SYNTHESIS, COMPOSITION AND MEDICAL APPLICATION OF CYCLIC PENTABORATE COMPOUNDS

Abstract: A cyclic pentaborate composition is provided which exhibits immuno-modulatory activity and anti-bacterial in animals including humans. This composition is prepared from a chemical reaction between boric acid and borax (sodium tetra-borate dodecahydrate) in the presence of cytosol fluid obtained from boric acid hypersensitized fungal or microorganism cultures. The cyclic pentaborate compositions of this invention have been useful in enhancing the antibody production in treated animals afflicted with AIDS, pneumonia, osteomyelitis, systemic lupus erythematosus, allergies, asthma, insecticidal intoxication, pre-eclampsia and a variety of other bacterial or viral infectious diseases.

Description: SYNTHESIS, COMPOSITION AND MEDICAL APPLICATION OF CYCLIC PENTABORATE COMPOUNDS The present invention relates to cyclic pentaborate compositions having immuno-modulatory activity in mammals. Human Immunodeficiency Virus Type 1 (HIV-1) is now generally accepted as the causative agent for AIDS. Since the first cases of AIDS were reported to the Center for Disease Control (CDC) in 1981, over 100,000 cases have been reported in the United States. Over fifty percent of these patients have already died. Based on estimates that one to two million Americans are currently infected with HIV but are asymptomatic, and that twenty percent to thirty percent of infected subjects will develop overt disease within five years, there will be about 270,000 total cases of AIDS by 1991, of which 3,000 will be children. The viral origina¬ tion of the disease has prompted the search for HIV-1 inhibitors. By virtue of their inhibitory effect on the virus replicative cycle, these inhibitors may be expected to prevent progression of disease and possibly promote recovery from the disease. In addition to this approach and because HIV results in an impaired immune system, a number of i munomodulating agents have also been investi- gated. It can be reasoned that an agent which has the potential to boost the anti-viral immune response should aid in the elimination of the virus. The validity of anti-viral therapy in HIV-1 induced diseases first became apparent when it was shown that suramin, a potent inhibitor of retrovirus associated reverse transcriptase, suppressed replication of HIV-1 in vitro and in vivo. However, no clinical or immunologic improvement was noted over a six-week treatment period, the time at which treatment was terminated due to toxicity of the drug. Even a longer treatment period (37 weeks) produced no clinical or immunologic improvement. The next compound tested, AZT (3'-azido-2' ,3 '-dideoxy- thymidine) , inhibited HIV-1 at a significantly lower con¬ centration with a substantially higher level of inhibition than that noted for suramin. Treatment of both AIDS and ARC patients with AZT for six weeks produced both clinical and immunological improvement, as well as a decreased frequency of opportunistic infections and mortality rate during a twenty-four week period of therapy. AZT, also referred to as Retrovir or zidovudine, is the only drug to date that has been approved for treatment of this disease. Although AZT is of benefit in some AIDS or ARC patients treated up to twenty-four weeks, the beneficial effects of this drug wanes over a six-month period. New drugs which show promise in ongoing clinical trials include ddC (2' ,3'-dideoxycytidine) , DDI (2'3'- dideoxyinosine and ddA (2' ,3'-dideoxyadenosine) . Of these three, DDI has shown little or no toxicity and has produced significant clinical or immunologic improvement. When destruction of the immune system has occurred, it may be warranted to combine anti-viral therapy with an immune response modulator such as interferon (HulFN-alpha,- beta or -gamma) or granulocyte-macrophage colony stimulat¬ ing factor (GM-CSF) . Usually, immuno odulators are given with an anti-viral agent. For instance, it has been shown that GM-CSF potentiates HIV-1 production in monocytes, but when used in combination with AZT, enhances the net anti- HIV activity of AZT. The present invention provides novel cyclic pentabor¬ ate compositions having both immunomodulatory and anti¬ bacterial activity. The cyclic pentaborate compositions of the present invention are useful for enhancing antibody production in a treated mammal, and combatting bacterial infections in a treated mammal. The cyclic pentaborate compositions can be administered in any number of pharma- ceutically acceptable routes, including for example orally, intravenously, topically, intracavity, rectally, locally or intramuscularly. One of the preferred cyclic pentaborate compositions of this invention (termed herein as L-51) is prepared from catalyzed reaction of boric acid and borax (sodium tetra- borate decahydrate) in the presence of cytosol fluid obtaned from fungal preparations having been hypersensitized by the step-wise addition of increasing concentrations of boric acid. Chemical analysis of L-51 in dry powder form shows it contains equimolar quantities of two major components, sodium pentaborate and boric acid, plus trace levels of CH and/or N, indicative of trace quantities of the cytosol fraction from the lysed hypersensitized fungal culture. In solution, only sodium pentaborate was detected by physicochemical analysis. The present cyclic pentaborate composition has been used to treat HIV-infected humans. These HIV-infected individuals included patients ranging from Walter Reed (WR) classifications of 2 through 6. In the majority of patients, the drug is given twice daily by the oral route for a period of four months. The dose of medication is adjusted to the lymphocyte counts, but ranges from 500mg to 2.5gms per day. Those patients who have developed symptoms consistent with ARC or AIDS have improved clinically to the point of returning to work. We have observed a group of patients who did not respond favorably long-term to treatment with the drug but whose quality of life is enhanced. This includes patients with: (1) Kaposi's Sarco a (KS) and/or marked neurologic symptoms; and (2) patients who have an absolute CD4 positive cell count of less than 40. A typical pattern is noted in all HIV-infected indi¬ viduals after initiation of the treatment protocol. Approximately three to four weeks post-treatment, all patients develop one or more of a number of symptoms, including loss of weight, night sweats, depression, minor peeling of skin from palms of their hands and bottoms of their feet, diarrhea or constipation, and headaches. During this same period, a decline in the total lymphocyte count and CD8 positive cells is generally noted. In addition, a decline in CD4 positive cells is also noted in many of the patients. At eight to twelve weeks post- treatment, the clinical symptoms noted above subside and a significant elevation of total lymphocytes and of CD8 positive cell numbers increase above the upper limit of normal for uninfected humans. In the majority of patients, the treatment can be terminated after four months. No relapses have been noted in thirteen patients studied nine months post-treatment. The present cyclic pentaborate compositions have also been administered with efficacy to a number of patients with diseases unrelated to AIDS. These include human patients with open and/or bacterially infected wounds, osteomyelitis, encephalitis and other bacterial and viral infections. In addition, efficacy has been noted with a number of human immunologic disease syndromes, including asthma, and systemic lupus erythematosus (SLE) . Administration of the present cyclic pentaborate compositions by the oral or intraperitoneal route into mice infected with Rauscher Murine Leukemia Virus (MuLV) leads to significant reduction of tumor mass as measured in spleen weight. Little or no toxicity is seen at concentrations of L-51 which inhibit tumor induced splenomegaly. An LD50 of 1.5 grams L-51/kg body weight has been determined in rats. L-51 has been tested for direct anti-bacterial activity, for virucidal activity and for in-vitro anti¬ viral activity. The viruses tested included human immunodeficiency virus (HIV) , herpes simplex virus (HSV) , cytomegalovirus (CMV) , Epstein Barr Virus (EBV) and fourteen species of bacteria. No significant anti-viral activity has been observed. In vitro anti-bacterial activity was noted when L-51 was screened against fourteen species of bacteria: Streptococcus pneu oniae, Streptococcus pyogenes, Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudo onas aeruginosa, Shigella depenteriae, Shigella flexneri, Salmonella typhi, Escherichia coli, Proteus vulgaris, Haemophilus influenzae, Brucella suis and Mycobacterium tuberculosis. Subsequent experiments revealed similar jLn vitro anti-bacterial activity in the presence of cyclic pentaborate only. The advantage of L-51 for treatment of infectious diseases includes expression of little or no observable toxicity, rapid response of a virus or bacterial infected host which have been unresponsive to other drugs and ability to treat chronic viral infections, such as HIV for a definite period of time without apparent relapse after cessation of the treatment. EXAMPLE 1 I. Synthesis of L-51 A. Rice Infusion Media 1. Remove discolored seeds from white rice. 2. Weigh 30 grams of clean white rice. 3. Add rice to 750ml of purified water at 96 * C. 4. Bring to a boil and boil for 4-8 minutes (5 minutes is preferred) . Operator care must be exercised during this phase to prevent boil-over. 5. Let rice settle for 10-15 minutes (15 minutes is preferred) before first filtration. 6. Obtain infusion media by vacuum filtration of the above material through Whatman #541 or similar filter paper. Change filter paper as required. 7. When all infusion has been filtered, filter again (according to step 6) for a second time. 8. Check pH (6.4±0.6 pH units is required to proceed) . If pH is outside this range, discard prep and start with step Al. 9. Bacteriologic controls: a. Brain and heart infusion broth (BHI) should contain no cloudiness after incubation time to 72 hours at 36'C when inoculated with lOul of rice infusion. B. Grape Must 1. Thoroughly wash a bunch of seedless grapes. Allow normal flora to repopulate surface of grapes by holding grapes in a non-sterile area for about 72 hours. 2. Check pH of the juice extracted from 3-4 representative grapes as described in step B4 (pH must be 3.7±0.6 pH units to proceed) . 3. If pH is not within specification leave grapes another 24 hours. Then check pH again, if not within specifications discard grapes. 4. Once pH is within specifications, rinse grapes (remove all stems at time of weighing) . 5. Weigh out 150 grams of grapes and place in a mechanical blender. Blend intermittently at a high speed for no more than 30 seconds. Collect grape juice and pulp. 6. Filter juice and pulp through a 420 micron mesh pre-filter and collect juice. Vacuum filter using Whatman #41 or similar filter paper. Change filter paper as required. 7. Collect a minimum of 35ml clarified juice. 8. Check pH (must be 3.7±0.6 pH units to proceed) . If pH is not within limits, discard juice and begin again at step Bl. 9. Bacteriological Controls: a. Brain and heart infusion broth (BHI) should contain no cloudiness after an incubation time of 72 hours at 36'C when inoculated with lOul of grape juice. C. Biomass Reaction 1. Heat 300ml of filtered rice infusion to 70'C in a 500ml beaker. Add 5.2gm of USP grade gelatin (300 bloom) . 2. Dissolve gelatin using a stirring hot-plate maintaining a 70'C temperature. 3. Transfer rice infusion-gelatin mixture to a 500ml Erlenmeyer flask. Heat to 70 * C. Stir for one minute (no foaming) . Add 30ml filtered grape juice. Assure temperature does not exceed 70'C. 4. Check pH (should be 3.9±0.5 pH units at 26 * C-30 * C before incubation begins). 5. Remove 4ml of biomass for quality control tests, seal flask with cap and incubate at room temperature for 72 hours. D. Hypersensitization Reactions l. After incubation at room temperature, warm the flask containing 300ml. biomass in a water bath at 50 * C until completely liquified. 2. Remove flask from the water bath; add 30ml of 1% (w/v) boric acid to biomass with vigorous mixing for 2-4 minutes. (Assure a vortex by proper setting of stir plate.) 3. Recap flask tightly and incubate for 24 hours at room temperature. 4. After incubation at room temperature, warm the flask containing 330ml of acidified biomass in a water bath at 50 * C until completely liquified. 5. Remove flask from the water bath; add 30ml of 2% (w/v) boric acid to the acidified biomass with vigorous mixing for 2-4 minutes. (Assure a vortex by proper setting of stir plate.) 6. Recap flask tightly and incubate for 24 hours at room temperature. 7. After incubation at room temperature, warm the flask containing 360ml of acidified biomass in a water bath at 50'C until completely liquified. 8. Remove flask from the water bath; add 30ml of 3% (w/v) boric acid to the acidified biomass with vigorous mixing for 2-4 minutes. (Assure a vortex by proper setting of stir plate.) 9. Recap flask tightly and incubate for 24 hours at room temperature. 10. After incubation at room temperature, warm the flask containing 390ml of acidified biomass in a water bath at 50'C until completely liquified. 11. Remove flask from the water bath; add 30ml of 4% (w/v) boric acid to the acidified biomass with vigorous mixing for 2-4 minutes. (Assure a vortex by proper setting of stir plate.) 12. Recap flask tightly and incubate for 24 hours at room temperature. 13. After incubation at room temperature, warm the flask containing 420ml of acidified biomass in a water bath at 50 * C until completely liquified. 14. Check pH (should be 4.1±0.5 pH units after the 4% boric acid addition) . 15. Store in 10-15ml aliquots at 2'C to 8'C. 16. Bacteriological Control: a. Count of microorganisms under a light microscope should be approximately 2-5 million per ml. E. Extract from Cvtosol of Microorcranism 1. Emulsify an aliquot of acidified biomass by warming and shaking at 50 * C until completely liquified. 2. Immerse sonication tip into the biomass contained in a 15ml glass beaker (temperature of biomass should be 30"C) and sonicate the mixture for 2 minutes ± 15 seconds with a power unit rating of 4% (at a setting of 2-3 on the power selector of a Model W-385 Ultrasonic Processor) . 3. Filter before cooling through a 0.7 micron paper filter. 4. Dilute 2ml of filtrate with 8ml of purified water. This product is now termed Suspension L. F. Chemical Reaction of Boron Compounds 1. Pulverize A.C.S. Reagent Grade boric acid crystals (99.8% purity) and sodium tetraborate decahydrate (99.5% purity) in a mechanical blender. 2. Add 500 grams of each powdered boron compound to a 3 liter glass container having an airtight sealing lid. Mix thoroughly by shaking and turning the jar vigorously for 10 minutes. 3. Add four separate 2.5ml aliquots of Suspension L to the powder mixture with thorough mixing (identical to the mixing in step F2) of 1 minute between each addition. After each Suspension L addition break up clumps by hand using a sterile glove or suitable mechanical device. Six minutes of mixing is given after final liquid addition. 4. Compact mixture to the bottom of the container, hermetically seal and incubate at room temperature away from any direct light source for 15-20 days. 5. Open jar and compact reactants once again. Incubate for 5-10 days. 6. Spread mixture onto a sterile non-porous tray, breaking moist paste into small fragments. 7. Allow paste to dry in a dehumidified area at room temperature. This will require approximately 5-8 days. G. Final Catalysis Reaction 1. Solution I—to 1000ml Of (20'C-24 * C) purified water, contained in a 3000ml container, add 480 grams of dried fragments with stirring. 2. Heat to dissolution point with stirring. 3. Add 40ml filtered undiluted Suspension L (prepared as in step El-3) to dissolved salt (Solution I) at 60 * C±5'C, stir 2 minutes and then heat the solution to 90'C. 4. Prepare Solution II by dissolving 160 grams boric acid in 1 liter purified water at boiling temperature in 2000ml container. 5. Add Solution II to Solution I at 90'C. Mix and bring to boil and hold for 5 minutes. 6. Filter the mixture through a Whatman GF/F (0.7 microns) filter paper. 7. Maintain temperature at 70'C and immerse a Ag/AgCl combination electrode into solution 7cm deep. Turn pH meter to position "MV" and allow to stabilize for 5-8 minutes before recording results. H. Final Preparation of L-51 1. Heat above solution to 90 * C±5'C then immediately filter through a 0.7 micron filter paper. Divide solution evenly into two 2 liter beakers. Concentrate by boiling liquid off to a final volume of approximately 350-450ml. Pour liquid in a non-stick tray. Dry until crystals form. Break up formed sheet of L- 51 into pieces, continue to dry overnight. Dry material in a 45'C oven. 6. Pulverize dry material (L-51) with mechanical blender intermittently at high speed. 7. Sieve dry powder to about 60 micron mesh size. 8. Fill capsules with L-51. EXAMPLE 2 Elemental Analysis The analyses were performed utilizing a CHN elemental analyzer, atomic absorption spectroscopy for boron and atomic emission spectroscopy for sodium. The elemental composition of L-51 was: Carbon - 0.015-0.065% Hydrogen - 3.75% Nitrogen - 0.02-0.04% Boron - 19.5% Sodium - 5.82% Oxygen - 70.93% An x-ray emission spectrum showed presence of only oxygen and sodium; however, this technique does not detect elements with an atomic number of less than six (i.e., boron and hydrogen) . Thus, material not accounted for in above elemental analysis was assumed to be oxygen (70.93%). A second elemental analysis performed by inductively coupled plasma spectroscopy showed boron (17.07%) and sodium (5.90%), which translated to a mole ratio of boron to sodium of 6.15. A series of physicochemical studies were performed. The infrared (IR) analysis (Figure 1) and U B NMR analysis (Figure 2) show that IR spectra and U B spectra matches the published spectrum for sodium pentaborate (Frey and Funck, Z. Naturf rsch 27b:101-125 (1972); Salentine, Inorσ. Chem. 22:3920-3924 (1983). In addition, low levels of CH containing material which appear to be mixtures were detected in two different lots of L-51. A solubility test for two different lots of L-51 and of sodium pentaborate NaB 5 0 8 "5H 2 0 was performed, which yielded the following results: L-51, Lot LM04209 - 0.13 g/g H 2 0 L-51, Lot BTR L-51007 - 0.13 g/g H 2 0 NaB 5 0 8 -5H 2 0 - 0.18 g/g H 2 0 Based on differences in solubility, x-ray powder crystallographic studies were performed. The x-ray powder crystallographic pattern data were compared to patterns published for borax, boric acid and sodium pentaborate. It was clear that there was no borax in L-51. However, a good fit was noted by superimposing the files for boric acid (referred to as hydrogen borate/sassolite) and that of sodium pentaborate (referred to as sborgite) (Figure 3) . In fact, there were no lines in the x-ray powder pattern between 10' and 45' two theta that were not matched with that noted for L-51. Therefore, in dry powder form, L-51 contains both sodium pentaborate and boric acid. These results were re-examined in light of the analytical data derived by inductively coupled plasma spectroscopy. Assuming that the H 2 0 content may vary, calculations were made assuming one mole H 2 0 per mole mixture (sodium pentaborate + boric acid) . Calculated for NaB 5 0 8 - 5H 2 0 + B (0H) 3 + H 2 0 B - 17 .29% Na 6. 13% Analytical Data B - 17 . 07% Na 5. 90% These results are low by 0.22% and 0.23%, respectively, suggesting that the water content is a little different from that calculated. These data are thus internally consistent with the mixture calculated. It was thus concluded that in powder form, L-51 contains equimolar quantities of sodium pentaborate (NaB 5 0 8 -5H 2 0) , boric acid [B(0H) 3 ] and water (H 2 0) . In addition, L-51 contains an organic component originally derived from the addition of boric acid treated fungal fermentation products which are added during the manufacture of the drug. The carbon values were 0.015 to 0.065%, and nitrogen values were 0.020 to 0.040%. Only sodium pentaborate is detected in a solution of L-51 dissolved in water. This is in keeping with the fact that borate compounds in solution rapidly establish an equilibrium of species whose identities and compositions are determined by such variables such as pH, temperature and concentration of reactions. With respect to sodium pentaborate, it is expected that other salt forms such as the potassium salt will also exhibit similar biological activity as the sodium form. EXAMPLE 3 Toxicity Studies The toxicity of L-51 has been ascertained in 14-16 gram Swiss mice challenged with 0.1ml of an intravenously administered preparation containing 10% Rauscher MuLV infected spleen homogenate generated by serial .in vivo passage of the virus. Three different routes of administration for L-51 were used, including intra- peritoneal (IP) , oral gavage (PO) and via drinking water. The body weights were recorded daily. Ribavirin was administered as a positive control at 150mg/kg IP. Five NIH Swiss mice were assigned to each of the following groups: 1. Phosphate buffered saline (PBS) administered by oral gavage bid. 2. Phosphate buffered saline (PBS) administered intraperitoneally bid. 3. Drinking water control. 4. 150mg Ribavirin/kg/day (divided twice daily) qd - 1 to +20 * administered intraperitoneally (IP) . 5. lOOOmg L-51/kg/day (divided twice daily) qd -1 to +20 administered intraperitoneally (IP) . 6. 500mg L-51/kg/day (divided twice daily) qd -1 to +20 administered intraperitoneally (IP) . 7. 250mg L-51/kg/day (divided twice daily) qd -1 to +20 administered intraperitoneally (IP) . lOOOmg L-51/kg/day (divided twice daily) qd -1 to +20 administered by oral gavage (PO) . 500mg L-51/kg/day (divided twice daily) qd -1 to +20 administered by oral gavage (PO) . 10. 250mg L-51/kg/day (divided twice daily) qd -1 to +20 administered by oral gavage (PO) . 11. 3mg L-51/ml administered in the drinking water qd -1 to +20. 12. 2mg L-51/ml administered in the drinking water qd -1 to +20. 13. lmg L-51/ml administered in the drinking water qd -1 to +20. * qd -1 to +20 = one dose of compound was administered one day preceding virus challenge and thereafter compounds were administered twice daily for 20 days. None of the treatments resulted in a loss of body weight in these toxicity-controlled mice. The least increase in body weights occurred in mice receiving Ribavirin and in the mice receiving L-51 via the drinking water at 3mg/ml. The only significant decreases (p<.05) in body weights for the toxicity control animals occurred with Ribavirin and with the three doses of L-51 administered IP and the lOOmg L-51/kg/day in the PO treatment group. These preliminary experiments show that L-51 is not demonstrably toxic and may be tolerated at higher doses. In fact, a previous experiment had indicated that the LD50 of L-51 in rats is 1.5 gm/kg body weight. EXAMPLE 4 Efficacy Study of L-51 Against Rauscher Murine Leukemia Virus (MuLV In Vivo Ten 14-16 gram Swiss mice injected with Rauscher MuLV were assigned to each of the groups listed as described in EXAMPLE 3 above. None of the virus-infected, drug treated mice had demonstrable decreases in body weight. Significant decreases (p<0.01) in spleen weights occurred with all compound treatments except the 250mg L-51/kg/day IP and the lmg L-51/ml of drinking water groups. The greatest reduction in spleen weights (78.0%) occurred in the Ribavirin treated mice. The greatest reduction in spleen weights occurring with L-51 treatment was 74.6% in the 3mg/ml of drinking water treatment group. The 3mg/ml of drinking water treatment group corresponded to an average daily dose of 1989mg L-51/kg of body weight. The second greatest reduction in spleen weights occurring with L-51 treatment was 66.1% in the lOOOmg L-51/kg/day PO treatment group. The efficacy ranking for each of the L-51 treatments based on a reduction in spleen weights is: 3mg/ml water > lOOOmg/kg PO > 500mg/kg PO > lOOOmg/kg IP > 2mg/ml water > 250mg/kg PO > 500mg/kg IP > lmg/ml water > 250mg/kg IP Within the L-51 administered IP groups, the only significant difference in dose levels was present with the lOOO g/kg/day group and the 250mg/kg/day group. Within the oral treatment groups, the lOOOmg/kg/day dose level reduced splenomegaly to a significantly greater extent than did the 500 or 250mg/kg/day dose levels. Within the drinking water-treated groups, the 3mg/ml dose level reduced splenomegaly to a significantly greater extent than did the 1 or 2mg/ml dose levels. The 3mg/ml of drinking water treatment group reduced splenomegaly to a significantly greater extent (p <0.05) than did any other L-51 treatment schedule except the lOOOmg/kg/day PO treatment group (p = 0.469). The lOOOmg/kg/day PO treatment also reduced splenomegaly to a significantly greater extent (p <0.05) than did any other L-51 treatment schedule except the 3mg/ml of drinking water treatment group. EXAMPLE 5 Biological Activity of Major Components Associated with L-51 EXAMPLE 4 shows that L-51 has in vivo immunomodulating activity reflected by decreased tumor burden measured by reduction of spleen weights in Rauscher Murine Leukemia Virus (MuLV) infected Swiss mice who were fed drinking water containing L-51. Therefore, this model was used to test the activity of each of the two major components associated with L-51, sodium pentaborate and boric acid. The results of this experiment are shown in TABLE 1. In summary, boric acid, sodium pentaborate and an equimolar mixture of the two, at a concentration of 4mg/ml of drinking water, produce approximately the same effects in terms of reduction of spleen weight (67.4-70.2%) . The best inhibition was noted with L-51 and the positive control drug Ribavirin, at 75mg/kg given IP, twice daily. L-51 and Ribavirin reduced spleen weight by 75.6% and 76.8%, respectively. Each of the two major components of L-51, sodium pentaborate and boric acid, also provide a significant degree of protection in the Rauscher MuLV infected mouse. However, the maximum degree of effectiveness was noted with the organically derived drug, L-51. TABLE 1 SPLEEN WEIGHTS IN RAUSCHER MuLV INFECTED SWISS MICE 67.6 57.5 30.1 67.4 52.0 19.6 70.2 55.2 34.1 75.6 53.8 42.2 76.8 * Expressed as the concentration of the drug in drinking water,' except Ribavirin which is expressed as the quantity of the drug per kilogram of body weight. EXAMPLE 6 Clinical Trials of AIDS Patients Treated with L-51 Seventeen AIDS patients were selected in a foreign country and placed on an L-51 treatment program. The analysis of an 18th AIDS patient was included in much of this study because blood was obtained at several intervals throughout this study. However, we failed to obtain a blood sample from this patient prior to initiation of the treatment regimen. Also, an HIV antibody negative individual served as an internal untreated negative control. The actual numbers in each WR stage are as follows: (a) WR 0, one individual; (b) WR 2-3, nine patients; (c) WR 4, two patients; (d) WR 5, two patients; and (e) WR 6, five patients. The basic protocol followed during this study was based on two assumptions: 1. Our working hypothesis was that L-51 was an anti-viral compound. Therefore, we assumed that, similar to the published AZT studies, virus cultures and p24 antigen results would become negative and hopefully CD4+ cells would increase in numbers. 2. We had no knowledge to guide us in the selection of patients, so we chose a range of patients from WR 2 through WR 6, including two patients with Kaposi's Sarcoma (KS) . In total we had ten patients with CD4+ cell counts of less than 400 but more than 50, four patients with CD4+ cell counts of less than 50, and four patients with CD4+ cell counts of more than 400. Our clinical observations can be summarized as follows: Fourteen patients have responded very well to the L-51 treatment. It should be pointed out that all fourteen went through a period ranging from week 6 through week 14 where they experienced a number of side effects. Some side effects were quite minor, including minimal weight loss, diarrhea and/or constipation, skin peeling from palms of their hands and/or feet, some rash, neural sensations in their extremities at times, a loss of appetite and periods of depression. In one of the patients, the weight loss was severe and he became deeply depressed; he vomited a great deal, became anemic and lost his appetite. However, after approximately fourteen to sixteen weeks, his appetite began to increase and he started feeling much better. His weight gain was dramatic (in excess of twenty-five pounds) from week 16 through week 32. An important observation with untreated AIDS patients is that once they start losing weight, they seldom or never regain weight. A group of patients appear to have been at a point of no return when treatment was begun. This is borne out in that three of the eighteen patients died. Four patients had CD4+ counts of less than 40. One of these patients refused or could not eat and died of malnutrition. Two other patients had KS. A fourth patient had neurological symptoms, a starting CD4+ count of 36, has not maintained a good diet and has not followed proper maintenance of his health in general. He is still living, but is in poor health. Therefore, patients in the following category may not be good subjects for treatment with L-51: 1. Patients who will not or cannot follow physician's directions such as good nutrition, maintaining a positive attitude, abstaining from drugs, alcohol and tobacco; 2. Patients who are diagnosed with advanced KS or possible other rare cancers, and/or with serious neurologic involvement; and 3. Patients who have severely depressed levels of CD4+ cells, i.e., less than 40. The scientific observations can be summarized as follows: Lymphocyte counts increased above both the starting levels of total lymphocytes in each of the fourteen patients who have experienced a favorable clinical outcome, as well as above the upper limit of normal values. In addition, the polymerase chain reaction (PCR) remained positive and serum antigen levels were negative when CD4+ cells became greater than 400. Virus cultures became negative in 5 of 7 cases when CD4+ cell counts exceeded 400. The subset of lymphocytes which increased most dramatically resided within the suppressor CD8+ cell population. In fact, when CD8+ cell numbers increased above the upper level of the normal range (862) , the patients consistently reported feeling better and were observed to improve clinically. There were no exceptions to this rule. Thirteen patients with pre-treatment CD4+ counts of greater than 50 were analyzed for these clinically observed changes. (One patient was not included since we did not have a zero time blood.) In general, it was observed that total lymphocytes and CD8+ cell counts increased, whereas CD4+ cell counts varied. The median values for these measurements confirm this information. Statistical analysis demonstrates that the absolute lymphocyte count and the CD8+ cell count elevations are highly significant. whereas CD4+ counts over a six-month period do not change significantly. An analysis of a group of seventeen untreated AIDS patients (Gerstoft et al.. Scan J. Infect. Pis. 19:503-509 (1987)) showed no significant changes in the lymphocyte or CD8+ cell populations, but a highly significant loss of CD4+ cells over a thirty-one month period. The CD4/CD8 ratio decreases in both studies, i.e., our study versus the Gerstoft study. However, the reasons for the decrease in the ratio are sharply contrasted between the two studies. In our studies, the ratios fall because of the increase of CD8+ cells, whereas in the latter study (Gerstoft) , the ratio decreases due to the loss of CD4+ cells. The patterns reported by Gerstoft et al. are typical. (For review, see de Martine and Parker, J. Clin. Lab. Anal. 3:56-70 (1989).) The final set of results derived from these patients was obtained from titrations of sequential serum samples from each patient for anti-HIV antibody using a commercial ELISA kit. Fourteen patients have been tested. Ten out of fourteen studied at this time have responded favorably to L-51 treatment and developed a two- to four-fold rise in antibody activity (i.e., anti-HIV activity as a result of, or concomitant with, their favorable clinical response to L-51 treatment. The one patient of the group who did not respond died and had experienced a two-fold decrease of antibody activity over a sixteen-week period. No significant fluctuation of antibody titer was noted in the other three patients. One of these patients had previously been treated with AZT, and there was a WR-2 classification and the other patient had extremely high levels of p24 antigen. Doses of the experimental drug, L-51, appear to modify the disease process at a dose of 2500mg/day. However, in response to the marked side effects and/or toxic reactions, the dose had to be reduced to smaller doses (500mg to 1500mg/day) in several patients. Those patients with CD4+ cell numbers of less than 40 and those with advanced KS did not respond favorably to the drug therapy. However, of the remainder, thirteen of the eighteen have become asymptomatic during the treatment and remained in apparent good health for five months following the conclusion of treatment. One patient formerly on AZT treatment developed Pneumocystis carinii pneumonia (PCP) after treatment was ended. She was successfully treated with IV Pentamidine and is presently experiencing significant weight gain after Pentamidine treatment. The most extraordinary result of this first Clinical Trial is that these patients have remained asymptomatic for four months after treatment was stopped, in contrast to the immediate relapse observed with people taken off AZT treatment. Our conclusion, based on this study as well as results derived from patients treated with L-51 prior to this study, suggests the potential mechanism of action for L-51 is that of a unique, but powerful, immunomodulatory agent. This mechanism may occur through the specific stimulation of a sub-population of CD8+ cells—namely, the CD8+ cytotoxic cell. In fact, patients such as WR 2 patients who usually start with high CD8+ numbers, first experienced a decrease in CD8+ cells. Therefore, it can be speculated that one of the two functional cell populations within the CD8+ subset, changes from a major suppressor cell population to a subset containing mostly cytotoxic cells. This proposed mechanism is in keeping with three recent reports that indicate the CD8+ cell is important in the maintenance of health in the AIDS patient (Walker et al., Science 234:1563 (1986); Kannagi et al., J. Immunol. 140:2237 (1989); Shearer et al., NCI, in press). In addition, another cell marker, HLA-DR, bears careful consideration, as this marker provided a good indication of return to health in ten of the fourteen patients in this initial drug treatment group. EXAMPLE 7 Treatment of Patients with Non-AIDS Illnesses Several hundred patients have been medicated with L-51 during the past six years. Follow-up physicals were performed on a select group of patients who have been treated with L-51 during the past three years. This select group included seven children and seven adults. The diagnoses of these patients included asthma, bronchial pneumonia, insecticidal intoxication, osteomyelitis, systemic lupus erythematosus, pre-eclampsia (premature labor at five months) , abscesses, allergies, mumps with neurologic symptoms and chicken pox. The medical summaries show that all fourteen patients are in excellent health at the present time. * * * The foregoing description of the invention has been directed to particular embodiments for purposes of explanation and illustration. It will be apparent, however, to those skilled in the art that many modifications and changes in the processes of preparing and implementing the described embodiments may be made without departing from the essence of the invention. It is Applicants• intention in the following claims to cover all equivalent modifications and variations as fall within the scope of the invention.

Claims: WHAT IS CLAIMED IS: 1. A cyclic pentaborate containing product prepared from a chemical reaction of sodium tetra borate decahydrate and boric acid in the presence of cytosol fluid obtained from boric acid hypersensitized fungal cultures and other micro¬ organisms. 2. A pharmaceutical composition comprising a therapeutically effective amount of the cyclic pentaborate containing product of claim 1, together with a pharmaceutically acceptable carrier. 3. A pharmaceutical composition comprising a therapeutically effective amount of boric acid and sodium pentaborate, together with a pharmaceutically acceptable carrier. 4. The pharmaceutical composition of claim 3 wherein the boric acid and sodium pentaborate are present in substantially equimolar amounts. 5. A method of enhancing antibody production in a mammal comprising administering to the mammal an immuno-modulating effective amount of a pentaborate containing composition of claims 2, 3, 4 or 12. 6. The method of claim 5 wherein the pentaborate containing composition is administered orally, intravenously, topically, intracavity, rectally, locally or intramuscularly. 7. The method of claim 5 wherein the treated mammal is one afflicted with an immune complex disease, osteomyelitis, AIDS, systemic lupus erythematosus, allergies, asthma, pneumonia, insecticidal intoxication, pre-eclampsia, bacterial or viral infectious diseases. 8. A method of treating AIDS in a human patient comprising administering to the patient an immuno- modulating effective amount of a pentaborate containing composition of claims 2, 3, 4 or 12. 9. The method of claim 8 wherein the pentaborate containing composition is administered orally, intravenously, topically, intracavity, rectally, locally or intramuscularly. 10. A method for producing a cyclic pentaborate composition comprising: reacting together sodium tetraborate decahydrate and boric acid in the presence of cytosol fluid obtained from boric acid hypersensitized fungal and microorganism cultures. 11. The method of claim 10 wherein the fungal and microorganism cultures are those native to grapes and rice. 12. A pharmaceutical composition comprising a therapeutically effective amount of a non-radioactive, pentaborate salt, together with a pharmaceutically acceptable carrier. 13. The pharmaceutical composition of claim 12 wherein the pentaborate salt is sodium pentaborate. 14. A method of enhancing antibody production in a mammal comprising administering to the mammal an immuno-modulating effective amount of a pharmaceutical composition comprising boric acid and a pharmaceutically acceptable carrier. 15. The method of claim 14 wherein the boric acid containing pharmaceutical composition is administered orally, intravenously, topically, intracavity, rectally, locally or intramuscularly. 16. The method of claim 14 wherein the treated mammal is one afflicted with an immune complex disease, osteomyelitis, AIDS, systemic lupus erythematosus, allergies, asthma, pneumonia, insecticidal intoxication, pre-eclampsia, bacterial or viral infectious diseases. 17. A method of treating AIDS in a human patient comprising administering to the patient an immuno- modulating effective amount of a pharmaceutical composition comprising boric acid and a pharmaceutically acceptable carrier. 18. The method of claim 17 wherein the boric acid containing pharmaceutical composition is administered orally, intravenously, topically, intracavity, rectally, locally or intramuscularly.

Inventors: BURK KENNETH H, CORONEL IGNACIO, DREESMAN GORDON R, GALVAN JUAN PABLO, RODRIGUEZ RAFAEL A, BURK, KENNETH, H., CORONEL, IGNACIO, DREESMAN, GORDON, R., GALVAN, JUAN, PABLO, RODRIGUEZ, RAFAEL, A.
Applicants: AMERICAN BIOTECH RESOURCES, TEXAS CPR INC, AMERICAN BIOTECH RESOURCES, INC., TEXAS CPR, INC.
Application year: 1990
Main classification: A61K33
IPC codes: A61K33