From: American Gastroenterology Association Digestive Disease Week meeting in Washington in May 1997

Title: Antioxidant drugs combined to alpha-interferon in chronic hepatitis C unresponsive to alpha-interferon: a randomized controlled multicenter study.

A. Bellobuono*, F. Vicari*, G. Ideo*, M. Andreoletti, F. Bissoli, C. Cestari, E. Colombo, P. Del Poggio, O. Fracassetti, S. Lazzaroni, A. Marelli, B. Paris, A. Prada, E.R aines, L. Roffi, P. Rossi. Multicenter Study Group; *Center for liver diseases "Crespi", Niguarda Ca' Granda Hospital, Milan, Italy.

In chronic hepatitis C (CHC) an oxidative damage has been reported and a treatment with anti-oxidant drugs combined to alpha Interferon (\alphaIFN) in patients resistant to \alphaIFN has been considered.

Aim: to evaluate the efficacy of anti-oxidant drugs in association to \alphaIFN in patients affected by CHC, resistant to a previous course of \alphaIFN.

Patients and methods: One hundred-twenty patients affected by biopsy-proven CHC have been enrolled in 12 Liver Centers and randomized to receive antioxidant drugs (N-Acetylcisteine 600 mg/day and Vitamine E 300 mg/day) in combination to IFN \alphaN3 (3-6 MU/tiw) for 6 months (group A), or IFN alone of the same type and doses (according to body weight < or > 60 Kg.) (group B). Serum ALT levels were evaluated monthly during and for at least 6 months after the end of treatment. Serum HCV RNA (by nested RT-PCR) was tested before and at the end of treatment. HCV genotyping was performed in pretreatment sera according to the method of Okamoto. The normalization of serum ALT at the end of therapy was considered as complete response, the maintenance of normal ALT for at least 6 months after the end of therapy was considered as sustained response. The clearance of HCV RNA at the end of therapy was considered as virological response. Chi square test was used for statistical evaluation.

Results: The prevalence of genotype 1 was 68.7% in group A, 70.6% in group B. The prevalence of cirrhosis was 22.5% in group A, 23.7% in group B. In both groups no virological response was found. Biochemical responses are summarized in the table:

Complete response Sustained response

antioxidants+ IFN 6/58 10.5% 1/58 1.7%

IFN alone 6/62 9.6% 0/62 0%

Conclusions: antioxidant drugs combined to \alphaIFN in patients affected by CHC resistant to \alphaIFN were found to be uneffective.

HepC BC

Antioxidants, Oxidative Stress, and NAC

by Carole Lemens and Craig Sterrit

Oxidative stress has emerged in recent years as a suspected component in the pathogenesis of HIV disease. Increasing numbers of researchers agree that even in the earliest stages of infection, a deleterious reductive-oxidative (redox) imbalance may occur, This means that increased damage causing reactive oxygen intermediates (also called "free radicals") are generated at the same time that stores of naturally occurring antioxidant reducing agents are depleted. The possible result is the uncontrolled presence of oxygen-containing molecules which may cause damage to cell membranes, proteins and nucleic acids, and alterations in the intra- and inter-cellular environments. The net effect of this damage has been termed oxidative stress.

Free radicals are oxygen atoms (or oxygen containing molecules) that are produced normally as intermediaries in cellular processes and respiration,and in the degradation of fatty acids. Free radicals are also generated by phagocytes (a type of immune cell) in the destruction of bacteria and virally infected cells, which leads some to speculate that increased oxidative stress occurs during inflammatory immune responses.

Antioxidants are compounds with a chemical affinity for free radicals. They exist in abundance and bond with free radicals before they can cause damage. Antioxidants are of five classes: enzymes, such s catalases, peroxidases, and superoxide dismustase (SOD); peptides, such as glutathione; phenolic compounds. like Vitamin E and plant flavonoids; nitrogen compounds. which includes various amino acids; and carotenoids, most notably beta-carotene.

Other agents may have antioxidant effects through replenishing mechanisms - Vitamin C, for instance, helps to recycle Vitamin E, and NAC (N-acetyl cysteine) provides an important component of glutathione. Low levels of free radicals are necessary for a number of important physiological functions including the inflammatory response, cell division, and white blood cell action against bacterial infection. Thus the importance of maintaining a system of checks and balances between antioxidants and free radicals and their compounds, where the balance is weighted on the side of antioxidants.

When the balance between free radicals and antioxidant supply is tipped, resulting oxidative stress can cause many problems -- either on its own or, in the case of HIV, increased viral replication. The most interesting evidence in HIV suggests that oxidative increases the HIV activation, which, in turn, increases free radical production and oxidative stress. But increased free radical production has been found in serious diseases, including cancer, HIV, and other viral diseases, and has been termed by a causative factor for disease and an effect of underlying pathology.

Limited scientific evidence links low levels of antioxidants (in particular vitamins C and E) with higher incidence of heart disease and cancer. There is no scientific evidence which proves that antioxidants improve disease progression or survival in people with HIV. Part of the confusion stems from the dilemma: are increased free radicals a cause or effect of disease? There is no solid evidence in either direction.

This article will focus on the function of glutathione (GSH), and the theory and evidence to date of the role of the GSH replenishing drugs NAC (N-Acetyl Cysteine) and Procysteine (OTC) in HIV disease.

NAC and Procysteine

NAC is a derivative of cysteine, an amino acid, which is essential for the synthesis of GSH in the body. It has been used as a possible treatment for HIV infection for over three years. In the U.S. it is available as the prescription aerosolized drug Mucomyst by Bristol Laboratories, a Division of Bristol-Myers and is used to treat acetaminophen (Tylenol) overdose and chronic bronchitis. In Europe where it has been used orally for decades it is marketed under the trade name of Fluimucil by Italy's Zambon group. The oral European version of NAC is available at buyers' clubs in the United States. Procysteine (OTC), a precursor of cysteine, is manufactured by Clintec Nutrition and is currently in use in phase II clinical trials for HIV infection. It is not commercially available. The rationale for the use of NAC in HIV treatment is based on evidence from in vitro studies that cells deficient in GSH are particularly sensitive to inflammatory cytokines (tumor necrosis factor alpha). Elevated levels of TNF are known to activate and increase replication. That is when TNF stimulates the nuclear transcription factor kB (NF-kB) in HIV-infected cells, virus transcription and replication is greatly increased. By increasing the levels of intracellular GSH, proponents of NAC therapy hope to offset TNF-mediated HIV replication. GSH is present in almost all human tissues. It is critical for a number of important cellular functions. Of primary importance, however, is its vital role as the principal intracellular defense against oxidation by free radicals and theircompounds. In their 1992 review article Staal et al reported that"adequate levels of GSH are required for mixed lymphocyte reactions, T-cell proliferation, T and B cell differentiation, cytotoxic T-cell activity, and natural killer cell activity. Decreasing GSH by 10 to 40 percent can inhibit completely T-cell activation in vitro. Thus, an intracellular GSH deficiency in lymphocytes has profound effects on immune functions." Abnormally low levels of GSH have been found in asymptomatic HIV-infected individuals. It has also been shown that concentrations of GSH within peripheral blood mononuclear cells (PBMCs) subsets (CD4 and CD8 T cells, B cells, and monocytes) within healthy controls have two distinct types of CD4 and CD8 cells containing high and low levels of GSH. Although the levels vary from individual to individual, the ratio of high GSH content to low GSH content CD4+ and CD8 cells remain constant. In asymptomatic HIV-infected individuals high GSH content T cells appear selectively depleted.

Droge reported that both humans with HIV infection and rhesus macaques with SIV infection have increased plasma glutamate levels, which even when minimally elevated cause a substantial decrease of intracellular cysteine levels (a major component of GSH). He noted that in clinical studies these levels in both HIV infected and uninfected controls corresponded to lymphocyte reactivity and CD4+ but not CD8 T lymphocyte counts. He reported further that the level of cellular cysteine affects both the "intracellular glutathione level and IL-2 dependent proliferation of T cells." He thus concluded "that the cysteine deficiency of HIV persons is, therefore, possibly responsible not only for the cellular dysfunction but also for the over- expression of tumor necrosis factor alpha, interleukin-2 receptor alpha chain, and B2-microglobulin."

Staal et al, among others, advocate for the use of NAC as a therapeutic agent in AIDS. The reasons they state: "NAC has antiretroviral effects in vitro, low toxicity in vivo, a long history of use in patients, can be given orally in a palatable form and is inexpensive."

Clinical Studies

The paucity of clinical trials is in stark contrast to the numbers of PWAs who take NAC and the sweeping recommendations drawn from in vitro studies.

Quay et al reported that oral doses of NAC can temporarily increase levels of cysteine and glutathione in some patients with HIV disease. The objective of the trial was to examine the mechanisms underlying the depletion of glutathione at different stages of HIV infection and the possibility of correcting the deficiency of cysteine and glutathione by the oral administration of NAC. The authors observed a decrease in both glutathione and cysteine in HIV-infected subjects. GSH depletion became more marked with disease progression. The observation of cysteine depletion led them to conclude that glutathione deficiency is the result of cysteine deficiency and not of increased consumption of glutathione by oxidative stress. Nine male patients at various stages of HIV infection were studied. Six uninfected volunteers served as controls. Blood was taken before and at several intervals after the oral administration of NAC at 30mg/kg body weight. Three to four-fold increases in both plasma and PBMC levels of cysteine were seen in all subjects, yet intracellular glutathione increased moderately only in subjects at the least severe stages of HIV infection. The authors felt that "since the rate of glutathione synthesis in PBMC is rather slow, a longer exposure to normal concentrations of cysteine may be required for a biologically meaningful increase in intracellular glutathione." The example of one patient who took 600mg of NAC three times daily resulted in a two-fold increase of the concentration of glutathione in PBMC. However, they stated that "whether a sustained increase in intracellular cysteine affects the function of PBMC or viral replication of HIV in the long term remains to be determined." It was further observed that levels of plasma concentration of the drug shown to be effective in inhibiting viral replication in vitro were "more than three orders lower" even afteringestion of large doses of NAC.

They conclude that "our results indicate that large doses of NAC should be administered repeatedly to achieve a sustained increase in intracellular GSH." and that "further studies should determine whether pharmacological modulation of the GSH status alters the natural history of HIV infection." The authors suggest that a tolerable daily dose for prolonged administration would be approximately 2 grams.

Side effects of oral administration of NAC are minimal, and are limited to nausea, vomiting or diarrhea and, rarely, bronchospasms in patients with asthma.

The Herzenberg Laboratory of Stanford University is now recruiting for an eight week trial of NAC. Its objective is to learn definitively whether oral NAC can replenish glutathione levels inside T cells of people with HIV. Participants must be HIV positive with a CD4 count of 500 orless. They will be assigned to take a total of 9600mg of NAC per day or placebo. For more information, call Debra Fial R.N., or Greg Dubs, Ph.D., at 415/863-8090.

A partially randomized placebo controlled, phase I trial of the oral form of Procysteine (OTC) in 36 asymptomatic patients with CD4 counts over 400 were enrolled in a one month safety and pharmacokinetics and dose ranging study. Participants were randomized to receive OTC 500, 1500 or 3000mg three times daily or placebo. Skowron et al reported no serious drug related toxicities. Mild gastric upset was seen at the highest dose during chronic dosing. The researchers noted that changes in CD4 and CD8 ratio and in CD4 percentages were greater in the treatedgroups in a dose dependent way; however, variability and the small number of patients in each group precluded statistical significance. Weight gain and a reduction in beta-2 microglobulin was seen in some patients on OTC; no significant changes in viral load were seen.

A Phase II/III double blind placebo controlled safety and efficacy study of oral OTC study is currently under way involving 168 patients with CD4 counts between 50 and 300 if symptomatic and between 50 and 200 if asymptomatic were randomized to receive oral OTC 1500mg, 3000mg, or placebo three times a day for one year. It would appear that this study may close due to restriction of funds from the parent drug company.

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HepC BC