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MONOCYTE AND GRANULOCYTE-MEDIATED TUMOR CELL
DESTRUCTION: A ROLE FOR THE HYDROGEN PEROXIDE
MYELOPEROXIDASE-CHLORIDE SYSTEM

Human monocytes and granulocytes play a key role as host defense and are capable of destroying a variety of targets including bacteria, fungi, viruses and nonmalignant or malignant cells. It appears that both oxygen dependent and independent mechanisms are important. The best characterized oxygen-dependent mechanism of cytotoxicity is the hydrogen peroxide (H2O2), myeloperoxidase (MPO) halide system described by Klebanoff. Using a model system consisting of purified MPO, a source of H2O2 and various halides. Klebanoff and co-workers have demonstrated the ability of this system to destroy a wide spectrum of target cells.

Recent attention has focused on the ability of intact leukocytes to use the MPO system to destroy tumor cell targets. Clark and Klebanoff demonstrated that human granulocytes could destroy a murine tumor cell by a mechanism dependent on H2O2, MPO and halide. In addition, it was recently demonstrated that human granulocytes could destroy a human T lymphoblast target (CEM) via the MPO system and suggested hypochlorous acid (HOCl) as the lytic mediator.

IN VITRO ACTIVITY OF "TX"

Direct activity of "TX" was assessed using cells, viruses, protozoa, bacteria and fungi. Solvent alone and where possible standard drugs and reference strains were used as controls.

At dilutions of less than 2,000 to 1, a myeloma cell line (x63 balb/c line) was killed within 48 hours.

At dilutions of less than 2,000 to 1 human spermatozoa were killed within 1 minute.

Direct biological activity of "TX" was tested on microorganisms which cause common infections in the community; and the activity was assessed on the basis of minimum inhibition concentration (MIC) as well as minimum bacterial concentration (MBC) after the standard overnight incubation period in culture. It is evident that, standard microorganisms that have been identified as resistant to conventional antimicrobials have uniform sensitivity to "TX" in the same manner as are sensitive organisms.

These observations indicated that "TX" is more active than conventional drugs against commonly encountered microorganism (In vitro) such as those causing common infections such as diarrhea (Salmonella spp, Shigella spp, enteropathogenic /entertoxigenic Escherichia coli), urinary tract infections (Neisseria gonorrhoea, Candida spp), respiratory tract infections (Klebsiella spp, Staphylococcus spp, Phialophora spp, Penicillium spp) and protozoal infections (Leishmania spp).

These microorganisms were uniformly sensitive to "TX" and the activity of the product on these microorganisms tested has no relationship with the resistance and sensitivity of conventional antibiotics on both gram positive and gram negative bacteria. It is important to note that some of the pathogens investigated are those that are routinely encountered in HIV – associated disease.

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TREATMENT OF MICE WITH INFECTED LEISHMANIA MAJOR::

Experimental cutaneous leishmaniasis was achieved when 1x107 promastigotes of Leishmania major strain NLB 144 were inoculated sub-cutaneously in the nasal dorsum of inbred balb/c mice weighing approximately 20g each. Mice treated with topical preparation responded better than those in any of the other treatment groups, and "TX" was superior to Pentostam in treating Leishmania major infection in mice.

IMMUNOMODULATORY ACTIVITY:

A group of 15 balb/c mice which had been used as negative controls (with "TX" but without infection) in the previous experiments on Leishmania infection were studied further to assess the effect of "TX" on immunological status of these mice.

Bone marrow examination revealed that there was an increase in new clones of lymphocytes in mice put on "TX" as compared to mice without any drug or those with Pentostam alone. This observation strongly suggests that "TX" is an immunomodulator, a property which is of critical consideration in the treatment of disease in an immunocompromised host and of autoimmune disorders. Studies in this area are still continuing.

The product "TX" is also active in vitro against Leishmania donovani, the
causative agent of visceral leishmaniasis.

TOXIDITY OF "TX"

No toxic effects have been observed in human subjects at their present
dosages for treatment periods of up to six years. The LD50 in rats is 700
mg/kg IP. For oral dosage the LD50) is greater than 5000mg/kg. There is no toxicity or irritation to skin, eyes or mucous membranes.

The notion that free radicals are toxic is completely wrong and must be
discarded. "TX" is completely non toxic as are endogenously produced free radicals because all normal cells contain protective enzymes such as SOD, glutathiones and catalase which are capable of protecting the cell from damage at any possible dosage level used therapeutically either endogenously generated or given as "TX" therapy.

It is interesting to note that in an analogous misconception it was thought that liquid bleach (HOCL) was too toxic to be used as an antimicrobial agent because of its effect on cells in vitro. However, when finally used for treatment of infections in the course of WWI it was found to be totally non-toxic to normal tissue in vitro and it became the most utilized antimicrobial substance through the war.

It has been demonstrated that neutrophils can use exogenously supplied free radicals to kill bacteria as in cases of chronic granulomatous disease wherein the defect is absence of the "respiratory burst".

It should now be no mystery as to why repeated attempts to use "free radical scavengers" such as superoxide dismutase and antioxidant vitamins have failed dismally to ameliorate auto immune disease. In fact recent studies have demonstrated that treatment with such compounds actually increase materially the incidence of neoplastic disease.

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WHAT "TX" CAN DO

When used topically "TX" is highly effective in essentially the entire spectrum of dermatological diseases including infectious dermatoses, noninfectious dermatoses, traumatic and toxic dermatological conditions.

When used vaginally "TX" is highly effective in the full spectrum of vaginal infectious disease, including those caused by viruses. In addition it is spermicidal and it is expected that ongoing studies will confirm that it is effective in the prevention of transmission of all venereal disease including HIV.

"TX" systemically is effective in a variety of infectious diseases including HIV infection, a broad spectrum of autoimmune disease and chronic fatigue syndrome. It is equal or more effective than chemotherapy in neoplastic disease without toxicity and with the ability to utilize long term therapy.

All forms of arthritis including rheumatoid and degenerative osteo- arthritis are placed in total remission after 2-12 weeks. Failures of therapy are rare.

The full spectrum of efficacy "TX" will not be enumerated here as
confirmatory clinical trials are not yet complete. However, the clear implication of the above statements is that the level of efficacy must generate a total reevaluation of prior scientific concepts. Observed characteristics of "TX" demonstrate the following:

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MODE OF ACTION

"TX" acts in vivo through the production of free radicals. Neutrophils and
other phagocytic 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 it is clear that these free radicals exert a strong direct microbiocidal effect even in the absence of leukocytes. Thirdly free radicals exert both a immunomodulatory and immunostimulatory effect by chemotactically signaling the immune system that a pathogen/leukocyte interaction is occurring.

Little is known how "TX" 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. "TX" 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
infected cells. It appears that "TX" also plays a role in cell membrane
stabilization.

OLD THEORETICAL MODELS

It is clear that the concept that free radicals are toxic must be discarded.

The concept of differential toxicity as a basis for therapeutics as in
chemotherapy and use of such drugs as AZT must be reevaluated.

The concept of immunosuppression as a rational or preferred treatment for autoimmune disease must be discarded.

The compartmentalization of disease and therapy must be reevaluated.

NEW THEORETICAL MODELS

Understanding the function of the immune system creates the understanding that a broad spectrum therapeutic is rational and scientifically based. Immunostimulation is preferred to immunosuppression.

There is more similarity to the various disease processes than previously
recognized. Many so called unrelated diseases have similar pathogenesis and differ primarily in the target organ attacked.

Increasingly likely model is the following:

1. Retroviral infection leading to short acting acute febrile illness. This is
followed by a variable period without symptoms which may be many years.

2. During this latent period the virus remains within the host and eventually succeeds in causing sufficient immune dysfunction within the host so as to protect itself. The result of this immune dysfunction is manifest disease in the host which varies according to the target organ damaged. As in AIDS it is T cells. As in arthritis it is joint cartilage.

3. Chronic long term immune system dysfunction may be the result of high viral load with multiple viruses remaining within the host over a long period of time. A constellation of degenerative process then occur which are associated with the aging process.

CONCLUSION

Much work remains to be done before we can say that we have most of the answers. However, it is clear that "TX" and it's implications demands the rethinking of many less than perfect old concepts in medicine. We can look forward to many new revelations as the slow process of drug evaluation and approval moves on.