Treatment Issues, Vol 11, No 3; March 1997
Dave Gilden and Jill Cadman

The paper's lead author, Leonore Herzenberg, Ph.D., of Stanford University, summarizes her research conclusions in this manner: "We are not saying that people should take NAC, just that the data are consistent with NAC being useful [in prolonging survival] and that people with HIV should avoid behaviors that deplete glutathione" such as chronic intake of alcohol or acetaminophen (Tylenol). Given limitations in the study's organization, the inferences drawn from it must remain tentative.

Glutathione is a key antioxidant compound required for the smooth functioning of all cells. It is composed of three amino acids: cysteine, glutamine and glycine. Besides acting as an antioxidant, glutathione is involved in protein synthesis, amino acid transport, and in the recycling of other antioxidants, such as vitamin C. Test tube studies2 have noted that lowering intracellular glutathione levels decreases cell survival and limits T-cell response to foreign antigens. At the same time, the cells become more easily activated and more liable to improper programmed cell death ("apoptosis") in response to inflammatory cytokines such as tumor necrosis factor alpha (TNFa). HIV replication also increases in these cell cultures. Other studies, though, have found no glutathione deficiency in people with HIV, hence belittling the compound's role in disease progression.3

The Stanford Study -- Glutathione and Survival

Just this month, Leonore and Leonard Herzenberg and colleagues at Stanford University published the findings from two analyses -- one an investigation of the correlation of glutathione level in CD4 cells with CD4 count and the other a trial designed to determine whether orally administered NAC replenishes low glutathione levels.1 Researchers then went on to retrospectively analyze the survival rate of the populations in both studies. The HIV-positive volunteers had to have CD4 counts under 500 (no lower limit) and to be free of active opportunistic infections or malignancies. Nearly all the 204 participants were men.

In the initial survey, glutathione levels in CD4 cells closely correlated with CD4 count. The average glutathione level in 79 uninfected controls was 28% above those of 107 people with HIV and CD4 counts above 200. Glutathione levels in the controls averaged 41% higher than the 97 HIV-positive volunteers with CD4 counts below 200. Two to three years later, the researchers found that these glutathione levels were highly predictive of survival in the stratum with CD4 counts below 200: Eighty-five percent of the 28 persons who started out with glutathione levels within the normal range were still alive 900 days out, compared to only about 18% of the 69 with below normal glutathione levels. (The small number of deaths in the 107 volunteers with initial CD4 counts above 200 made it impossible to find any correlation between glutathione and survival in that stratum).

Reviewing the findings, Dr. Herzenberg maintains that glutathione levels are a more important predictor of survival than CD4 count. She commented, "If you simply look at people with T-cells under 200, then T-cell count is the risk factor. However, if you take into account glutathione levels, that steals the significance of T-cells."

The Stanford Study -- Glutathione Replenishment

Dr. Herzenberg went on to state that she does not know if restoring glutathione levels will necessarily increase survival. Low levels of glutathione are associated with other illnesses and may be merely an incidental effect of HIV infection rather than the cause of poor survival. Although the Stanford researchers warn against taking substances such as alcohol or acetaminophen that deplete glutathione stores, the long-term effect of these substances on disease progression remains undetermined.

The second part of the Stanford study took the first steps in determining the benefits of administering NAC in an effort to restore glutathione levels. Dr. Herzenberg maintains that the trial can be viewed as a Phase I safety study and showed that there were no harmful effects to the participants at the highest dosages. Really, though, the trial was so loosely organized that it could be better characterized as a pilot or feasibility study that would serve as a preface for more rigorous investigation.

Participants started at 8,000 mg of NAC per day but had the option of reducing this amount. Final daily doses ranged from 3,200 mg to 8,000 mg, with a median of 4,400 mg. Dr. Herzenberg now thinks that these extremely high doses, prompted by previous studies showing low absorption of oral NAC,4 probably are not necessary. The common dosages of one to two grams a day should be sufficient.

The 53 trial participants came from the survey participants with subnormal glutathione. They all took part in the initial eight-week randomized, placebo-controlled phase of the study. By the end of this first phase, those who received NAC had largely restored their blood glutathione concentrations, with the average level increasing to 113% of baseline. In contrast, the placebo group's average glutathione level remained virtually unchanged. All subjects completing the eight-week phase were offered open-label NAC for an additional six months.

Although the researchers considered NAC safe, there was nonetheless a substantial number of dropouts. Among the 37 participants with CD4 counts below 200 taking either placebo or NAC, nine left the trial, mainly in the first week, because of nausea, digestive upset or rash, and three decided against entering the open-label continuation phase after being on placebo for eight weeks. Twenty-five others, 13 from the NAC arm and 12 from the placebo arm, did continue, but most of those did not complete the extra 240 day follow-up period. By day 200, half the remaining participants had dropped out. (For the trial group as a whole, median follow-up was similar, 24 weeks.)

Dr. Herzenberg claims that the high drop-out rate was not because of side effects or disease progression. She says that some participants simply did not like the taste of NAC, which was administered as 800 mg effervescent tablets. She also contends that since adverse events were evenly divided between the NAC and placebo groups, the filler present in both the real NAC and placebo tablets was probably to blame.

When checking up on survival two to three years after the baseline readings, the investigators found that among those in the lower CD4 stratum, the 25 participants who received NAC had a statistically significant better survival rate than 19 similar individuals who either did not enter the continuation phase or never entered the trial in the fist place although eligible to do so. Dr. Herzenberg argues that "the survival curve for subjects who took NAC is displaced towards higher survival for about the same length of time as the subject took NAC." To be more precise, no one died in the NAC group in the first year whereas four people did in a "no-NAC" comparison group. After that, the survival curves for the two groups descended at roughly the same rate through the second year.

But this comparison is dubious. It is based on measuring the experience of the 25 who continued in the trial to 19 persons, dubbed the "no-NAC" group, who either dropped out in the first weeks or refused to start once they were admitted. One would suspect that this "no-NAC" group had some distinguishing characteristics, probably related to greater illness in the first place. After extensive investigation, the Stanford investigators have not identified any such characteristic, however.

The PNAS paper only gives a sketchy account of the NAC trial. There are no analyses of CD4 count trends (which apparently were flat for the NAC takers in at least the first eight week randomized portion of the trial, but this has little significance), opportunistic infections or adverse events, as is usually the case in reporting the results of clinical trials. A formal, full trial report is only now in preparation for publication. Also, viral load assays are currently being run on stored plasma samples. A third planned paper will describe the relation between glutathione levels, NAC treatment and viral load.

Dr. Herzenberg affirms that the trial data indicate that improved survival is linked with restoring glutathione levels, but she acknowledges the need for prospective long-term trials of NAC. A formal trial would monitor differences in CD4 counts, viral loads or opportunistic infection occurrence in a single population, some of whom were randomly assigned to receive NAC while others took a placebo. Sophisticated testing is not likely to occur in the foreseeable future for this inexpensive over-the-counter product. "We would like very much to continue, but we can't find a sponsor," she says.

NAC obviously is not the equal of the protease inhibitors, but it could well provide some extra benefit. While waiting for further information, Dr. Herzenberg recommends using NAC as adjunctive therapy. There are no tests commercially available to determine glutathione levels in a given individual. Since NAC is a low cost, readily available and non-toxic compound, Dr. Herzenberg nonetheless reasoned, "Why not take it?"

Other Studies are Conflicting

As noted above, many researchers in addition to the Herzenbergs have postulated the use of glutathione replacement therapy in the treatment of HIV infection to enhance the immune system. A continuous slow stream of papers come out on the subject, and the Stanford study is not the only recent report.

Wulf Droge of the German Cancer Research Center in Heidelberg was the first investigator of glutathione deficiency in people with HIV5 and the original proponent of NAC for restoring glutathione levels. But Dr. Droge soon became concerned that NAC might promote HIV replication. On the one hand, even a moderate deficiency might be harmful to the immune system and contribute to the pathology of HIV infection. On the other, superoptimal levels of the antioxidant might suppress vital immune cell activity.

Dr. Droge undertook two studies to determine whether this was the case. Both studies revealed that NAC does not increase viral replication and at high dosages may have an inhibitory effect. But he warned that the high level of NAC required for such inhibition could be disruptive to immune cell metabolism, which requires a certain level of oxidation, and cautioned against taking the 3,000 to 8,000 mg doses used in the Stanford study. He advised against attempting to conduct trials that looked at short-term effects on HIV replication rather than long-term improvements in immune functioning.

Last May at a conference in Paris, Dr. Droge presented a review of his work on glutathione and NAC.6 He argued that abnormally low cysteine and glutathione levels coincide with a decline in CD4 counts and were both a consequence and cause of HIV infection, tipping the balance in favor of the virus. He also described his institute's experience with administering NAC to patients for up to four years. Doses of NAC were adjusted in each patient to maintain normal plasma cysteine levels and ended up ranging from 600 to 3,000 mg/day. The possible influence of NAC on the progression of disease in the German patients was not described in this presentation.

A second warning7 was published recently by a group from Johns Hopkins University looking at the effects of NAC and OTC. (OTC is another source of cysteine for glutathione. It had no effect on CD4 count or viral load in a six week dose-ranging trial in 24 HIV-positive volunteers with CD4 counts over 400 -- although it may have raised glutathione levels at the highest dose.8)

The Johns Hopkins study found that NAC enhanced HIV replication in cell cultures consisting of resting peripheral blood mononuclear cells (PBMCs) pretreated with NAC or OTC before infection. The source of HIV was a latently infected lab cell line added to the PBMCs. The enhanced HIV activity seemed to arise from an increase in cell-to-cell transmission from the infected cells to the PBMCs via cell clumping. Similarly, HIV could be more frequently detected in a mouse model of HIV infection when the mice were pretreated with OTC and then inoculated with HIV. These models differ in a number of ways from giving NAC to people with highly active HIV infections. The viral load data from the Stanford study will help clarify their relevance.

The Johns Hopkins group concluded by recommending that HIV-infected people receiving NAC should also receive appropriate antiviral therapy since adding AZT to the cell cultures abrogated NAC's apparent HIV-promoting effect. In the Stanford test of NAC, everyone was receiving a stable nucleoside analog regimen (protease inhibitors were still unavailable at that time).

Finally, a Swedish group last August published the results of a true randomized, placebo-controlled trial of NAC in HIV infection.9 Forty-five volunteers with CD4 counts above 200 were assigned to receive either 800 mg of NAC per day or placebo for a period of 16 weeks. Also included was a comparison group of 16 HIV-negative volunteers. The HIV-negative group had higher cysteine but not significantly higher glutathione concentrations than the HIV-positive trial participants. Conversely half of the volunteers with HIV had elevated TNFa levels. The group as a whole also evinced increased oxidative stress (as measured by the investigators' assays of free radical production by neutrophils).

Plasma cysteine levels did return to normal in the NAC group while remaining low in the placebo group. Tumor necrosis factor levels also declined in the NAC group. Otherwise there was no appreciable benefit: CD4 counts declined slightly in the placebo group while remaining stable in the NAC group, but this difference was not statistically significant. Also, glutathione levels remained unchanged, as did the investigators' measure of oxidative stress.

The Swedish authors ended by calling for further controlled studies at higher dosages and longer periods of time. As indicated by even the recent reports, NAC and glutathione have a controversial history in HIV, and it may turn out that higher doses, though perhaps not as high as those used at Stanford, may have a role in preserving immune system function. That remains an open question, though. So does the niche NAC could fill in the era of highly active antiviral therapies -- reducing HIV levels by more than 99% could go a long way to relieving oxidative stress all by itself.

It is probably not practical or ethical at this point to run simple active compound versus placebo trials of long duration with an eye toward collecting information on clinical endpoints or disease progression (see Spring Cleaning in Trial Land in this issue). For NAC, there is no financial incentive to do so, either. Still, it would be enlightening to track viral load and immune function in people taking NAC plus antivirals compared to others with similar anti-HIV regimens without NAC. Such an observational study need not be inordinately expensive and would yield important further information on the benefits of altering the cellular environment while treating HIV.

References:

1 Herzenberg L et al. Proceedings of the National Academy Of Sciences, USA. March 4, 1997; 94(5):1967-72.

2 Roederer M. Novel HIV Therapeutic Strategies (symposium). Cambridge Healthtech Institute. Nov. 20-21, 1996.

3 Pirmohamed M et al. AIDS. May, 1996; 10(5):501-7.

4 Kalebic T et al. Proceedings of the National Academy of Sciences, USA. Feb., 1991; 88(3):986-90.

5 Eck HP et al. Biochemistry Hope-Seyler. Feb., 1989; 370(2):101-8.

6 Droge W et al. Oxidative Stress and Redox Regulation (symposium). Paris, France, May 21-24, 1996.

7 Chen P et al. AIDS. Jan., 1997: 11(1):39-41.

8 Kalayjian RC et al. Journal of Acquired Immune Deficiency Syndromes. Apr., 1994; 7(4):369-74.

9 Akerlund B et al. European Journal of Clinical Pharmacology. August, 1996; 50(6):457-61.

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