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

Immunodeficiency Virus Disease

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 Journal of Acquired Immune Deficiency Syndromes and Human Retrovirology
12:153-157 © 1996 Lippincott-Raven Publishers, Philadelphia

Double-Blmd Placebo-Controlled Pilot Trial of Acemannan

in Advanced Human Immunodeficiency Virus Disease

Julio S. G. Montaner, John Gill, Joel Singer, Janet Raboud, Ric Arseneau,
Brian D. M:cLean, Martin T. Schechter, and John Ruedy

Summary: We assessed the safety and surrogate markers' effect ofacemannan

as an adjunctive to antiretroviral therapy among patients with advanced HIV
disease receiving zidovudine (ZDV) or didanosine (ddl) in a randomized, dou-
ble-blind, placebo-controUed trial of acemannan (400 mg orally four times
daily). Eligible patients of either sex had CD4 counts of50-300/|xl twice within
1 month of study entry and had received 26 months of antiretroviral treatment
(ZDV or ddl) 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/p.l 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 be-
tween 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 pharmacoki-
netics. Twenty-four patients, 11 receiving placebo and 13 receiving aceman-
nan, 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
HI V disease. Acemannan showed no significant effect on p24 antigen and
quantitative virology. Acemannan was well tolerated and showed no signifi-
cant pharmacokinetic interaction with ZDV. Key Words: Acemannan—
Zidovudine—Didanosine—Surrogate markers—Pharmacokinetics.

Acemannan has been shown to induce interleu-
kin-1 (IL-1) and prostaglandin E; production by hu-
man peripheral blood cells in vitro (1). Exposure of
lymphocytes to acemannan is associated with a
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 Bur-
rard St., Vancouver, BC, V6Z 1Y6, Canada.
Manuscript received June 30, 1995; accepted February 2,
1996.
concentration-related increase in alloantigenic re-
sponses and increase in major histocompatibility
(MHQ/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 ^g/ml acemannan, little
or no HIV-1 RNA could be detected. Preliminary
studies have suggested a beneficial effect of ace-

153
 154 J. S. G. MONTANER ET AL
mannan in the treatment of cats with feline leuke-
mia 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. re-
ported a beneficial effect of acemannan on CD4
counts and HIV-related symptoms in a group of pa-
tients 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 un-
dertook the present study to assess the safety and
surrogate markers' effect of oral acemannan among
patients with advanced HIV infection receiving
concurrent antiretroviral therapy.
PATIENTS AND METHODS
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/pJ 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 ran-
domized, the protocol was amended to allow inclusion of pa-
tients with a minimum of 6 months of previous didanosine (ddl)
therapy at a stable dose in the previous month. This amendment
was necessary to maintain the protocol's consistency with con-
temporary practice (6). Eligibility criteria also included a granu-
locyte count >1 x 109/L; a white blood cell count > 1.5 x 109/L;
a platelet count >75 x 109/L; a creatinine level <265 |jiM; a
calcium level <2.74 mM; bilirubin <34 p,M; transaminase, alka-
line phosphatase, or GOT 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.
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 cen-
ter in blocks of four by computer-generated randomization
codes. Compliance was monitored by pill counts of returned
bottles at each visit.
Follow-Up
Eligible patients completed a medical history (including a Kar-
nofsky score) and physical examination. Laboratory tests includ-
ing hematology, blood chemistries, immune profile, urinalysis
Journal of Acquired Immune Deficiency Syndromes and Human Retrovirology, Vol. 12, No. 2, 1996
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 clas-
sified according to AIDS Clinical Trial Group (ACTG) criteria.
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 antiret-
roviral therapy. The primary analysis was an efficacy analysis.
To be e valuable, 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% reduc-
tion 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 immu-
nopathology laboratories involved in the study participated in the
National Quality Control Programme from the Laboratory Centre
for Disease Control (LCDC), Health and Welfare, Canada.
Pharmacokinetics
To assess the possible influence of acemannan on the absorp-
tion 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 fast-
ing 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 ofacemannan 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 adminis-
(ration. Blood samples were centrifuged s£l h after collection at
1,000 g for 15 min at room temperature, and plasma was there-
after transferred to screw-capped polypropylene tubes which
were stored at -20°C. Measurements were made by radioimmu-
noassay (RIA) of ZDV (9) and its glucuronide on each sample,
and population pharmacokinetics profiles were separately deter-
mined for each patient.
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
 ACEMANNAN IN ADVANCED HIV
addition, a rapid though transient increase occurs in CD4 count
after the initiation of ZDV or after substitution of ddl for ZDV
(6-10,12). Consequently, only patients who had been receiving
therapy for at least 12 weeks were included in the primary effi-
cacy 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 (AUG) 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 nonpara-
metric equivalents were used when appropriate to compare these
measures of change between groups.
RESULTS
Sixty-three subjects, 33 receiving placebo and 30
receiving acemannan, were randomized between
M:arch 1991 and April 1992. All participants were
men, and 14 had AIDS at enrollment; 57 patients
were receiving ZDV and six were receiving ddl (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 en-
try in the two treatment groups. Baseline character-
istics were not statistically different between study
groups (Table 1). The study was terminated in Sep-
tember 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
TABLE 1. Baseline characteristics by treatment group
Placebo Acemanan
Parameter (n = 33) (n = 30)
Age (mean yr) 39.1 41.1
Sex (Male %) 100 100
Homosexual men (%) 93.9 90.0
AIDS (%) 27.3 16.7
Months of ZDV/ddI (mean) 15.6 15.7
CD4 (mean) 165 147
Karnofsky (mean) 87.8 87.0
Journal of Acquired Immune Deficiency Syndromes and Human Retrovirology, Vol. 12, No. 1, 1996
155
compared between treatment groups before and af-
ter 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, dur-
ing 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 be-
tween treatment groups (WUcoxon rank-sum test, p
=0.55 and 0.11, respectively). The median declines
were 61 and 0 cells a year among placebo- and ace-
mannan-treated patients (Wilcoxon rank-sum test,
p = 0.11), respectively. We noted similar results
with normalized AUG and change in CD4 count rel-
ative to baseline and when we restricted the analy-
sis to study participants whose antiretroviral ther-
apy was not changed during the study. No statisti-
cally significant differences were detected with
regard to mean levels of CD4 percent, CD4/CD8
ratio, P24 antigen, or pz-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 X 10 , 1 x
106, 2 X 105, 4 x 104, and 2 x 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 X 10 , but the actual
value assigned to patients with negative culture was
not important as long as it was <2 X 10 , 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 re-
suits (Fisher's exact test, p = 0.68).
In all, 30 patients, 19 receiving placebo and 11
receiving acemannan, had pharmocokinetic deter-
minations at baseline and 4 weeks. AUG was cal-
culated 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
 756 J. S. G. MONTANER ET AL

180-1

16(H

140

I-
z 120
3
0
0 100
^t
Q
u 80
PLACEBO

60 ACEMANNAN

40

20

0
0 16 24 32 40 48
Placebo N=33 31 28 26 25 22 16
Acemannan N=30 26 24 21 18 18 13

FIG. 1. Mean CD4 count (and 95% confidence intervals) by treatment group and week of follow-up, including all patients.

4 weeks (p = 0.05) but that the mean change from DISCUSSION

baseline to 4 weeks did not differ between treatment
groups (p = 0.88). The findings regarding glucuro-
nide-ZDV (GZDV) and the sum ofZDV 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 pla-
cebo 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 devel-
opment of AIDS-defining illnesses and non-AIDS-
defining illnesses. No serious drug-related adverse
effects were attributable to the study drug. How-
ever, 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.
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 pre-
vent the decline in CD4 count characteristic of HIV
disease progression during the study period. Fur-
thermore, 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 pro-
gression. This contrast can be partially explained by
the fact that we limited our study to patients who
were clincally stable on nucleoside therapy. How-
ever, we believe this was necessary to avoid the
potential confounding effect of introducing new an-
tiretroviral agents on CD4 count or other surrogate
markers (6-10,12-14).
Our pilot study had a relatively small sample size

Journal of Acquired Immune Deficiency Syndromes and Human Retrovirology, Vol. 12, No. 2, 1996
 ACEMANNAN IN ADVANCED HIV
and consequently limited power to detect small dif-
ferences 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 ex-
elude a meaningful change in a positive direction at
any timepoint in either treatment group. The esti-
mates 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, ad-
junctive 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 Ca-
nadian HIV Trials Network and Carrington Laborato-
ries,; 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 pa-
tients 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 secre-
tarial assistance.
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