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Double-Blind Placebo-Controlled Pilot Trial of Acemannan in Advanced Human Immunodeficiency Virus Disease

Montaner, Julio S. G.; Gill, John; Singer, Joel; Raboud, Janet; Arseneau, Ric; McLean, Brian D.; Schechter, Martin T.; Ruedy, John

Journal of Acquired Immune Deficiency Syndromes and Human Retrovirology: June 1st, 1996 - Volume 12 - Issue 2 - p 153-157
Clinical Science

Summary: We assessed the safety and surrogate markers' effect of acemannan as an adjunctive to antiretroviral therapy among patients with advanced HIV disease receiving zidovudine (ZDV) or didanosine (ddI) in a randomized, double-blind, placebo-controlled trial of acemannan (400 mg orally four times daily). Eligible patients of either sex had CD4 counts of 50-300/μl twice within 1 month of study entry and had received 26 months of antiretroviral treatment (ZDV or ddI) at a stable dose for the month before entry. CD4 counts were made every 4 weeks for 48 weeks. P24 antigen was measured at entry and every 12 weeks thereafter. Sequential quantitative lymphocyte cultures for HIV and ZDV pharmacokinetics were performed in a subset of patients. Sixty-three patients were randomized. All were males (mean age 39 years). The mean baseline CD4 counts were 165 and 147/μl in the placebo and acemannan groups, respectively; 90% of the patients were receiving ZDV at entry. Six patients in the acemannan group and five in the placebo group developed AIDS-defining illnesses. There was no statistically significant difference between the groups at 48 weeks with regard to the absolute change or rate of decline at CD4 count. Among ZDV-treated patients, the median rates of CD4 change (ACD4) in the initial 16 weeks were - 121 and - 120 cells per year in the placebo and acemannan groups, respectively (p = 0.45); ACD4 from week 16 to 48 was 0 and - 61 cells per year in the acemannan and placebo groups (p =.11), respectively. There was no statistical difference between groups with regard to adverse events, p24 antigen, quantitative virology, or pharmacokinetics. Twenty-four patients, 11 receiving placebo and 13 receiving acemannan, discontinued study therapy prematurely, none due to serious adverse reactions. Our results demonstrate that acemannan at an oral daily dose of 1600 mg does not prevent the decline in CD4 count characteristic of progressive HIV disease. Acemannan showed no significant effect on p24 antigen and quantitative virology. Acemannan was well tolerated and showed no significant pharmacokinetic interaction with ZDV.

Address correspondence reprint requests to Dr. Julio S. G. Montaner, Co-Director, Canadian HIV Trials Network, St. Paul's Hospital/University of British Columbia, 667-1081 Burrard St., Vancouver, BC, V6Z 1Y6, Canada.

Manuscript received June 30, 1995; accepted February 2, 1996.

Acemannan has been shown to induce interleukin-1 (IL-1) and prostaglandin E2 production by human peripheral blood cells in vitro (1). Exposure of lymphocytes to acemannan is associated with a concentration-related increase in alloantigenic responses and increase in major histocompatibility (MHC)/human leukocyte antigen (HLA)-Dr class II expression, as well as in natural killer cell activity. Kahlon et al. demonstrated that acemannan causes a concentration-dependent reduction in the amount of human immunodeficiency virus-1 (HIV-1)-specific RNA detectable in vitro (2). When infected cells were treated with 62.5 μg/ml acemannan, little or no HIV-1 RNA could be detected. Preliminary studies have suggested a beneficial effect of acemannan in the treatment of cats with feline leukemia virus, an oncogenic retrovirus (3).

Acemannan has been shown to be nonmutagenic and devoid of significant toxicity in animals at a variety of oral and parenteral doses. A multiple-ascending dose tolerance study of 40 normal healthy males showed no toxicity when acemannan was given orally in doses as high as 3,200 mg/day for 6 days (4). More recently, Clumeck et al. reported a beneficial effect of acemannan on CD4 counts and HIV-related symptoms in a group of patients treated for 24 weeks with a combination of zidovudine [ZDV (AZT)] and oral acemannan. No significant toxicity was associated with acemannan administration in their study (5). We therefore undertook the present study to assess the safety and surrogate markers' effect of oral acemannan among patients with advanced HIV infection receiving concurrent antiretroviral therapy.

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Patients of either sex were eligible to participate in this study if they were aged 16-75 years, had a mean CD4 count of 50-300 cells/μl based on two counts made 7-14 days before enrollment, at least 6 months of previous ZDV therapy at a dose of 300-600 mg/day and at a stable dose in the previous month, and a life expectancy of at least 6 months. After 40 patients had been randomized, the protocol was amended to allow inclusion of patients with a minimum of 6 months of previous didanosine (ddI) therapy at a stable dose in the previous month. This amendment was necessary to maintain the protocol's consistency with contemporary practice (6). Eligibility criteria also included a granulocyte count >1 × 109/L; a white blood cell count >1.5 × 109/L; a platelet count >75 × 109/L; a creatinine level <265 μM; a calcium level <2.74 mM; bilirubin <34 μM; transaminase, alkaline phosphatase, or GGT less than three times the upper limit of normal; and prothrombin time, partial prothrombin time, and bleeding time all within normal range. The study was approved by institutional review boards at both participating centers, and all subjects gave written informed consent.

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Treatment Regimen

In this 48-week randomized, double-blind, placebo-controlled pilot study, patients were randomly assigned to receive 100-mg capsules of acemannan or an identical placebo at a dose of four capsules four times daily (q.i.d.). Patients were stratified by center in blocks of four by computer-generated randomization codes. Compliance was monitored by pill counts of returned bottles at each visit.

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Eligible patients completed a medical history (including a Karnofsky score) and physical examination. Laboratory tests including hematology, blood chemistries, immune profile, urinalysis and virology, and an electrocardiogram (ECG) were performed at the screening visit. Blood levels of ZDV were measured at baseline and at weeks 4 and 24. Quantitative peripheral blood mononuclear cell (PBMC) cultures were performed on a subset of 23 unselected patients at baseline and at 12, 24, 36, and 48 weeks, according to standardized protocol previously reported (7).

Patients were examined weekly for the first month and monthly thereafter. Hematological and immunological tests were performed every 4 weeks; blood chemistry and urinalysis were performed every 8 weeks. The CD4 count was determined twice at baseline and once every 4 weeks thereafter. At weeks 24 and 48, duplicate measurements were made 1 week apart to minimize the effect of T-cell count variability. Adverse events were classified according to AIDS Clinical Trial Group (ACTG) criteria.

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Sample Size

Our primary objective was to evaluate the effect of acemannan given as an adjunctive therapy on the rate of change in CD4 counts in a 48-week period in patients receiving standard antiretroviral therapy. The primary analysis was an efficacy analysis. To be evaluable, a patient had to be receiving acemannan or placebo for at least 16 weeks. Assuming that the mean 1-year decline in CD4 count would be 80 among patients with 36 weeks of exposure to ZDV and CD4 counts <400 and an SD of 50 cells (8), 26 patients per group were required to detect a 50% reduction in mean decline. Therefore, we sought to recruit 60 patients, 30 in each treatment arm.

We minimized CD4 count variability by using standardized conditions with regard to blood testing, including time of the day, venipuncture technique, and handling of laboratory specimens. Duplicate measurements also were performed at the beginning, middle, and end of the study, and the mean of the duplicate measurements was used to calculate slopes. Finally, both immunopathology laboratories involved in the study participated in the National Quality Control Programme from the Laboratory Centre for Disease Control (LCDC), Health and Welfare, Canada.

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To assess the possible influence of acemannan on the absorption of ZDV, pharmacokinetics studies were made at baseline and at weeks 4 and 24 in a subset of 40 patients. Patients were to take their last dose of ZDV before midnight. A heparinized fasting blood specimen was obtained at 8:00 a.m. the following morning and the usual dose of ZDV was then given at week 0 without acemannan or placebo and at weeks 4 and 24 with four 100-mg capsules of acemannan or placebo. A second timed blood specimen was taken 45-75 min after drug administration, and a third timed specimen was taken 90-150 min after drug administration. Blood samples were centrifuged ≤1 h after collection at 1,000 g for 15 min at room temperature, and plasma was thereafter transferred to screw-capped polypropylene tubes which were stored at -20°C. Measurements were made by radioimmunoassay (RIA) of ZDV (9) and its glucuronide on each sample, and population pharmacokinetics profiles were separately determined for each patient.

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Analytic Rationale

From previous experience, the drug was not expected to have an effect on CD4 count until after 3 months of therapy (5). In addition, a rapid though transient increase occurs in CD4 count after the initiation of ZDV or after substitution of ddI for ZDV (6-10,12). Consequently, only patients who had been receiving therapy for at least 12 weeks were included in the primary efficacy analysis. Furthermore, to minimize the confounding effect that could be introduced by changes in antiretroviral therapy during the course of the trial, we conducted separate analyses restricted to patients in whom antiretroviral therapy was not altered during the study.

The linear slope of the CD4 count between 16 and 48 weeks of therapy was prospectively chosen to be the primary index of change. However, we also planned to examine other measures as well, such as normalized area under the curve (AUC) of CD4 over time and change in CD4 counts from baseline to the end of 48-week study therapy (13). Two sample t tests or their nonparametric equivalents were used when appropriate to compare these measures of change between groups.

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Sixty-three subjects, 33 receiving placebo and 30 receiving acemannan, were randomized between March 1991 and April 1992. All participants were men, and 14 had AIDS at enrollment; 57 patients were receiving ZDV and six were receiving ddI (one in the placebo group and five in the acemannan group) at entry. The frequency of HIV-associated symptoms, such as fatigue, headache, fever, chills, and gastrointestinal complaints, were similar at entry in the two treatment groups. Baseline characteristics were not statistically different between study groups (Table 1). The study was terminated in September 1992.

As shown in Fig. 1, there were no statistically significant differences between treatment groups with regard to mean absolute CD4 count over time on an intention-to-treat basis. Similarly, there were no statistically significant differences when the analysis was restricted to patients who completed 16 weeks of the study therapy and who did not change antiretroviral therapy while in the study (data not shown).

The annualized rate of change in CD4 counts was compared between treatment groups before and after 16 weeks on study drug. Similarly, median rate of CD4 count change (25th and 75th percentiles) was -101 (-159, 23) and -110 (-250, -33) for placebo and acemannan groups, respectively, during the first 16 weeks of the study and -61 (-157, 43) and -17 (-56, 85) for placebo and acemannan groups, respectively, between weeks 16 and 48. These values were not statistically different between treatment groups (Wilcoxon rank-sum test, p = 0.55 and 0.11, respectively). The median declines were 61 and 0 cells a year among placebo- and acemannan-treated patients (Wilcoxon rank-sum test, p = 0.11), respectively. We noted similar results with normalized AUC and change in CD4 count relative to baseline and when we restricted the analysis to study participants whose antiretroviral therapy was not changed during the study. No statistically significant differences were detected with regard to mean levels of CD4 percent, CD4/CD8 ratio, P24 antigen, or β2-microglobulin over time between treatment groups.

Viral burden was assessed quantitatively for a subset of 23 patients, 12 receiving placebo and 11 receiving acemannan. Virus was cocultured with various numbers of the patients' cells: 2 × 106, 1 × 106, 2 × 105, 4 × 104, and 2 × 103. Patients were considered negative with respect to viral culture if the virus did not grow for any of the tested numbers of cells. Two patients were negative at baseline but had no follow-up cultures. Log-transformed levels of viral burden were compared between treatment groups at baseline and 12, 24, 36, and 48 weeks of follow-up by Wilcoxon rank-sum tests. There were no statistically significant differences at any of the follow-up times. Patients with negative culture were assigned the log value of 1 × 102, but the actual value assigned to patients with negative culture was not important as long as it was <2 × 103, since nonparametric tests were used. The patterns of missing data and intermittent results were similar between treatment groups. Eight of the 12 patients on the placebo arm (66%) and 6 of 11 patients on the acemannan arm (55%) had intermittent virologic results (Fisher's exact test, p = 0.68).

In all, 30 patients, 19 receiving placebo and 11 receiving acemannan, had pharmocokinetic determinations at baseline and 4 weeks. AUC was calculated based on the levels determined at time 0, 45, and 90 min after ZDV administration. An analysis of variance (ANOVA) indicated that there was a significant decrease in ZDV levels from baseline to 4 weeks (p = 0.05) but that the mean change from baseline to 4 weeks did not differ between treatment groups (p = 0.88). The findings regarding glucuronide-ZDV (GZDV) and the sum of ZDV plus GZDV were similar. However, these differences were present at baseline and there was no indication that absorption decreased to a greater extent over time in the acemannan group as compared with the placebo group. Further pharmacokinetics studies were available for 11 patients, four receiving placebo and seven receiving acemannan at 24 weeks. ANOVA showed no statistically significant differences for any of the outcome variables.

There was no statistically significant differences between groups with regard to Karnofsky scores, frequency of HIV-associated symptoms, or development of AIDS-defining illnesses and non-AIDS-defining illnesses. No serious drug-related adverse effects were attributable to the study drug. However, 24 patients discontinued study medications prematurely, mostly due to self-withdrawal (n = 19). Reasons for withdrawal and time to withdrawal were not statistically different between treatment groups.

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Our results demonstrate that at the dose tested acemannan is well tolerated and has no significant pharmacokinetic interaction with ZDV. Adjunctive treatment with acemannan, however, failed to prevent the decline in CD4 count characteristic of HIV disease progression during the study period. Furthermore, acemannan showed no significant effect on other surrogate markers, such as P24 antigen or quantitative virology.

Our results confirm previous reports indicating that acemannan is generally safe and well tolerated by HIV-infected persons (5). However, we failed to confirm any beneficial effect of oral acemannan on clinical or surrogate markers of HIV disease progression. This contrast can be partially explained by the fact that we limited our study to patients who were clincally stable on nucleoside therapy. However, we believe this was necessary to avoid the potential confounding effect of introducing new antiretroviral agents on CD4 count or other surrogate markers (6-10,12-14).

Our pilot study had a relatively small sample size and consequently limited power to detect small differences between treatment groups. However, our study did have adequate sample size to rule out an increase in CD4 counts in the acemannan group at any time after initiation of treatment. This approach was pursued, based on the previous report of the beneficial effect of acemannan on CD4 cell count and HIV-related symptoms in patients treated for 24 weeks with ZDV and acemannan in combination (5). The 95% confidence intervals in our study exclude a meaningful change in a positive direction at any timepoint in either treatment group. The estimates of change at any timepoint are biased in a positive direction because patients who withdrew from study therapy did not return for continuing CD4 determinations (15). Furthermore, the lack of beneficial effect on CD4 counts in our study should be contrasted with the effect of other therapeutic strategies which can lead to a rapid and relatively sustained increase in CD4 counts (6,10,16).

We conclude, that although well tolerated, adjunctive oral acemannan at daily doses of 1,600 mg for 48 weeks cannot prevent the decline in CD4 count characteristic of progressive HIV disease among patients with advanced disease. Because of the limited power of our study, we cannot rule out the possibility that rates of CD4 count decline may differ between treatment groups. However, our data have adequate power to allow the conclusion that CD4 count did not increase above baseline at any time after initiation of acemannan therapy.

Acknowledgment: This work was supported by the Canadian HIV Trials Network and Carrington Laboratories,; J.S.G.M. is a National Health Scholar from the NHRDP, M.T.S. is National Health Scientist from the NHRDP; J.R. is a postdoctoral fellow with NHRDP. We acknowledge the invaluable cooperation of the many patients and their treating physicians who volunteered their time to ensure the completion of this study. We also thank Deborah Hamann-Trou and Kelly Hsu for secretarial assistance.

FIG. 1

FIG. 1

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Acemannan; Zidovudine; Didanosine; Surrogate markers; Pharmacokinetics

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