OxyFile #31

                        AIDS NEWS SERVICE
                   Michael Howe, MSLS, Editor
                     AIDS Information Center
                VA Medical Center, San Francisco
                     (415) 221-4810 ext 3305
                         April 15, 1994

                     OZONE THERAPY (Part I)

          Ozone Therapy: The Science Behind the Scandal

     Ozone therapy, involving doses of the reactive oxygen gas,
 has long been in Europe a popular alternative treatment for a
 variety of ailments.  While health authorities chide
 practitioners for using this "unproven" therapy, reports
 continue to describe favorable results.  Scientists also
 continue to investigate the potential of ozone therapy to 
 eliminate disease-causing organisms from the bloodstream.  In 
 the mid-1980s, German researchers began using a process called
 autohemotherapy to test the use of ozone on blood infected with
 HIV and hepatitis B and, in 1986, a biotech company called
 Medizone International was created to follow up on the approach.

     Since then, Canadian and American scientists have confirmed
 ozone's direct antiviral effects, and its ability to boost key
 parts of the immune system.  Last May, a Canadian study reported
 that ozone completely inactivated SIV, the simian equivalent of
 HIV, in monkey blood.  The implications for safeguarding the
 blood supply are clear, although the therapeutic potential is
 not.  Nevertheless, according to Medizone, preliminary trials
 are  being conducted at five centers in Italy using an 
 ozone/oxygen mix to treat patients with HIV and hepatitis B.  A 
 great deal of research remains to be performed on ozone, but 
 advocates predict that because ozone cannot be patented, it will 
 not attract financial backing for the scientific studies needed 
 to win FDA approval.  Longevity (04/94) Vol. 6, No. 5, P. 54.

 Frankum B.  Katelaris CH.  Ozone Therapy in AIDS--Truly
 Innocuous? [letter].  Med J Aust. 1993 Oct 4;159(7):493.

 Carpendale MT.  Freeberg J.  Griffiss JM.  Does Ozone Alleviate
 AIDS Diarrhea?  J Clin Gastroenterol. 1993 Sep;17(2):142-5.

     Five patients with acquired immune deficiency syndrome
 (AIDS) or AIDS-related complex (ARC) and intractable diarrhea
 were treated with daily colonic insufflations of medical ozone
 (oxygen/ozone mixture) for 21-28 days. The daily dose of ozone
 (O3) ranged from 2.7 to 30 mg. Three of the four patients whose
 diarrhea was of unknown etiology experienced complete
 resolution, and one patient had marked improvement. The fifth
 patient, whose diarrhea was due to Cryptosporidium, experienced
 no change. No consistent change in the absolute number of helper
 (CD4) or suppressor (CD8) lymphocytes was detected, and no
 obvious changes were seen in the PO2 or the results of routine
 hematologic and blood chemistry studies. Patients had mild to
 moderate local discomfort during ozone administration early in
 the course of treatment, but no adverse systemic effects were
 observed. The results of this series suggest that medical ozone
 administered by rectal insufflation is simple, safe, and
 effective. Should this simple treatment be used routinely to
 treat chronic intractable ARC/AIDS diarrhea?

 Carpendale MT.  Griffiss J.  Is There a Role for Medical Ozone 
 in the Treatment of HIV and Associated Infections?  In: Ozone in
 Medicine.  Proceedings of the Eleventh Ozone World Congress,
 August 29-September 3, 1993, San Francisco, CA.  PP. M-1-32-M-1-
 45.  International Ozone Association, Pan American Committee, 31
 Strawberry Hill Ave., Stanford, CT 06902-2608.

     Medical Oxone inactivates many pathogenic viruses including
 HIV in vitro.  Pilot studies in man suggest positive benefits in
 the early stages of HIV infection (t-4 cells greater thanf 400).
 
 These include incrased T4 and T8 cells, normalizing of T4:T8
 ratio, and a general feeling of wellbeing and minimal evidence
 of infection.  Improvement also occurs in AIDS patients (T4
 cells less than 200) but less evidence of T4 cell resurgence.  
 These studies indicate that at least in vitro there is a good 
 safety margin between the ozone dose required to inactivate HIV 
 and the earliest suggestion of suppression of lymhocytes.  In 
 fact, the lymphocytes are being stimulated at doses that 
 completely inactivates HIV.  More work needs to be done to 
 clarify the most effective dosage and means of treating HIV 
 infections with medical ozone.

 LoLordo, Ann.  AIDS Treatment Documentary Premieres Amidst
 Controversy.  PR Newswire, San Francisco, 08/30/93.

     The medical world [criticized] a documentary about an
 unapproved medical treatment called ozone therapy, which may
 allegedly deter cancer and AIDS.  Canadian filmmaker Geoffrey
 Rogers' "Ozone and the Politics of Medicine" [described] a
 potential breakthrough drug that is dismissed by health
 officials, although millions of patients in Europe have already
 used it.  Rogers [included] scientific evidence that ozone can
 inhibit cancer cells and inactivate viruses.  A recent study by
 the Canadian military and the International Red Cross discovered
 that monkeys injected with blood plasma tainted with SIV, the
 primate equivalent of the AIDS virus, died within two weeks.
 Monkeys receiving ozone injections, however, remained healthy
 and were not infected.  The Food and Drug Administration has
 condemned ozone therapy, and even labeled its use as health care
 fraud.  The drug gained national attention [in July, 1993] when
 the famous New York doctor Robert Atkins lost his medical
 license over a complaint about the use of ozone therapy.  Dr.
 Atkins' license was subsequently reinstated. 
 
 Wolfstadter HD.  Sacher J.  Hopfenmuller W.  Stange R. 
 Retrospective Benefit Following Individualized Naturopathic
 Therapy in HIV-patients at Different Stages.  Int Conf AIDS.
 1992 Jul 19-24;8(3):147 (abstract no. PuB 7588). 

     OBJECTIVE: To assess the long-term efficacy and benefit of a
 complementary treatment regimen, we investigated on laboratory
 findings and clinical outcome in a cohort of 175 out-patients
 (CDC II-IV E) successively treated since 1986. METHODS AND
 PATIENTS: The therapeutic regimen comprised autologous ozone
 transfusions, homeopathy, phytotherapy, therapy with enzymes,
 mineral-, vitamin- and trace element substitution, nutritional
 management, correction of intestinal dysbacteria and
 psychophysical means, set up on an individualized basis. No
 conventional antiviral therapy was given. Patients (all male
 homosexuals) were divided into 5 groups (Gr. I-V) according to
 their CD4 lymphocyte counts at entry into therapy (Gr.I n = 22,
 CD4 0-50; Gr. II n = 12, CD4 51-100; Gr. III n = 17, CD4 101-
 200; Gr. IV n = 81, CD4 201-500; Gr. V n = 53, CD4 greater than
 500 [/microliters]) and 15 hematological and biochemical
 parameters were evaluated with individual regression analysis
 according to the length of observation of patients (min.
 obs.time in Gr. I-III 3 months, min. obs.time in Gr. IV and V 6 
 months).  Moreover we studied the incidence and severity of 
 opportunistic infections and overall QOL during the observed 
 period. RESULTS: Patients in Gr. I presented a median loss of 
 CD4 lymphocytes per month of 0.54 cells/microliters(range -42.0 
 to 4.50, median obs.time 8 months), Gr. II median loss 3.65 
 cells/microliters (range -5.9 to 8.8, median obs.time 10.5 mo.), 
 Gr. III median loss 4.98 cells/microliters (range -13.5 to 11.0, 
 median obs.time 16.8 mo.). In Gr. V, apparently due to the 
 earlier stage of disease, no clear statistical trend of helper-
 cell deterioration could be observed. Patients in Gr. IV, with 
 an approved indication for antiviral therapy, presented a median 
 loss of CD4-cells of 4.47/microliters (range -17.2 to 37.5, 
 median obs.time was 25.4 mo.).
 
     Compared to CD4 lymphocyte deterioration given in the
 literature for patients under antiviral therapy, 52% of our
 patients in Gr. IV exceeded these values, while 24.6% remained
 below. No substantial adverse events or side effects accompanied
 the therapies, thus we found QOL generally increased.
 CONCLUSIONS: Our results suggest that patient performance under
 a combined and individualized naturopathic regimen might be to
 some extend improved with respect to data collected from cohorts
 in the literature. Further investigation including controlled
 clinical trials on different aspects of the single therapies is
 necessary. 
 
 Brown, David.  A New Look at Alternative Therapies.  Washington
 Post (Health), 06/23/92, P. 8.

     The National Institutes of Health will soon examine
 alternative therapies more closely because of the possible
 efficacy of the treatments.  John C. Pittman, a physician in
 Raleigh, N.C., discontinued his ozone gas therapy for AIDS
 patients after the North Carolina Board of Medical Examiners
 told him they were looking into his controversial practices. 
 However, an advisory board at the NIH last week expressed
 interest in Pittman's work and requested more information on his
 claim that three out of 25 patients with HIV had overcome the
 virus after having the highly reactive gas inserted into their
 blood.  Ed McCabe, author of a book on unconventional uses of
 oxygen, also told an NIH panel how ozone treatment had
 significantly improved the conditions of 300 HIV-positive
 patients.  In ozone therapy, a blood sample can be treated with
 the gas and returned to the patient, or a small volume of gas
 can be inserted directly into the vein.  Advocates say the 
 procedure should be done twice daily for three weeks to treat 
 HIV infection.  The Office for the Study of Unconventional 
 Medical Practices, established after the 1992 federal budget 
 requested that NIH spend at least $2 million on such an effort, 
 will attempt to determine which treatments may be promising and 
 can be tested in conventional experiments.

 Hooker MH.  Gazzard BG.  Ozone-Treated Blood in the Treatment of
 HIV Infection [letter; comment].  AIDS. 1992 Jan;6(1):131.

 Carpendale MT.  Freeberg JK.  Ozone Inactivates HIV at
 Noncytotoxic Concentrations.  Antiviral Res. 1991 Oct;16(3):281-
 92.

     The inactivation of human immunodeficiency virus (HIV) and
 cytotoxic properties of ozone-treated serum and serum-
 supplemented media were examined. The titer of HIV suspensions 
 in human serum was reduced in a dose-dependent manner when 
 treated with total reacted ozone concentrations at a range of 
 0.5 to 3.5 micrograms/ml-1. Complete inactivation of HIV 
 suspensions was achieved by 4.0 micrograms/ml-1 of ozone in the 
 presence or absence of H-9 cells. In contrast, cellular 
 metabolism, as measured by MTT dye cleavage, and DNA 
 replication, as measured by BUdR incorporation, were enhanced in 
 H-9 cells grown in media treated with quantities of ozone that 
 completely inactivate HIV.  The permissively HIV-infected cell 
 line HXB/H-9 was cultured in ozone-treated media for six days 
 with culture supernatants being sampled and assayed on alternate 
 days for HIV p24 core protein.  HIV p24 was reduced in all 
 treated cultures compared to control cultures, with an average 
 reduction of 46% [p24].

 Wells KH.  Latino J.  Gavalchin J.  Poiesz BJ.  Inactivation of
 Human Immunodeficiency Virus Type 1 by Ozone in vitro.  Blood.
 1991 Oct 1;78(7):1882-90.

     A device was designed to deliver a constant source of given
 concentrations of ozone to fluids containing human
 immunodeficiency virus type 1 (HIV-1). Ozone was found to
 inactivate HIV-1 virions in a dose-dependent manner. Greater
 than 11 log inactivation was achieved within 2 hours at a
 concentration of 1,200 ppm ozone. Similar concentrations of
 ozone had minimal effect on factor VIII activity in both plasma
 and immunoaffinity-purified preparations of factor VIII treated
 for the same time period. The data indicate that the antiviral
 effects of ozone include viral particle disruption, reverse
 transcriptase inactivation, and/or a perturbation of the ability
 of the virus to bind to its receptor on target cells. Ozone
 treatment offers promise as a means to inactivate human
 retroviruses in human body fluids and blood product
 preparations.

 Garber GE.  Cameron DW.  Hawley-Foss N.  Greenway D. Shannon ME.
 The Use of Ozone-Treated Blood in the Therapy of HIV I=fection
 and Immune Disease: A Pilot Study of Safety and Efficacy [see
 comments].  AIDS. 1991 Aug;5(8):981-4.

     The use of ozone therapy is reported to be effective in a
 variety of viral illnesses, including HIV disease. We performed
 a phase I study of ozone blood treatments in 10 patients in whom
 no significant toxicity was observed. Three patients with
 moderate immunodeficiency showed improvement in surrogate
 markers of HIV-associated immune disease. A phase II controlled 
 and randomized double-blinded study was initiated comparing
 reinjection of ozone-treated blood, and reinjection of
 unprocessed blood for 8 weeks, followed by a 4-week observation
 period. Ozone had no significant effect on hematologic,
 biochemical or clinical toxicity when compared with placebo. CD4
 cell count, interleukin-2, gamma- interferon, beta 2-
 microglobulin, neopterin and p24 antigen were also unaffected by
 both treatment arms. In conclusion, ozone therapy does not
 enhance parameters of immune activation nor does it diminish
 measureable p24 antigen in HIV-infected individuals. 

 Mayer C.  Soyka.  Naber D.  [Paranoid hallucinatory psychoses in
 an HIV infected patient on ozone therapy].  Nervenarzt. 1991
 Mar;62(3):194-7.

 Roder W.  Muller WE.  Merz H.  [Is Ozone Suitable for
 Sterilization of HIV infected Bones?]  Unfallchirurg. 1991
 Jan;94(1):50-1.

     HIV infection can be transferred by blood, blood products
 and organ transplantation. In traumatic surgery allogeneic bone
 transplantation is commonly used for reconstruction in severe
 bone injuries. This technique has been abandoned since the
 appearance of reports of infections with HIV. In an experimental
 in vitro study we showed that ozone treatment cannot inactivate
 HIV in bone for transplantation.

 Wagner K.  Mayers D.  Toro L.  Baker JR Jr.  The Effect of Ozone
 (03) on Lymphocyte Populations in Normal and HIV1-Infected
 Blood. 
 Int Conf AIDS. 1989 Jun 4-9;5:656 (abstract no. C.587).

     OBJECTIVE: Measure the effect of varying 03 concentrations
 on lymphocytes in whole blood from an uninfected and a Walter
 Reed Stage 2 HIV1 patient. METHODS: Heparinized whole blood
 samples were exposed in triplicate to (03) of 20, 40, and 60
 ug/ml with an oxygen control. Coded, blinded blood samples were
 gently agitated for 10 min. and incubated for 1 hr. at 27 C.
 Mononuclear cells were separated using Ficoll-hypaque gradients
 and stained for FACS analysis using labelled monoclonal
 antibodies. RESULTS: 03 had no effect on lymphocyte populations
 of the uninfected donor as numbers of total T cells, CD4, CD8,
 and B cell subpopulations did not change. In contrast, there
 were marked changes in the lymphocyte populations of the HIV
 positive donor with increasing (03). CONCLUSIONS: Ozone, at
 concentrations previously shown to inactivate HIV1, may alter
 lymphocyte surface markers in HIV1 infected patients. Further
 studies are indicated to examine this effect.

 ****************************************************************
 [Editor's Note:  This is the text of a letter from Medizone
 International Inc. in response to inquiries regarding ozone.]

 --------------------------------------------------------------
 Medizone International Inc
 123 East 54th Street,
 Suite 2H,
 New York
 NY 10022
 (212) 421-0303
 Fax: (212) 888-2798

 June 29, 1993

 Dear xxxx,

 Thank you for your letter. At present Medizone International Inc
 and Medizone Canada Ltd are awaiting US Food and Drug
 Administration and Canadian Health and Welfare approval,
 respectively, to commence human clinical trials for the use of
 the Medizone (R) (ozone/oxygen) drug in the treatment of
 Acquired Immune Deficiency Syndrome (AIDS). The following is a
 brief overview of Medizone International Inc's research to date.


 Acquired Immune Deficiency Syndrome (AIDS) is a condition
 described in 1981 and found to be caused by a retrovirus (HILV-
 III/HIV). To date, treatment only affords temporary suppression
 of the virus. 

 Since the identification of Acquired Immune Deficiency Syndrome
 (AIDS), researchers have employed many modalities to treat
 patients with HIV-related disease. In Europe, one modality
 employed has been an ozone/oxygen mixture. The mixture is
 introduced into fixed volumes of the patient's blood 'ex vivo'.
 This procedure has been entitled autohemotherapy, but may be
 referred to more descriptively as extracorporeal circulation.
 Anecdotal reports on the results of this work are extremely
 encouraging. However, in view of the fact that no controlled
 trials have been performed, these results must be carefully
 evaluated.

 In March 1986 Medizone International Inc was created 
 specifically to scientifically evaluate this treatment and bring 
 the technology to market. A series of studies were undertaken to
 establish:  

 a) the safety of extracoporeal circulation with an ozone/oxygen
 (Medizone(R)) mixture in a variety of animal models (toxicity
 studies);

 b) the effect(s) of ozone/oxygen mixture (Medizone(R)) on a 
 human HIV target cell line, HUT-78;

 c) the anti-retroviral activity of ozone/oxygen (Medizone(R)) on
 HIV 'in vitro';

 d) the effect of ozone/oxygen (Medizone(R)) in human peripheral
 blood 'ex vivo';

 e) the effect of ozone/oxygen (Medizone(R)) on exogenously HIV-1
 'spiked' Factor VIII preparations.

 The studies and results to date include:

 a) A preliminary rabbit animal model treated with (ozone/oxygen)
 Medizone(R) in a manner analogous to the proposed human
 treatment regime at the Long Island College of Pharmacy
 suggested no toxicity at concentrations up to ten times the dose 
 proposed in man. 

 b) A limited feline model toxicity study performed at the 
 Cornell Feline Health Centre, Cornell Veterinarian College, 
 Ithaca, to investigate the relative toxicity of Medizone(R) has 
 yielded no detectable toxic effects.

 c) Cell-free HIV treated with (ozone/oxygen) Medizone(R) 
 resulted in 100% inactivation of the virus while maintaining 
 HUT-78 viability. These studies were performed at the State 
 University of New York at Syracuse under the auspices of Dr 
 Bernard Poiesz.

 d) Implementation of a patented hollow fibre technology has
 demonstrated Medizone(R)'s ability to *reduce* intracellular
 viral expression by greater than 99% while maintaining target
 cell viability. 

 e) Treatment of human peripheral blood with Medizone(R) revealed
 hemolysis and coagulation changes well within the standard for
 re-infusion of packed human blood. These studies were performed
 at the Mount Sinai School of Medicine in New York, under the
 auspices of Dr Michael Greenburg.

 f) Published results (Blood, Vol. 78(7):1882, 1991) involving 
 the treatment by Medizone(R) of Factor VIII preparations 
 exogenously 'spiked' with HIV-1 yielded a minimum (ten) log 
 diminution of viral load while maintaining 90% biological 
 activity of this blood component.

 g) Investigation with Visna Virus and Feline Intestinal
 Peritonitis Virus, two lipid enveloped viruses, have been
 inactivated with measurable lipid peroxides derived from
 Medizone(R) treatment.  

 h) 'In vitro' inactivation by Medizone(R) of a variety of Simian
 Immune Deficiency (SIV) variants studied through a multi-agency
 Canadian government collaboration have paralleled those results
 published by Poiesz et al. 

 The hypotheses underlying ozone's virucidal activity are based
 upon the drug's propensity toward lipid peroxidation. Those
 viruses which are lipid-encapsulated (ie. lentivirus family) are
 highly susceptible to the direct oxidative effect of ozone, and
 are thereby inactivated.

 Data indicate the differential effect on lipid envelope viruses
 versus those whose lipid capsid composition is minimal.

 We postulate that ozone will inactivate cell-incorporated 
 viruses by at least two discreet mechanisms:

 1) Due to the high degree of lipid peroxidation catalysed by
 ozone interaction(s), viral binding to specific receptors (ie.
 HIV to CD4A receptor), whose membranous nature (both viral coat
 and receptor) implies a finite composition of lipid [including
 polyunsaturated fatty acids (PUFA)], may indeed be ozone
 sensitive. Investigations with Rhodamine-labelled HIV,
 challenged with ozone sensitized HIV virions, have suggested
 alterations in receptor/ligand binding capacity yielding
 diminished viral binding. This data suggests that ozone,
 delivered by hollow fiber technology at antiviral
 concentrations, does impair HIV's ability to bind to CA4A + 
 target cells.

 2) It has been demonstrated that target cells with pro-viral DNA
 incorporated into its genome have decreased titers of certain
 protective enzyme systems with respect to oxidative
 perturbations. In particular, superoxide distumase (SOD),
 catalase (CAT) and glutathione peroxidase (GSHPx) levels are
 diminished in a number of virally transformed cell lines. Such
 decreases may render these cells selectively sensitive to the
 oxidative effects initiated by ozone. It should be noted that
 ozone's effects are instantaneous with regard to peroxidation
 and the products of this reaction with cellular membrane lipids
 (hydroperoxides) are relatively stable and can participate in a
 host of oxidative (including free-radical) propagating
 reactions. It is our intention to generate, via ozone's direct
 activity and product(s) derived through lipid interactions, data
 to support: 

 a) inactivation of those viruses [ie. HIV, Hepatitis B, non-A 
 and non-B (Hepatitis C)] associated with transfusion associated
 diseases while maintaining the replacement value of the blood
 fractionate of interest (ie. plasma proteins, packed red cell
 preparations and platelets).

 b) reduction of cell incorporation by virus through impairment 
 of viral-receptor binding; 

 c) inactivation of cell-incorporated virus render them non-
 viable while maintaining normal target cell viability. 

 The results of experimental work have demonstrated non-toxicity
 in treating; a preliminary animal rabbit model, human HIV target
 cells, a limited feline model, human peripheral blood, and
 Factor VIII preparations exogenously 'spiked' with HIV-1, all
 with ozone/oxygen mixtures (Medizone(R)). Anti-retroviral was
 demonstrated at concentrations maintaining HUT-78 viability, as
 well as Factor VIII biological activity, respectively.

 On June 2nd, 1993, Medizone International Inc announced the
 successful completion of the first two phases of a Canadian
 research project that has demonstrated preliminary scientific
 evidence supporting the use of the company's blood
 decontamination technology in a live, primate (monkey) model.

 In making the announcement, Medizone president, Dr Joseph S
 Latino said, "To date, the research program has successfully
 demonstrated that monkeys receiving blood fractioned plasma,
 purposely infected with a highly virulent strain of Simian
 Immunodeficiency virus (monkey equivalent to HIV), but treated
 with Medizone's process, did not demonstrate any signs of
 infection over the course of the study (35 days). However, all
 animals receiving similarly infected products without the
 intervention of Medizone's contamination technology died within
 12-14 days."

 This research project was under the direction of an 
 international collaborative team of scientists representing the 
 Canadian Red Cross, Canadian Departments of Defence and 
 Agriculture, Cornell University Veterinary Medical College, and 
 Medizone Canada Ltd. 

 Please do not hesitate to contact me should you require any 
 additional information.

 Your sincerely,

 Katherine M Kalinowski
 Corporate Secretary