenya, the 9^th largest medical research center in the world, is being considered as a potential partner in order to erect a fulfillment center for the receipt of treatments from Immune Technologies, “LLC”.  They will handle the distribution of some of our products at the start of the launch through their ministers of health and religious organizations throughout Africa.  The Company is also working on building a distributor network in order to best position its products as well as co-marketing and co-promotional opportunities with HIV/AIDS diagnostic companies.  Instruction materials for the use of the product, marketing collateral for the awareness of the product, and information technology for the Company is currently being planned in preparation for the launch of Alphamir^TM.

j.          Regulatory Affairs

The Company has received approval for sale and distribution of its products for HIV/AIDS and Rheumatoid Arthritis.  The Company is continuing clinical trials as required by the Kenya Pharmacy & Poisons Board.  Immune Technologies has contracted with a nationally recognized law firm for life sciences intellectual property to review our current patents and work with the Company on future patents we intend to file.  Immune Technologies will also be working with the law firm’s FDA regulatory experts on making sure we are in compliance with all state regulations and Title 21 Code of Federal Regulations/Food and Drugs as well as all other federal regulations. 

Immune Technologies plans on working with a contract research organization that coordinates clinical trials in order to be sure that data being documented in the ongoing trial in Kenya will comply with FDA or FDA equivalent requirements.  This data will be used when we are ready to submit an Investigational New Drug (IND) application to the United States Food and Drug Administration.  In addition, Immune Technologies plans on expanding registration and/or licensing our products for uses outside of HIV/AIDS and Rheumatoid Arthritis.

Once we have adequate funding from investors, Immune Technologies plans on leasing a 12,500 square foot facility to meet the expected production requirements.  Product will then be sent to pharmaceutical facilities for final formulation and packaging.  Immune Technologies plans on recruiting experienced individuals, some of which have been identified, who have managed pharmaceutical companies, preferably with HIV/AIDS experience.  They will work with the Company to present our data to the US FDA.

APPENDIX   A:    Summary of Safety & Toxicity Studies and Clinical Trials

Summary of Toxicity Results:  Alphamir^TM is a safe and non-toxic substance.  Minimum Inhibition Concentration assays, Minimum Bacterial Concentration assays, acute systemic toxicity, dermal toxicity, oral toxicity, ocular irritation, primary skin irritation, vaginal and rectal irritation, AMES Mutagenesis assays, Micronuclei in Mouse Bone Marrow, and other topical studies were performed in order to substantiate the effectiveness, safety, and tolerability of Alphamir^TM.  The safety profile was performed over a period of 6 years between 1988 and 1994 in 4 laboratories located in the United States, Cuba, and Kenya.  Three of the facilities were medical academic institutions and the fourth was a hired professional contract research organization. 

The following studies were performed by:  North American Science Associates Incorporate (NAmSA) of L.P. One, 9341 Hazel Circle, Villa Park, CA 92667 performed the Following Tests:

Acute Systemic Toxicity (June 8, 1988 – June 11, 1988)

A 1% (w/v) solution was prepared by diluting Alphamir^TM in a solvent.  Healthy, young white mice ranging in body weight from 17 to 23 grams were used to test animals.  Two groups, each consisting of 5 mice, were used.  One group was injected (IP) with 500 mg/kg via 50 ml/kg with Alphamir^TM, while the other group was injected with a Blank.  After injection, the animals were observed immediately and at 4, 24, 48, and 72 hours.  Initial and final body weights were recorded as well as mortalities and/or reactions.  None of the animals treated with Alphamir^TM showed a significantly greater reaction than the animals treated with the Blank. 

Dermal Toxicity Study in the Rabbit of KE-091/ATX (June 8, 1988 – June 15, 1988)

Alphamir^TM was evaluated for dermal toxicity in accordance with the guidelines of the Consumer Product Safety Commission Title 16, Chapter II, Part 1500.  Five of the ten rabbits in the study received a series of epidermal abrasions with a sterile needle; the skin of the other five rabbits remained intact. 

The test mixture, a 1% (w/v) solution of the test article in a solvent, was then topically applied, via saturated two layer thick gauze, to the skin at a dose of 2.3 ml/kg of body weight.  The 10 healthy rabbits were of the New Zealand White variety, identified by ear tag or tattoo and weighing 2.5 to 2.9 kg, were obtained from a USDA licensed supplier and acclimated to the laboratory.  The animals were observed for up to 7 days for any signs of toxicity.  Under the conditions of this study, Alphamir^TM would be considered dermally non-toxic to the rabbit; the LD50 was greater than 2.3 ml/kg of body weight of a 1% (w/v) solution of the test article in the solvent.  There was no death observed during the test period.  There was slight dermal irritation noted at the beginning of the test period and completely disappeared by Day 6 of the study.  Parakeratosis was noted in the test area of all animals on Days 6 and 7.  The mean body weight gain appeared normal.  Upon necropsy, parakeratosis was noted in the test area of all animals and there were no macroscopic changes of the viscera noted.

Ocular Irritation in the Rabbit Using KE-091/ATX  (June 8, 1988 – June 11, 1988)

Tests were performed to evaluate for Ocular Irritation in the rabbit according to the guidelines of the Consumer Product Safety Commission in order to determine the potential ocular tissue irritation of a single test of Alphamir^TM instillation.  Six healthy rabbits of the New Zealand White albino variety, identified by ear tag, were selected from the stock colony.

The eyes were judged free of irritation by examining with a pen-light and under UV light after instillation of 2% fluorescein stain.  A 1% (w/v) test solution was prepared by diluting to volume, 1.0 g of the test material in 100 ml of solvent.  A 0.1 ml portion of the 1% (w/v) solution was instilled into the lower conjunctiva sac of one eye of each rabbit.  The lids were held closed for one second.  The opposite eye of each rabbit received 0.1 ml of the solvent without Alphamir^TM, and served as a comparative control.  Eyes were examined and the ocular reaction scored according to the “Illustrated Guide for Grading Eye Irritation by Hazardous Substances”.  At 24, 48, 72 hours post dosing, the eyes were examined with a pen light and re-examined with UV light following fluorescein staining of the cornea.  Under the conditions of this test, KE-091/ATX was considered non-irritant to ocular tissue in the rabbit.  There was no significant difference between the treated eye and the comparative control eye.

Primary Skin Irritation Test in the Rabbit Using KE-091/ATX (June 9, 1988 – June 13, 1988)

Tests were conducted to evaluate for primary skin irritation in the rabbit according to the guidelines of the Consumer Safety Commission, Title 16, Chapter II, Part 1500.  The purpose of this test was to determine the dermal irritation potential of KE-091/ATX to intact and abraded skin of the rabbit.  Six healthy rabbits of the New Zealand White variety, identified by ear tag, were obtained from USDA licensed suppliers.  Approximately four hours prior to application of KE-091/ATX, the backs of the animals were clipped free of fur.  Each rabbit received epidermal abrasions with a sterile needle at one test site while the skin at the opposite site remained intact.  A 1% (w/v) test solution was prepared by diluting to volume, 1.0 g of the test material in 100 ml of solvent.  A 0.5 ml portion of the 1% (w/v) solution was applied to each site (two sites per rabbit) by introduction under a double gauze layer to an area of skin approximately 1” square.  The patches were covered with a no reactive tape and the entire test site was wrapped with a binder.  After 24 hour exposure, the binders, tape and test material were removed and the skin evaluated.  The test material residue was removed with 70% isopropyl alcohol.  An evaluation was also made at 72 hours after application.  The Primary Irritation Index of 1.0 for the test material falls within the slight irritant category.  According to the FHSA Regulations, a material with a P.I.I. score less than 5.00 is generally not considered a primary irritant to the skin.  Therefore, under the condition s of the test, KE-091/ATX would be considered a slight irritant to the skin.

Oral LD50 Study in the Rat of KE-091/ATX (August 9, 1988 to August 26, 1988)

An acute oral LD50 study was conducted in 40 albino rats.  The purposes of the test were to define the dose at which death would occur in 50% of the animals subjected to a single oral administration of KE-091/ATX.  A 40% (w/v) solution was prepared by dissolving 20.0 g of KE-091/ATX in 50 ml of solvent resulting in a solution with a concentration of 400 mg/ml.  This was the test solution. 40 albino rats, of the Sprague-Dawley strain, weighing approximately 110-140 g prior to dosing, and equally divided by sex, were obtained from Harlan Sprague Dawley, Incorporated.  After a minimum of 7 days acclimation, dosing commenced in a step wise fashion.  5 male and 5 female rats per group were orally intubated at doses of 2,000, 2,510, 3,160, and 5,010 mg/kg body weight.  At each phase of the study, rats were fasted overnight and then given a single oral dose via blunt tipped cannula.  Rats were observed immediately, at 4 hours and on a daily basis.  The rats were observed for 14 days for signs of illness and death.   No adverse post-dose observations were noted.  Necropsy findings appeared normal and showed no adverse macroscopic changes.  The mean body weight gain appeared normal.  The oral, single dose, LD50 of KE-091/ATX was determined to be greater than 5,010 mg/kg body weight in the rat.

The following studies were performed by:  Developmental and Cell Biology, University of California, Irvine

Effect of Daily I.P. KE-091/ATX on Survival, Body Weight, and Rectal Temperature of Female Sprague-Dawley Rats (June 18, 1989 to August 17, 1989)

Six weeks old female Sprague-Dawley rats, obtained from Simonsen Laboratories, were given a dose range of KE-091/ATX I.P. daily for eight days.  The doses used were 19.5 mg (214 mg/kg), 1.95 mg (21.4 mg/kg), 195 ug (2.14 mg/kg), 19.5 ug (214 ug/kg) in 0.2 cc of diluents.  One group of rats was given only 0.2 cc control/solvent.  Body weights were measured on days 1 and 6 of the tests while rectal temperatures were determined on days 1,2,4,6, and 8.  The values found are given as the Mean + S.D. for each group.  Each group contained 5 rats for a total of 25 rats used for the experiment.  All the rats showed significant weight gains during the course of the test (87.5+3.6 mg on day 1 to 112.5.2 on day 6.  No statistically significant difference in weight on day 6 was found between any of the groups.  Rectal temperatures between the different groups were not significantly different on any of the days studied nor were there significant differences on different days within the same group of rats.  All of the animals survived the duration of the test.  The results show that the rats can tolerate daily I.P. of KE-091/ATX at a dose of 214 mg/kg that is almost 25 times higher than that proposed for AIDS patients (8.7 mg/kg).  This relatively high dose has no effect on rectal temperature, which remains unchanged, and on body weight, which increased by 28% in six days.  

Preliminary Studies to Determine the Dose Response of Female Sprague-Dawley Rats to I.P of KE-091/ATX (June 18, 1989 to August 17, 1989)

Preliminary studies were done with six-week-old female Sprague-Dawley rats to determine their survival rate and rectal temperature and body weight response to increasing doses of I.P. KE-091/ATX.  Individual rats were used for each dose [ 38 mg (380 mg/kg), 63 mg (586 mg/kg), 95 mg (883 mg/kg), and 136 mg (1.265 g/kg) in 0.5 ml I.P/daily for three days].  Another set of rats were divided into individual sets and given 95 mg (883 mg/kg) in increasing volumes of a solvent carrier (0.25 cc, 0.50 cc, 0.75 cc, and 1.00 cc) daily for three days.  In both groups body weight was determined on days 1 and 3.  Female rats given KE-091/ATX I.P. doses up to 63 mg (586 mg/kg) in 0.5 cc survive for at least 3 days.  Those given high doses die within 48 hours.  Relatively little change in body weight is seen in the 3 day period survivors.  Rectal temperature dropped to below 35 Degrees Celsius in the 63 mg KE-091/ATX treated rat. 

As might be expected, none of the 95 mg (883 mg/kg) treated rats survived 48 hours, regardless of diluents volume.  The results show that female rats tolerate a higher dose level [63 mg (586 mg/kg)] than male rats.  The mg KE-091/ATX level tolerated by rats is at least 38 (380 mg/kg). 

The following studies were performed by:  Kenya Medical Research Institute

Toxicity and Tolerability of KE-091/ATX in Mice (November 5, 1991 – March 27, 1992)

Three solvents of the chemical were used to evaluate a total of 186 Balb/c mice of 8-10 weeks old and weighing an average of 20g.  The KE-091/ATX was dissolved in one of the three suitable solvents and a total of 0.4 ml injected intraperitoneally.  The drug was formulated in such a way that the desired dose was contained in 0.4 mls of solvent.  After injection, the animal was kept under constant observation for one hour, then every half an hour for 5 hours.  The animal was then observed daily for a further 12 weeks.  In each case, a set of 6 mice was used for each dose of a formulation.  A set of 6 mice was similarly injected with the solvent to serve as controls.  The result was an LD50 of KE-091/ATX in mice has been calculated to be 700 mg/kg.  The minimum tolerable dose is 600 mg/kg.

Activity:  Minimum Inhibition Concentration and Minimum Bacterial Concentration (African Journal of Health Sciences Vol. 1 No. 4 November 1994)

Tests indicated that KE-091/ATX is more active than conventional drugs against commonly encountered microorganisms (in vitro) such as those causing common infections as diarrhea (Salmonella spp, Shigella spp, enteropathogenic/enterotoxigenic Escherichia coli), urinary tract infections (Neisseria gonorrhea, Candida spp), respiratory tract infections (Klebsiella spp, Staphylococcus spp, Phialophora spp, Penicillium spp) and protozoal infections (Leishmania spp).  These microorganisms were uniformly sensitive to KE-091/ATX, and the activity of the product on these organisms tested has no relationship with the resistance and sensitivity of conventional antibiotics on both gram positive and gram-negative bacteria.

The following studies were performed by:  Department of Pharmacology and Toxicology  Calle 200 y 21, Atabey, Playa, Apartado postal 16042 , La Habana, Cuba:

LD-50 Toxicity in a Single Dose of KE-091/ATX Orally Administered to Mice (January 4, 1994 to February 10, 1994)

The study of acute toxicity is a procedure in order to evaluate the adverse effects that might happen due to the administration of a single dose or multiple doses of a substance in a short period of time (24 hours).  14 Balb/c mice (7 males and 7 females) received KE-091/ATX orally in a single dose by means of cannulas adapted to syringes of 1ml graduated in 0.02 ml.  The exact dose was calculated taking into account that the volume to be administered should not exceed 0.025 ml/g (25 ml/kg) of body weight.  The dose that was given was 5,500 mg/kg.  They were registered regarding time, duration and intensity.  All mice were observed during the first hour and at 2,4, and 6 hours of post-administration.  In the following 14 days, this was done 1 time a day.  All animals that died during the observation period were registered.  The principal signs of toxicity were observed in the one male  (no females died) that died.  Those signs were excitation, hemiplegia, eyes redness, dyspnea and clonic-tonic fits, which approximately started 15 minutes after the product was administered.  The rest of the animals only developed excitation and transitory piloerection.  Due to the immediate character and severity of the toxicity signs shown in the only mouse that died which are not characteristic of this kind of administration in substances with a similar solubility to the one present in KE-091/ATX, it was considered that this mouse presented a systemic absorption.  It was not possible to identify ulcerations or any other damage in mucous epithelium during post-mortem examination. 

Medium lethal dose was not obtained because of only one male animal died at 4 hours after a single oral dose larger than 5 g/kg.  Substances with a LD50 larger than 5 g/kg orally administered to rodents are considered as non-toxic or moderately toxic substances.  Thus, this pharmaceutical preparation of KE-091/ATX is practically non-toxic when orally administered.

The following studies were performed by:  Center For the Research and Development of Medicines (CIDEM) Calle 200 y 21, Atabey, Playa, Apartado postal 16042 , La Habana, Cuba:

Irritability of Suppositories and Vaginal Ovals and Topical Solution Formulated with KE-091/ATX (completed April 1, 1994)

Tests were performed to evaluate the possible irritant and toxic effects in skin and vaginal, rectal, and ocular mucosa in rabbits. 

Vaginal Irritability:  18 Female virgin albino F rabbits from CENPALAB were used, with a body weight of 2 and 2.5 kg and were equally divided into three groups:

1) Administered with KE-091/ATX; 2) Placebo administered; 3) Control without treatment.  There was no irritation at the level of vaginal mucosa since it is within the range of minimum irritation.

Rectal Irritability:  18 male albino F rabbits were used with a body weight of 2 and 2.5 kg and were equally divided into three groups: 1) KE-091/ATX suppository administered; 2) Placebo suppository administered; 3) Control without treatment.  There was no irritation at the level of rectal mucosa since it is within the range of minimum irritation.

Dermic Irritability:  6 male albino F rabbits with a body weight of 2.5 and 3 kg were used.  They were debilitated 24 hours before the test in the dorsal part of the trunk.  The topical solution was directly administered from the flask, with any solvent in a volume of 0.5 ml at each site of assay.  It was then covered with a porous gauze plaster of approximately 25 mm X 25 MM and mixed at the place with adhesive band during 4 hours.  After this, the pastier was removed and each site was washed with distilled water; the site was marked for further observations; which were made at 1, 24, 48,72 hours after removing the pastier.  The irritation degree was evaluated according to the classification system of Draize scale based on the apparition of edema and erythema observed at each administration site.  There was no dermic irritation as the edema and erythema formation was non-existent.

Ophthalmic Irritation:  6 male albino F1 rabbits were used with a body weight of 2.5 to 3 kg and had received a thorough ocular examination with an ultraviolet light lamp.  The topical solution was administered without any solvent in the conjunctival sack of the right eye in each animal in a quantity of 0.1 ml; the left eye was taken as a control.  Both eyes of each animal were observed at 1,2,4,24,48,72,96 and 168 hours after the administration of the medicine.  The evaluation was made by Garcia et al., method based on the Draize scale.  Slight alterations were observed in the evaluated animals, but this irritation diminished during the course of the assay.  The response degree obtained according to the classification system was 10.4.  This value classifies the product as a slight irritant.

The following studies were performed by:  Center of Pharmaceutical Chemistry, Department of Pharmacology and Toxicology Calle 200 y 21, Atabey, Playa, Apartado postal 16042, La Habana, Cuba:

AMES Mutagenesis Test of KE-091/ATX (December 24, 1993 to February 12, 1994)

The purpose of the AMES test is to determine if a substance has mutagenic potential.  The mutagenic effect is fundamentally due to the analogy of physical or chemical agents for nucleic acids and it may determine genetic mutations at sub-toxic concentrations.  The genic mutation assay in bacteria, AMES Test, is the most frequently used for the evaluation of mutagenic properties in chemical products.  Five strains of Salmonella typhimurium were used (TA 1535 HisG, TA 1537 His C, TA 1538 His D, TA 98 His D, TA 100 His G).   At doses of 5,000 and 20,000 ug/pl, the product was toxic with the strain tested and after checking there was no mutagenic effect in the dose of 500 ug/pl, it was decided to test a dose range between 1,000 and 3,000 ug/pl in the final study. 

The results of the tests show that KE-091/ATX does not show a mutagenic effect at any of the studied concentration for all strains included in AMES Test.  Because of the phenotypic characteristics of Salmonella typhimurium stocks selected in the AMES Test, KE-091/ATX does not present a mutagenic effect of the kind of mutations due to a frame shift running nor to changes of base pairs.  KE-091/ATX metabolites coming from the extrinsic enzymatic action (supernatant of homogenate of rat liver treated with Arocolor) do not present a mutagetic activity in any of the studied concentrations. 

Though KE-091/ATX is a compound of anti-microbial action, it was possible to analyze its effects regarding retrogressive mutation in bacteria, at the interval of doses frequently used in Ames Assay (10 to 5,000 ug/plate).  Toxic concentration values found in some strains do not correspond to the reported inhibitory concentrations for KE-091/ATX in several pathogenic bacteria.

Toxicity by a Single Dose of KE-091/ATX Topically Administered in Mice:  LD-50 Topical (January 9, 1994 to February 22, 1994)

Tests were performed to study the acute toxicity in order to evaluate adverse effects that might happen due to the administration of a single dose or multiple doses of substance in a short period of time (24 hours).  10 Balb/c mice (5 females and 5 males) with average weights between 20 and 24 g and age of 6 weeks were administered KE-091/ATX (1g/ml) topically in a single dose in the dorsal area of the animal.  An area of approximately 2 cubic cm was previously shaved.  100 ul of neat KE-091/ATX was spread to each animal on the selected site.  All mice were frequently observed the day of the administration (each 2 hours) and in the following 14 days 1 time a day.

No toxicity sign was observed during a period of 15 days in animals of both sex, after being topically administered with a larger dose of 5000 mg/kg.  There was no sign of dermic irritation during the time of observation.  Therefore, KE-091/ATX does not show any toxicity sign due to its topical administration in single doses to mice of both sexes.

LD-50 Toxicity in a Single Dose of KE-091/ATX I.P. Administered to Mice (January 24, 1994 to February 18, 1994)

Tests were performed to study the acute toxicity in order to evaluate the adverse effects that might happen due to the administration of a single dose or multiple dose of a substance in a short time period (24 hours) when it is intraperitonial (I.P.) administered.  42 (21 females and 21 males) Balb/c mice age 6 weeks and weighing between 20 and 24 g were split into 3 groups of 14 with half the mice being male and the other half female.  The exact dose was calculated considering that the volume to be administered should not exceed 0.050 ml/g (50 ml/kg) of body weight.  The first group received 1000 mg/kg, second 1500 mg/kg, and the third 2000 mg/kg.  All animals were observed for the entire first hour after administration and afterwards every 2 hours and in the following 14 days once a day. 

The main signs of toxicity found in the animals were difficulty in walking, piloerection and swollen abdomens in the ones that died.  In previous studies, it was confirmed the absence of toxic effects in the solvent administered to mice using this technique.  The Minimal Toxic Dose value of the solvent was 14,000 mg/kg of body weight.  During necropsy of the dead animals, an oily deposit was observed in the abdominal cavity of a similar volume to the one administered (not absorbed).  LD50 was calculated using the Probits method, with the mortality data obtained during the whole time of observation to be 1909.4 mg/kg for females with a confidence limit at 95% (17.29.6-2107.9 mg/kg) and 2307.6 mg/kg for males with a confidence limited of 95% (1011.4 – 5265.1 mg/kg).  The LD50 obtained for KE-091/ATX administered to mice of both sex are characteristic of a moderately toxic substance, regarding the effects due to an acute administration.  A slight sensitivity is observed in males, but it might be due to the number of animals used.  The characteristic of late deaths after 48 hours elapsed, without any other dramatic sign of toxicity suggest that it may exist a controlled liberation of the active principle at intraperitoneal oily deposit or that the damages provoked by a single administration result in late consequences.  The target organ was not known.

Micronuclei Assay (IP) in Mouse Bone Marrow for KE-091/ATX (March 3, 1994 – April 8, 1994)

Micronuclei tests were performed in mouse bone marrow to evaluate if KE-091/ATX when administered intraperitoneally to Balb/c mice is able to produce chromosomic aberrations, if obtained a significant increase in the formation frequency of micronuclei in bone marrow erythrocytes.  20 male Balb/c mice 7 weeks old with an average weight between 16 and 24 g were treated with 2 administrations separated 24 hours a part and then were killed 6 hours after the second administration.  The product was administered using the intraperitoneal technique using 1ml syringes and broken equally into 4 groups: a) no KE-091/ATX; b) 100 mg/kg positive control; c) KE-091/ATX 500mg/kg; d) KE-091/ATX 700 mg/kg.  Each animal was killed by cervical dislocation and after this; both femurs were removed and mixed with 1ml of Bovine Fetal Serum, to extract the cells form the bone marrow.  It was centrifuged at 1,000 rpm for 5 minutes and then the cell pellet was re-suspended.  They were placed in plates previously washed with sulfocromic mixture and a solution of methanol and clorhidric acid at 5%.  Two plates were made for each animal.  Plates were fixed with methanol for 10 minutes.  After 24 hours, they were dyed with Giemsa at 5% for 16 min.  The species of Balb/c mice used in this study of micronuclei in mice bone marrow is sensitive to known mutagens and significantly yields response with an increase in micronuclei % and a decrease in proportion of polychromatic and normochromatic erythrocytes.  It should be noted that the loss of nucleated cells is an indication of cytoxicity.

The intraperitoneal treatment with two levels of dosage of KE-091/ATX in solvent, corresponds to 2 to 3 times the therapeutic concentration, does not cause mutagenic effects when statistically evaluating the appearance of micronuclei and the erythrocyte composition of normo and polychromatic cells.

Assay of Micronuclei in Mouse Bone Marrow for KE-091/ATX (February 2, 1994 – March 7, 1994)

Tests were performed to determine if KE-091/ATX, when topically administered to Balb/c mice, is able to produce chromosomic aberrations if a significant increase is obtained in the formation frequency of micronuclei in erythrocytes of bone marrow.   30 female Balb/c mice weighing between 16 to 20 g that were 7 weeks old were divided into 3 groups of 10 and given 2 administrations, 24 hours apart, with dosages of a) control, b) 500 mg/kg, and c) 1000 mg/kg.    The product was topically administered, in the dorsal region, previously debilitated in an approximate area of 4 cm. 

An abrasion was made to half of the animals in each group in the site where the product was administered, following a recommended procedure to avoid dermic lesions.  All the animals were killed 24 hours after the second administration by cervical dislocation. 

After this, both femurs were removed and perfunded with 1ml of Bovine Fetal Serum, to extract the cells from the bone marrow.  It was centrifuged at 1,000 rpm for 5 minutes and then the cell pellet was re-suspended.  They were spread in sheets previously washed with sulfocromic mixture and a solution of methanol and clorhidric acid at 5%.  Three replications were made (sheets) per animal.  Sheets were fixed with methanol for 5 minutes, 24 hours after they were dyed with Giemsa at 5% for 16 minutes. 

The species of Balb/c mice used in this study of micronuclei in mice bone marrow is sensitive to known mutagens and gives a significant response with an increase in the % of micronuclei and a decrease in the proportion of normo and polychromatic erythrocytes.  The topical treatment with two levels of dosage of KE-091/ATX in solvent carrier, which corresponds to 2 and 5 times of the therapeutic concentration (and of 100 to 200 times the therapeutic dose), even in intact skin as in skin with abrasion, do no cause mutagenic effects when statistically evaluating the surging of micronuclei and the erythrocyte composition of normo and polychromatic cells.  The carrier solvent, used as a control since it is also a carrier of pharmaceutical formulation for use in humans, does not present any mutagenic property for this method of administration and for the dose used.

APPENDIX B           Phase II Clinical Trials HIV/AIDS 1999-2001

Dose Range Determination of Trioxolane (KE091/ATX) for Efficacy, Safety and Tolerance in Individuals with HIV/AIDS – Performed at Kenya Medical Research Institute ( protocol # 557 in 1999)

The most recent study was an open label, randomized dose ranging clinical trial for efficacy, safety and tolerability of Trioxolane (KE091/ATX) in asymptomatic HIV-1 individuals with CD4 counts of 100-500 cells/ul of whole blood.  In this study, 48 patients with proven HIV-1 status were recruited.  The study consisted of three arms at buccal dose levels of 100mg (Group 1), 200 mg (Group 2), and 400 mg (Group 3) daily for a period of 4 months.  The patients CD4+ T cell counts were divided into low and medium, 100-250 cell/ul and 251-500 cell/ul of whole blood respectively before random allocation to the 3-treatment dose levels.  Efficacy was determined largely on the basis of changes in the levels of CD4+ T cells as well as those of viral loads.  Toxicity was assessed by monitoring liver, renal, and bone marrow functions.  Follow-up was done every two weeks.  At each follow-up, all toxicity and efficacy assessments were done.  Because of costs and the known slow pace of change, samples for the analysis of viral loads were taken every month, but those of two monthly intervals were analyzed.  CD4+ T cell levels were determined every two weeks.

A total of 48 individuals were recruited into the formal study.  Five of them could not complete the study for reasons not related to the medication.  Two were due to relocation of their work place following posting by their employers; two were due to the frequent travels in and out of the country that could not offer sufficient time for regular follow-up and visits.  One simply indicated that he was too busy to adhere to follow-up timetable.  However, they still insisted that they wanted to continue with the medication.  They were then given medication, but were withdrawn from the study.

A total of 43 out of 48 subjects had complete examinations (clinical) and full laboratory results for various measurements completed and assessed at baseline and at each of the eight follow-up visits up to 16 weeks.  There were 17 patients in Group 1 of whom 14 completed the study, 16 in Group 2 of whom 15 completed and 15 in Group 3 of whom 14 completed the study.

CD4+ T cell levels showed better increase over the base line in those on daily doses of 100 mg.  The levels tended to plateau during the third month of treatment at an average of 30% increase over the baseline.  Some had increases of more than 60 percent over the baseline.

On the average, 50 percent of the patients on 100 mg had significant increase in CD4+ T cells while it was 47 percent in those on 200 mg daily dose and 14 percent in those on 400 mg daily dose.  At this stage, it is difficult to tell whether the cells we are observing and registering at subsequent follow-ups are the ones that were observed at the beginning.  Obviously, some cells will have undergone apoptosis while others will have achieved senescence and died.  Others will have been recruited and matured.

Some cells have been observed to rapidly mature and strongly express CD4 markers on their surface upon starting treatment.  These cells are neither naïve nor memory/activated.  These can serve as prognostic cells, and which had been dubbed Koech’s Prognostic Cells (KPCs).

It was, therefore, planned to examine the sub-populations of CD4+ cells through the assessment of the level of maturity of these CD4+ T cells, as it was believed that they were at different stages of development. This was going to be achieved by using variously labeled monoclonal antibodies CD45 RO (for memory cells), CD45RA (for naïve cells) and CD4 PerCP (for possible identification of CD4_ cells which had been dubbed KPCs). These cells are the R3 or the CD4 DIM. It was now possible to confirm Koech's earlier observations that these cells were indeed real and could be identified and or isolated using CD45RO PE and CD45RA FITC. These cells are vital in the prognosis of response to treatment.

A reduction of 50 percent or more of the baseline in viral load was considered significant while an increase of 100 percent or more was considered significant in the other direction.

Trioxolane at 100 mg/day given in 4 divided doses had a good effect on the viral load reduction of 66.7% at 2 months. This degree in viral load reduction dropped by 33.3% by 16 weeks. Similarly, the observed significant reduction in log viral load dropped by over 50% at 16 weeks. At 200 mg, given in 4 divided doses, Trioxolane showed a reduction in viral load of 46% at 2 months. This degree of reduction dropped by 29% by 16 weeks. At 400 mg similarly administered, the viral load drop was only 30% at 2 months, but this drop was insignificant. At 16 weeks the number of patients with this minimal drop in viral loads rose to 64%. But only one person showed any significant drop in the log viral load drop at both 4 weeks and 16 weeks follow-ups.

In the case of weight gain, the lower dose showed a consistent increase between 1 month and 16 weeks follow-ups than any of the two high doses. The higher doses showed inconsistent changes in weight. No adverse effects have been encountered in any of the doses. The drug, KE-091/ATX when given in four equal doses at a daily dose of 100 mg, is able to significantly increase the levels of CD4+ T cells. From the foregoing, the data suggest that the lower dose appears to cause a better drop in viral loads than the higher dose. However, even this lower dose showed a better effect at 2 months than at 16 weeks. This suggests that a much lower dose than 100 mg per day might be what is required for a possible sustained viral load reduction. The drug is able to cause significant viral load reduction. The 100 mg daily dose was found to be better than 200 mg or 400 mg daily dose. Further follow-up led to the conclusion that a daily dose of 100 mg gave better results and hence, all follow-up as well as subsequent patients were put on 100 mg daily dose beginning August 2000. Follow-up continues. Some of the data have so far not been analyzed. Patients continue to receive the drug and funds are being sought for Phase III.

Click Here for Profiles of Viral Loads in KE091/ATX HIV/AIDS Study

Because of great variation in the results arising from the determination of viral loads, necessitating repeats, future samples for viral load were collected for different time intervals, accumulated and run at the same time. This will reduce variations. The results obtained so far justify the need for facilitating wider use of this drug in the clinical management of HIV/AIDS.

APPENDIX  C.  Approval

APPROVAL OF ALPHAMIR™ AND ARTHROMIR™

REPUBLIC OF KENYA

MINISTRY OF HEALTH

PHARMACY AND POISONS BOARD

JOSEM TRUST HOUSE
MASABA ROAD
P.O. Box 27443
NAIROBI

Ref. No. …………………………………….                                              …………………………… 20 ….

MS 9/1/33

24th May, 2001

Mirable, Inc.
C/o Mirable Africa Limited
P.O. Box 25410
NAIROBI

APPLICATION FOR REGISTRATION OF DRUGS

Your letter dated 7th March 2001 is acknowledged and the contents therein. The following comments are made about your products.

13946                  Alphamir Solution

The Board has carefully considered your request for the fast tract registration of this product and has the following observations to make.

This product still requires further development. You ate therefore Required to continue studying it until such time that enough clinical Information is generated and forwarded to the Pharmacy and Poisons Board for consideration. Because of this reason, provisional Registration of two years is granted. During this period, you are required to provide more clinical information generated from Approved health institutions.

This information will be submitted every year, failure to which registration will be revoked. You are directed to treat this product as a prescription only medicine. If the patients are not able to afford the product arrangements should be made to access the product.

DR. K. C. KOSKEI
REGISTRAR, PHARMACY & POISONS BOARD

cc: Permanent Secretary
Director of Medical Services

APPENDIX  C:  APPROVAL OF ALPHAMIR™ AND ARTHROMIR™

REPUBLIC OF KENYA

MINISTRY OF HEALTH

PHARMACY AND POISONS BOARD

JOSEM TRUST HOUSE
MASABA ROAD
P.O. Box 27443
NAIROBI

Ref. No. …………………………………….                                                          …………………………… 20 ….

MS 9/1/33

24th May, 2001
Mirable, Inc.
C/o Mirable Africa Limited
P.O. Box 25410
NAIROBI

APPLICATION FOR REGISTRATION OF DRUGS

Your letter dated 7th March 2001 is acknowledged and the contents therein. The following comments are made about your products.

13945                   Arthromir Solution

The Board has carefully considered your request for the fast tract registration of this product and has the following observations to make.

The information given on this product is still preliminary. The number of patients involved is small. However, it appears that the product might be of immense use to the patients with rheumatoid arthritis. Because of this reason, provisional Registration of two years is granted. During this period, you are required to provide more clinical information generated from Approved health institutions. This information will be submitted every year, failure to which registration will be revoked. You are directed to treat this product as a prescription only medicine. If the patients are not able to afford the product arrangements should be made to access the product.

DR. K. C. KOSKEI
REGISTRAR, PHARMACY & POISONS BOARD

cc: Permanent Secretary
Director of Medical Services

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APPENDIX D:  Patent