A Phase I/II study of a Ganoderma lucidum extract in patients with chronic hepatitis B
Shufeng Zhou1, Yihuai Gao2, Guoliang Chen3, Xihu Dai4, and Jingxian Ye5
1Division of Pharmacology and Clinical Pharmacology, Faculty of Medicine and Health Science, the University of Auckland; 2Landcare Research Auckland, Private Bag 92170, Auckland, New Zealand; 3Shanghai Academy of Agricultural Sciences, Shanghai, China; 4Fuzhou General Hospital of Nanjing Military Region of the Peoples’ Liberation Army, Fuzhou, Fujian; 5Hospital of Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian, China.
Correspondence author: Dr Shufeng Zhou, MD, PhD
Division of Pharmacology and Clinical Pharmacology, Faculty of Medicine and Health Science, Auckland University, Auckland, New Zealand; Ph: 0064 9 3737599 (Ex 6414); Fax: 0064 9 3737556; Email: email@example.com
Running title: Phase I/II study of Ganopoly in cancer patients
The polysaccharide fractions and triterpenes isolated from Ganoderma lucidum have shown protection effects on the liver in animal studies. This double-blind, randomized and multi-centered study aimed to evaluate the safety and effect of a G. lucidum extraction, Ganopoly, in chronic hepatitis B. Ninety patients with chronic hepatitis B, hepatitis B viral (HBV) DNA positivity and aminotransferase elevation were included in this multicenter prospective randomized phase I/II study. Patients were randomized to be given Ganopoly (n = 60) or placebo (n = 30) for 12 weeks, then followed up for 13 weeks. Effect of therapy on levels of HBV DNA and aminotransferase activities in serum and hepatitis B e antigen (HBeAg) status were investigated. There were 78 assessable patients who entered the trial for efficacy and safety; 13 of 52 (25%) patients receiving Ganopoly responded by reducing HBeAg and HBV DNA, compared to one of 26 (4%) in the control group (P < 0.05). Among those with serum aspartate aminotransferase (AST) values < 100 U/L (n = 29), 41% (12/29) responded and among those with AST values > 100 U/L (n = 23), 65% (15/23) responded. Within the 6-month study period, 33% (17/52) of treated patients had normal serum alanine aminotransferase (ALT) values, and 13% (7/52) had cleared hepatitis B surface antigen (HBsAg) from serum, whereas none of the controls had normal ALT values or had lost HBsAg. Eight of 60 patients in Ganopoly group and 4 of 30 in the controls were unable to be followed up due to lost or withdrawal. Our study indicates that Ganopoly is well tolerated and appears to be active against HBV in patients with chronic hepatitis B.
Key words: Ganoderma lucidum, Ganopoly, Hepatitis, Liver function.
Hepatitis B virus (HBV) infection often progresses to chronic hepatitis, cirrhosis, and possibly hepatocellular carcinoma (Hoofnagle and di Bisceglie, 1997). Treatment of various cytokines (particularly interferons) and nucleoside analogues results in a sustained loss of HBV DNA and hepatitis B e antigen (HBeAg) in about 20-45% of the patients (Perrillo and Mason, 1994; Hoofnagle and di Bisceglie, 1997). However, treatment with nucleoside analogues such as famciclovir and lamivudine may induce drug-resistant virus mutations in the polymerase gene (Colacino and Staschke, 1998). Nevertheless, further improvements in the treatment of chronic hepatitis B are still needed.
It has been found that recovery from HBV infection depends on the activity of the patient’s immune system (Chisari and Ferrari, 1995). Elimination of HBV is mediated mainly by a vigorous virus antigen-specific, human leukocyte antigen class I-restricted cytotoxic T lymphocyte (CTL) response (Rehermann et al., 1996 a; 1996c). Cytokines such as interleukin-12 (IL-12) plays a key role in the development of a specific CTL response. Many traditional medicines have been shown to regulate the immune system beneficially – one of which, Ganoderma lucidum has been used as a tonic and remedy for many centuries (Shiao et al., 1994; Wasser and Weis, 1999). It has demonstrated efficacy in various chronic diseases, such as chronic hepatopathy, hypertension, diabetes and cancer (Gao, 2000). The polysaccharides and triterpenes isolated from G. lucidum are the major active components. These compounds have immune-modifying activity and antiviral effects (Eo et al., 1999a; Eo et al., 1999b; Eo et al., 2000), which indicate a possible role for G. lucidum in the treatment of chronic hepatitis B.
G. lucidum products are used as an adjunct to or as the sole therapy in Asian countries for the treatment of liver disease, and some open non-randomized clinical studies have been published in oriental languages (e.g. Chinese or Japanese). No clinical trials of G. lucidum used to treat chronic hepatitis B have been reported in English journals (Gao, 2000). Ganopoly, an aqueous extract of G. lucidum by patented technique, has been marketed as an over-the-counter product in New Zealand, Australia, Hongkong and Taiwan. We undertook this double-blind, controlled and randomized clinical study of Ganopoly to investigate its efficacy and safety in adults with chronic hepatitis B.
Materials and Methods
Patients were screened for eligibility using the following assessments: medical and hepatitis history; physical examination, vital signs, hematological profile, clinical chemistry, urinalysis, and use of concomitant medications. Male and female patients aged 18 to 70 years with chronic hepatitis B were eligible for enrolment. They were HBsAg- and HBV DNA-positive in serum; elevated serum alanine transaminase (ALT) levels (=1.5 times the upper limit of normal documented for at least 24 weeks before initiation of treatment); and had chronic hepatitis B without cirrosis, which was determined by the local pathologist. If a patient was biopsied 6 to 12 months before starting treatment and the pathologist discovered significant bridging fibrosis, the patient was rebiopsied to rule out potential progression to cirrhosis. The presence of hepatitis B viremia was confirmed at least 3 times in serum samples obtained 6 weeks before initiation of treatment and at baseline.
Patients were excluded if they were positive for hepatitis C surface antigen or antibody to the human immunodeficiency virus. Further exclusion criteria were evidence or history of any autoimmune disease; clinically significant hematologic, hepatic, metabolic, renal, rheumatologic, neurological or psychiatric disease; anaphylactic reactions; clinically significant cardiac or cardiovascular abnormalities; chronic obstructive pulmonary disease; abnormal carbon monoxide diffusion capacity; organ grafts; severe or disabling illnesses; systemic bacterial or fungal infection; clinically significant bleeding disorders; evidence of malignant neoplastic disease within 5 years; average daily intake of alcohol exceeding 50 g of ethanol; drug abuse within the previous year; or treatment with systemic interferons or systemic antiviral agents within 3 months. Patients who were unable to discontinue concomitant medication were excluded.
One hundred and two subjects were screened from January 1998 to December 1999, and 90 patients met the criteria and were enrolled into the study (Table 1). The ethics committees at the participating centers approved the study, and all patients gave written informed consent before enrollment.
Eligible patients were randomly assigned to receive either Ganopoly or placebo (the same shape, colour and size of capsules) at a dose of 1 capsule, three times daily orally before meals for 12 weeks. Each capsule contained 600 mg extract of G. lucidum or placebo. The drug and placebo were provided by Encore International Co., Auckland, New Zealand. Patients were also instructed to follow the diet recommended by the Chinese Infection Disease Association.
Patients were monitored during the treatment. At its completion, patients were assessed for vital signs; weight; concomitant medication or illness; clinical chemistry; hematological profile; lipid profile; urinalysis; serum testosterone; pregnancy test; and adverse events.
The efficacy parameter was serum HBV DNA level at the end of treatment (12 weeks), which was measured using a liquid hybridization assay (Abbott Labs, Chicago, IL) with a detection limit of 1.7 pg/ml. Patients were considered as responders to the study if there was either a loss of detectable serum HBV DNA or a 50% or greater decrease in the level of HBV DNA as compared to the baseline. ALT levels and changes in viral markers (HBsAg, HBeAg, anti-Hbe and anti-HBs) were also monitored, using commercially available immunoassay kits. The safety variables included a standard clinical laboratory screen, plasma lipids (triglycerides, total cholesterol, HDL cholesterol, and LDL cholesterol), body weight, blood pressure, adverse events, and hypoglycemic events. Clinical laboratory test results were categorized as normal, high, or low based on laboratory reference ranges. Adverse events were defined as events reported before treatment that were exacerbated during treatment or as events reported during treatment or within 7 days after the study drug was stopped. A serious adverse event was defined as any event, whether treatment related or not, that posed a medically significant hazard to the patient was fatal, life-threatening, or permanently disabling, or required hospitalization.
Changes between baseline and the last treatment (last visit) were evaluated for all efficacy variables, with the last observation carried forward. Data compiled at baseline and 12, and 24 weeks were analysed by two-way analysis of variance by ranks. Median values of quantities, such as age and days since diagnosis, were compared using Wilcoxon’s rank-sum test. Test results for treatment group effects were considered statistically significant if P < 0.05. For clinical laboratory tests, an unpaired Student’s t-test examining the mean change from baseline to the end of study was performed.
As shown in Table 1, there were 49 men and 11 women in the Ganopoly group, with a median age of 47.2 yrs. In 30 patients receiving placebo, there were 26 men and 4 women, with a mean age of 45.4 yrs. Twelve patients were not assessable for response and adverse effects because they were lost to follow-up or refused further therapy before 12 weeks of treatment. Patients in both groups were similar with respect to age, sex, serum HBV DNA and ALT level, and liver histology.
The results indicate that 78 patients entered the trial were assessable; whereas 8 of 60 patients in Ganopoly group and 4 of 30 in the controls were unable to be followed up due to lost or withdrawal. Twenty-five percent (13/52) of patients receiving Ganopoly responded by reducing HBeAg and HBV DNA, compared to 4% (1/26) of controls (P < 0.05). Serum HBV DNA levels decreased significantly after 12 weeks of Ganopoly therapy with respect to the baseline values (Figure 1; P < 0.05). After the follow-up (week 24), serum HBV DNA levels remained significantly lower compared to the baseline. Only one control-group patient (1/30) had significant reduction of HBV DNA level. Among those with serum aspartate aminotransferase (AST) values < 100 U/L (n = 29), 41% (12/29) responded and among those with AST values > 100 U/L (n = 23), 65% (15/23) responded. Within the 6-month study period, 33% (17/52) of treated patients had normal serum alanine aminotransferase (ALT) values, and 13% (7/52) had cleared hepatitis B surface antigen (HBsAg) from serum, whereas none of the controls had normal ALT values or had reduced HBsAg.
Safety results for all patients were recorded for the 12 treatment weeks and the follow-up periods. Six side effect events (2 dry mouth, 2 nausea and 2 vomitting) were recorded, all being mild-to-moderate. Ganopoly was generally well tolerated and was not associated with hematologic or biochemical toxicity. Toxicity was generally mild (grade 1).
This study was designed to investigate the anti-HBV effects of Ganopoly in patients with active chronic hepatitis B. The results showed Ganopoly significantly decreased HBV DNA levels and serum ALT levels in patients with chronic hepatitis B after 12-week treatment, and this reduction remained after 12 weeks of follow-up. The responder rate for Ganopoly treatment was comparable with that from treatment with interferons. Ganopoly was well tolerated. These findings indicate the potential use of herbal medicines in the treatment of chronic hepatitis B. Recently, there has been increasing interest in the application of herbal medicine to treat patients with chronic liver disease (Vickers, 2000). Complementary to this are attempts to identify an anti-HBV remedy from edible mushrooms, in particular from Ganoderma species, which are highly ranked in oriental traditional medicine. There are increasing investigations in the field of anti-viral polysaccharides and triterpenes from edible mushrooms, but reliable and objective comparative data on the anti-HBV activity of G. lucidum preparations as over-the-counter nutritional supplements are not available. To the best of our knowledge, this study (as a double-blind and randomized clinical trial) showed the anti-HBV efficacy in patients for the first time.
The mechanism of anti-HBV activity of Ganopoly is unknown. Preclinical studies have shown the anti-viral action of polysaccharides and triterpenes of G. lucidum (Eo et al., 1999a; 1999b; 2000). This is possibly related to the inhibitory effect on the DNA polymerase of viruses. However, the stimulatory effects of active components from G. lucidum cannot be excluded. For example, there is evidence indicating that the /font>-D-glucans induce biological response by binding to membrane complement receptor type three (CR3, aM/font>2 integrin, or CD11b/CD18) on immune effector cells such as macrophage (Konopski et al., 1994; Muller et al., 1996; Battle et al., 1998; Mueller et al., 2000). The /font>-D-glucan binding site (lectin site) of CR3 has been mapped to a region of CD11b located C-terminal to the I-domain and its distinct metal ion-dependent adhesion site for the many protein ligands of CR3 such as iC3b, ICAM-1 and fibrinogen (Diamond et al., 1993; Lee et al., 1995; Thornton et al., 1996). The binding may induce the activation of immune effector cells and thus enhance the CTL reponse to HBV. Triterpenes have shown direct anti-viral activity in vitro. More studies are required to identify more-active components from G. lucidum, and its mechanism of action.
Table 1. Demographic, histological and biochemical profiles of patients with chronic hepatitis B.
|Characteristic||Ganopoly group||Control group|
|No. of patient||60||30|
|Age (yrs)||47.2 ? 3.8 (32-61)||45.4 ? 9.1 (19-54)|
|ALT (/font> ULN)||3.1 ? 1.2||3.2 ? 1.1|
|HBV DNA (pg/ml)||123 (10-500)||132 (20-480)|
|Duration of disease (yrs)||6.2 ? 3.5 (1-18.2)||6.5 ? 3.7 (0.9-14.1)|
Figure 1. Serum HBV DNA levels at the start, 12 and 24 weeks in patients with chronic hepatitis B
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