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Rooted in Research
While the focus of Biomox's business is predominantly on manufacturing, the company maintains a strong link with research. Thanks to its exclusive contract with the University of Pretoria, Biomox can call on the research skills of about a dozen postgraduate researchers at the University who investigate new extraction methods and active ingredients for Biomox. 'In return, we assist the students with their projects,' notes van Brummelen, 'and have even employed two of the students who recently completed their Masters degrees with distinctions.' Biomox also has a contract with the Technikon Pretoria, and conducts clinical trials in collaboration with several departments at the Tshwane University of Technology (TUT).

Product testing is one of the areas in which we see potential growth for Biomox and its research partners in the near future.

Internationally Patented Product
One of the focuses of the research done by Biomox Pharmaceuticals since its inception in 1993, was the support of the immune system. In this respect, Biomox developed a product, which has been patented internationally and tested extensively in several clinical trials and presented papers on work done at several international conferences.

List of scientific papers and conference presentations

Tenth International Conference on AIDS, Yokohama, Japan, 7-14 August 1994 (Equimmune in Treatment of HIV Infected Patients.)
12th World AIDS Conference Geneva, (Switzerland), June 28-July 3, 1998 (New Immune Supportive Therapy in HIV+ Patients. A South African Study.)
12th World AIDS Conference Geneva, (Switzerland), June 28-July 3, 1998 (2) (Glutathione, cysteine and homocysteine levels in HIV+ patients, possible immune supportive treatment.)
The 7th International Congress on Amino Acids and Proteins Vienna, Austria, August 2001 (L-Methionine; Immune Supportive Supplement in HIV+ patients: A South African Study.)

Scientific papers and conference presentations

1. Tenth International Conference on AIDS, Yokohama, Japan, 7-14 August 1994

Equimmune in Treatment of HIV Infected Patients
Bissbort SH, Davis H, van Brummelen R, Miller Steven

Objective:
The main objective was to test Equimmune; a recently formulated immunostimulant, in HIV infected and AIDS patients.

Methods:
An open, multi-centre, prospective, clinical trial with 103 patients with laboratory confirmed HIV positivity and a CD4 count at entry lower than 650/ml was done. The patients were evaluated at four-weekly intervals over a 6-month period. The four main variables recorded were CD4 count, CD4%, S-b2-Microglobulin and Karnofsky score. The change in variable over time was evaluated statistically by means of the Sign test and the Wilcoxon Signed Ranks test.

Results:
A rise in CD4 count (p<0.0001), as well as in CD4% (p<0.0001) were observed after only 3 months, this proved to be highly significant. There was an associated improvement in the Karnofsky score (p<0.0001), with a significant decrease in the S-b2-Microglobulin levels (p<0.0026). No major side-effects were reported.

Conclusion:
Equimmune seems to have a definite beneficial effect for the HIV infected patient as an immune stimulant.

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2. 12th World AIDS Conference Geneva, (Switzerland), June 28-July 3, 1998

New Immune Supportive Therapy in HIV+ Patients
A South African Study

Summary:
L-Methionine is an essential amino acid and the initial metabolite in several fundamental biological processes including protein synthesis, transmethylation and transsulphuration and together with its derivatives, play a primary role in normal growth and development (Finkelstein; 1990). In this study a specific biochemically formulated, internationally patented L-methionine combination (LMC) was tested for its’ possible role as an immune supportive therapy for HIV+ patients.

Introduction:
S-adenosyl-L-methionine (SAM) is the product of methionine adenosylation, a reaction catalysed by the enzyme methionine-adenosyltransferase. SAM can provide a methyl group to a variety of substances with S-adenosylhomocysteine (SAH) formed as a product (Mudd et al; 1995). SAM is thus the methyl donor to molecules such as hormones, neurotransmitters, nucleic acids, proteins, phospholipids (at cellular level) and certain drugs (Friedel et al; 1989). This methylation plays an important role in several reactions in the body, such as in the immune system. In certain genetic cases of severe combined immunodeficiencies, especially in the ADA deficient form, it could be shown that the mechanism of impaired immune function is, amongst others things, due to a lack of methylation capacity (Herschfield & Mitchell; 1995). In a deficiency of SAM or a low ratio of SAM in respect to SAH, a T-cell deficiency can develop (Surtees et al; 1990).

Methionine also enhances the synthesis of glutathionine. Glutathionine, a triptide involved in intracellular defence mechanisms, is depleted in patients infected with HIV. Results provide evidence that the depletion of glutathionine also leads to a methionine depletion, which in turn injures the methylation processes (Lertratanangkoon et al; 1996). This was confirmed by a study which indicated low concentrations of methionine in the plasma of HIV infected patients (Muller et al; 1996). Glutathione protects against two kinds of metabolic stress; 1) it can non-enzymatically reduce substances such as free radicals and 2) through the enzyme glutathione S-transferase participates in with detoxification of many substances (Mathews & van Holde; 1993). Glutathionine itself has been found to be important as a mediator of normal immune responsiveness and to have possible antiviral; activity (Ho & Douglas; 1992, Kalebis et al; 1991). Absorption of oral glutathione, however, may be relatively poor due to gastrointestinal enzymatic degradation. Supplementation of the precursor N-acetyl-cysteine (NAC) to increase glutathionine levels in HIV infected people has also been described with some success (Roederer et al; 1991, Droge et al; 1992).

This study therefore investigated the possibility of using LMC as an immune supportive treatment in HIV + patients as a safe affordable option for early stage treatment.

Materials and methods:
A randomised, double blind, placebo controlled study, testing LMC in approximately 400 HIV+ patients, not yet on anti-viral treatment; CD4 count between 200 and 500, was done. The trial was approved and supervised by three different ethical committees and an independent trial monitoring committee. All patients were closely supervised on a 6-weekly basis. Parameters measured included; CD4 count, total lymphocyte count, viral load, several clinical, as well as mechanistic parameters. The difference in the change from the baseline (active – placebo) was determined for each parameter. The study is ongoing.

Results and conclusions:
Already within 3 months, significant trends are noted. The CD4 count of the patients on the active therapy, presented with a slower rate of decrease, compared to the placebo group, mean difference (MD) in the change from baseline; 15.1/cmm and 95% confidence interval (CI); -8.2 to 38.4/cmm, with several patients in the active group showing definite increases. The active group also presented with a meaningful higher total lymphocyte count (MD; 137/cmm and 95% CI; 7.24 to 267/cmm). This positive trend is further reflected in the viral load levels (MD; -8264/ml and 95% CI; -25579 to 9051/ml), as well as clinical parameters such as weight and feeling of well-being; as evaluated by means of a modified MOS health rating, thus confirming the immune supportive effect of this therapy.

Preliminary results indicate the differences between the two groups to be on the increase. Further long term studies are currently underway to elucidate the full effect of this therapy. These results, however, already indicate this new immune supportive therapy as a safe, affordable alternative to use as an early stage therapy, alone or in combinations with anti-viral therapy.

References:
Dröge W, Eck HP, Mihm S: HIV-induced cysteine deficiency and T-cell dysfunction – a rationale for treatment with N-acetylcysteine. Immunology Today 1992, 13 (6):211-214

Herschfield MS, Mitchell BS: Immunodeficiency diseases caused by adenosine deaminase deficiency and purine nucleoside phosphoylase deficiency. In The Metabolic Basis of Inherited Disease, 7th ed. Edited by Scriver CR, Beaudet AL, Sly WS, Valle DV: Mc Graw-Hill Inc.; 1995: 1744-1745

Finkelstein JD. Methionine metabolism in mammals. J Nutr Biochen 1990;1:228-37

Friedel HA, Goa KL, Benfield P. S-Adenosyl-L-Methionine. A review of its pharmacological properties and therapeutic potential in liver dysfunction and affective disorders in relation to its physiological role in cell metabolism. Drugs 1989;38:389-416.

Kalebic R, Kinter A, Poli G, Anderson ME, Meister A, Fauci AS: Suppression of human immunodeficiency virus expression in chronologically infected monocytic cells by glutathione, glutathione ester, and N-acetylcysteine. Proc Natl Acad Sci USA 1991, 88:986-990

Lertratanangkoon K, Orziszewski RS, Scimeca JM. Methyl-donor deficiency die to chemically induced glutathione depletion. Cancer Res 1996;56(5):995-1005

Mathews CK, van Holde KE: Metabolism of sulphur-containing amino acids. In Biochemistry, The Benjamin/Cummings Publishing Company, Inc. 1993;711-712

Mudd SH, Levy HL, Skovby F: Disorders of transsulfuration. In the Metabolic Basis of Inherited Disease, 7th Ed. Edited by Scriver CR, Beaudet AL, Sly WS, Valle DV: McGraw-Hill Inc.; 1995:1279-1281

Muller F, Svardal AM, Aukrust P, Berge RK, Ueland PM, Froland SS. Elevated plasma concentration of reduced homocysteine in patients with human immunodeficiency virus infection. Am J Clin Nutri 1996;63(2):242-8

Roederer M, Raju PA, Staal FJT, Hertzenberg LA, Hertzenberg LA: N-Acetylcysteine inhibits latent HIV expression in chronically infected cells. AIDS Res Human Retroviruses 1992, 7(6):563-567

Roederer M, Raju PA, Staal FJT, Ela SW, Hertzenberg LA, Hertzenberg LA: Cytokine-stimulated human immunodeficiency virus replication is inhibited by N-acetyl-L-cysteine. Proc Natl Acad Sci USA 1990, 87:4884-4888

Surtees R, Hyland K, Smith I: Central-nervous-system methyl-group metabolism in children with neurological complications of HIV infection. Lancet 1990, 335:619-621

(Change in 3 months)	LMC Group	Placebo	95% Confidence Interval
Total lymphocyte count	+26.6	-110.6	7.24 to 267.0
CD4 Count	-14.3	-29.4	-8.20 to 38.4
Viral load	+0.1	+0.2	-0.21 to 0.12
MOS Health rating	9.7%	3.6%	11.06 to 1.89

Table 1

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3. 12th World AIDS Conference Geneva, (Switzerland), June 28-July 3, 1998

Glutathione, cysteine and homocysteine levels in HIV+ patients, possible immune supportive treatment.
R. van Brummelen, E. Erasmus*, D.P. Knoll*, L.J. Mienie* and S. Miller**
Biomox Pharmaceuticals, Pretoria, South Africa
* Department of Biochemistry and Microbiology
Potchefstroom University, Potchefstroom, South Africa
** The Studio, Johannesburg, South Africa

Summary: Glutathione (GSH) and subsequently its precursors; cysteine (CYS) and homocysteine (HCYS), seem to play an important role in the immune system of the HIV+ patient. The determining of these imbalances and the consequent treatment thereof, could play an important role in the supportive management of the patient. We investigated these parameters in patients enrolling for a randomised, double blind, placebo controlled South African based clinical trial, testing a possible new immune supportive therapy, aiming to address these imbalances, in HIV + patients (CD4 count 200-500).

Introduction: Several studies presented at the Oxidative Stress and Redox Regulation Conference, May 1996, as well as several recent publications seem to indicate decreased GSH levels in HIV patients tested in Europe, implicating GSH as an important role player in the regulation of the immune system (Skurnick et al; 1996, Delmas-Beauvieux et al; 1996, Barbaro et al; 1996). Findings suggest that GSH redox disturbances may be of pathogenic importance (Aukrust et al; 1996).

A most recent publication by Herzenberg LA et al implicates GSH levels as predictive of survival in HIV patients (Herzenberg et al; 1997). Results also seem to suggest that GSH could inhibit the reverse transcriptase process of HIV-1 and so directly influence virus levels (Kameoka et al; 1996, Sen & Packer; 1996). GSH was also shown to be effective in reducing the proviral DNA load in the first period of infection in murine AIDS (Rossi et al; 1996). In still another study it could be shown that exogenous GSH strongly suppresses the production of p24gag protein, as well as the virus infectivity (Palamara et al; 1996). All this serves to highlight the importance of determining the GSH levels in HIV patients and the possible role formulations addressing these deficiencies, could play in the supportive treatment of this disease. Already one study indicated the importance of antioxidant treatment in HIV patients to revert the impaired proliferative activity of their CD4 cells (Cayota et al; 1996).

Our aim was to do a comparative study to establish GSH levels and its precursors; CYS and HCYS, in HIV+ patients enrolling n a South African based study, as well as a HIV- control group, to see whether the results of previous studies indicating decreased GSH levels could be confirmed in our population group. Also investigating other important precursors such as CYS and HCYS; with the aim of eventually using these results as basis for developing a new immune supportive treatment.

Material and methods: Patients enrolling for a double blind, placebo controlled clinical trial, testing a possible new immune supportive therapy in HIV+ patients (CD4 count 200-500) and a HIV – control group were used. The CYS, HCYS and GSH levels of all patients were measured in the plasma and as total blood values and compared to a control group. The samples were all collected in specially prepared sample Electrospray ionisation mass spectrometry was carried out using a VG Quattro II quadrupole (Micromass, UK). Data was acquired in the neutral loss mode of operation, scanning over the relevant mass range in the first mass spectrometer and keeping the second mass spectrometer static, monitoring the collisional-induced dissication (CID) neutral fragments at 102, 131, 177 and 191. These neutral fragments allowed for the selective detection of butylated amino acids, GSH, CYS and HCYS respectively. Quantification was carried out using the corresponding deuterated isotopes of the metabolites and calibration curves were obtained using fixed concentrations of the isotopes and various concentrations of GSH, CYS and HCYS. The study is ongoing.

Results and conclusions: The plasma GSH levels were found to be decreased in the HIV+ group; 3.5µmol/l, compared to that of the control group; 16.6µmol/l. the mean difference (MC) in baseline values (HIV group – Control group), was –13.1µmol/l, with the 95% confidence interval for difference (CI); 19.1 to –7.18. Differences were also found in the GSH precursors levels between the two groups. The plasma CYS (MD; -205µmol/l, CI; -333 to –76.2) and HCYS (MD; 42,5µmol/l, CI;-75 to –9.93) levels were found to be significantly lower in the HIV+ group. These differences were also reflected in the total blood values of CYS(MD; -81µmol/l, CI; -152 to –10.6) and HCYS (MD; -8.83µmol/l, CI –16.1 to –1.57) (tab.1). The immunological parameters of the patients will be further monitored in a long term follow-up, as the new formulation attempt to address these imbalances.

	pGSH	pCys	pHom	TCys	tHom
n - Control	44	44	44	48	48
- HIV	41	41	41	164	164
Mean - Control	16.6	359.1	58.2	198.0	26.6
- HIV	3.5	154.3	15.7	116.7	17.7
Median – Control	8.8	287.3	35.1	158.6	25.1
- HIV	2.6	122.2	9.0	63.6	8.5
SD - Control	18.8	357.9	102.6	134.7	19.8
- HIV	3.6	215.5	21.7	237.5	23.1
Mean difference	-13.1	-205	-42.5	-81.4	-8.83
95% CI of difference	-19.1 to -7.18	-333 to -76.2	-75.0 to -9.93	-152 to -10.6	-16.1 to -1.57
p-value	<0.05	<0.05	<0.05	<0.05	<0.05

Table 1

These results confirm previous studies showing decreased GSH levels in HIV+ patients and presents new evidence implicating GSH as an important role player in HIV and disease progression. These results are of importance, not only in evaluating the South African HIV+ patient, but also in better understanding the mechanistics of the disease on a biochemical levels, to so better equip us in dealing with the disease. Preliminary results with a new internationally patented therapy developed to address some of these deficiencies, is showing great promise a possible immune supportive treatment. Further studies to help elucidate the full effect of this therapy is already in progress.

References: Aukrust P, Svardal AM, Muller F, Lunden B, Nordoy I, Froland SS. Marked disturbed GSH redox status in CD$5RA + CD4 + lymphocytes in human immunodeficiency virus type 1 infection is associated with selective depletion of this lymphocyte subset. Blood; 88(7):2626-33, 1996

Barbaro G, Di Lorenzo G, Soldini M, Parrotto S, Bellamo G, Belloni G, Grisorio B, Barbarinin G. Hepatic GSH deficiency in chronic hepatitis C: quantitative evaluation in patients who are HIV positive and HIV negative and correlations with plasmatic and lymphocytic concentrations and with the activity of the liver disease. Am J Gastroenterol; 91(12):2569-73, 1996

Cayota A, Vuillier F, Gonxalec G, Dighiero G. In vitro anti oxidant treatment recovers proliferative responses of anergic CD4 lymphocytes from human immunodeficiency virus infected individuals. Blood; 87(11):4746-53, 1996

Delmas-Beauvieux MC, Peuchant E, Couchouron A, Constans J, Sergeant C, Simonoff M, Pellegrin JL, Leng B, Conri C, Clerc M. The enzymatic antioxidant system in blood and GSH status in HIV infected patients: effects of supplementation with selenium or betacarotene. Am J Clin Nutr; 64(1): 101-7, 1996

Herzenberg LA, De Rosa SC, Dubs JG, Roederer M, Abderson MT, Ela SW, Deresinski SC, Herzenberg LA. GSH deficiency is associated with impaired survival in HIV disease. Proc Natl Acad Sci USA; 94(5): 1967-72, 1997

Kameoka M, Okada Y, Tobiume M, Kimura T, Ikuta K. Intracellular GSH as a possible direct blocker of HIV type 1 reverse transcription. AIDS Res Hum Retroviruses; 12(16):1537-41, 1996

Rossi L, Schiavano GF, Chiarantini L, Magnani M. Inhibition of murine AIDS by reduced GSH. AIDS Res Hum Retroviruses; 12(14):1373-81, 1996

Sen CK, Packer L. Antioxidant and redox regulation of gene transcription. Faseb J; 10(7):709-20, 1996

Skurnick JH, Bogden JD, Baker H, Kemp FW, Sheffet A, Quattrone G, Louria DB. Micronutrient profiles in HIV-1 infected heterosexual adults, J Acquir Immune Defic Syndr Hum Retroviral; 12(1):75-83, 1996

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4. 7th International Congress on Amino Acids and Proteins Vienna, Austria, August 2001

L-Methionine; Immune Supportive Supplement in HIV+ patients: A South African Study
Dr R van Brummelen (BPharm, MSc, PhD)

Objectives: To develop a safe, affordable immune supportive therapy for HIV+ patients.

Design: A randomised, double blind, placebo-controlled study, testing an internationally patented L-Methionine combination (LMC), in approximately 400 HIV+ patients; not yet on anti-viral treatment (CD4 count 200 to 500).

Methods: Parameters measured included; CD4 count, total lymphocyte count, viral load, several clinical, as well as mechanistic parameters. The difference in the change from the baseline (Active – Placebo) was determined for each parameter. The study is ongoing.

Results: Within 3 months, significant trends are noted. The CD4 count of the patients on the active therapy, presented with a slower rate of decrease, compared to the placebo group, mean difference (MD) in this change from baseline; 15.1/cmm and 95% confidence interval (CI), this was confirmed by the total lymphocyte count values. After 12 months the placebo group was placed on active, causing the difference to disappear.

Conclusions: Although further trials are needed, these results already indicate L-Methionine as an important role player in the immune system of patients with impaired immune function.

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