Clinical Trial report on treatment of chronic Hepatitis B using Immune-Assist™ brand Dietary Supplement as adjunct with lamivudine [EPIVIR™]

Wang Ruwei, MD Ji Peijun, MD, Li Shuifu, MD, Xia Jinxing, MD1
Xie Jianjun, MD2
Wang Ruwei, MD, Zhan Hongpeng, MD, Sun Huiling, MD, Lei Lixing, MD
Zhang Guoyong, MD3
Li Songhua, MD4
Yu Jin, MD5
Wang Yin, MD6
John C. Holliday7

1. The National Medicine Academy of Zhejiang Province in Lishui City, PRC
2. The Sino-Japanese Exploitation Academy of Plant Resources in Lishui City, PRC
3. The People’s Hospital in Lishui City, PRC
4. Daogen Medical College, Japan, Jiangjin Guochi Internal Medicine Hospital PRC
5. The Chinese Medicine Hospital of Hangzhou City, PRC
6. Surgical Institute in Zhejiang, PRC
7. Aloha Medicinals Inc, Maui, Hawaii, USA

Sponsored by:
Hygienic Food Academy of the Disease Control Center of Nanjing, PRC

Abstract: Presented here are the results of a study conducted on 60 patients with chronic hepatitis B over a two year period, evaluating the value of adding proprietary Immune-Assist™ brand of medicinal mushroom polysaccharide mixture as an adjunct to conventional therapy in order to achieve greater effectiveness than can be provided by the conventional therapy alone.

Introduction:
In recent years it has been found that the polysaccharides extracted from edible fungi have various biological activity functions, including immune enhancement. The city of Lishui, which lies in the south-west of Zhejiang province, is one of the major mushroom cultivation districts in China and has rich resources of these edible fungi. It is a long-standing tradition for the populace of this area to use the edible fungi as part of their medicine. While researching the traditional medicine and folk recipes of the people of this area, there is found a widespread belief that edible fungi can protect the liver, reduce obesity and improve overall immunity. In accordance with this traditional belief, this study was conducted using modern extraction methods in the preparation of a mushroom polysaccharide mixture according to the formula of Immune-Assist Critical Care Formula™, a proprietary health supplement developed as a joint project between the Government of Zhejiang province and a privately held American company, Aloha Medicinals Inc. of Maui, Hawaii. This mixture is manufactured and distributed in the United States of America by Aloha Medicinals Inc under the Hawaiian Health Products brand name. The formula is composed of alcohol precipitated hot water extracts of six species of medicinal mushrooms: Lentinula edodes, Agaricus blazei, Grifola frondosa, Coriolus [Trametes] versicolor, Ganoderma lucidum and two types of Cordyceps sinensis extracts, one from the mycelium and one from the culture broth of liquid-fermented Cordyceps sinensis. We tested this mixture concurrently with the accepted drug lamivudine in the cure of chronic hepatitis. With lamivudine therapy alone, the rate of hepatitis Be antigen converting from positive to negative is 10%-20% with a full years’ course of therapy, and this antibody sero-conversion to negative increases by continuous use of lamivudine continuously year after year. Because of this long period of treatment, there is concern that the virus will induce genetic variation, as well as some patients discontinuing the therapy after time due to economic hardship. From February 2000 to August 2001, our research group used this mushroom polysaccharide formula and lamivudine together to treat hepatitis B for 9 months. From April in 2001 to May in 2002, the research group used this mushroom polysaccharide tablet and lamivudine together to treat hepatitis B compounded with hyperlipidemia with good results. The PRC government clinical trial sanction number for this trial is 99-118. Each tablet contains 500 mg total active ingredients derived from the sixe species of mushrooms, of which 400 mg consists of amylase reactable polysaccharides. The primary research results are as follows:

Key words: Immune-Assist, mushroom polysaccharide, medicinal mushrooms, Hepatitis, Hyperlipidemia, HbsAL/HbeAg, lamivudine

Formula and preparation method
1.1Formula: Each tablet contains 500 mg mixture of active ingredients consisting of equal parts of the following: Lentinula edodes polysaccharides (Lentinan), Grifola frondosa polysaccharides (Maitake D-Fraction), Trametes versicolor protein-bound polysaccharides (PSK and PSP), Cordyceps sinensis Polysaccharides and exo-polysaccharides, Agaricus blazei Polysaccharide and Ganoderma lucidum polysaccharides. These polysaccharides were extracted from full spectrum mycoproducts grown by sterile tissue culture using a proprietary two step extraction and purification process consisting of extraction with hot water, repeated 4 times at 98-99 degrees C, concentration of the water extract portion under reduced pressure, then addition of 4 times the volume of pharmaceutical grade ethanol, which causes the purified polysaccharides, protein bound polysaccharides and heteropolysaccharides to precipitate from the solution, thus separating the alcohol soluble portion. The precipitated polysaccharide compounds are then collected and spray dried. This purified protein-polysaccharide complex is blended with pharmaceutical binders and excipients to manufacture tablets as per usual protocols used in tableting.

Quality control standard
2.1 Character: The tablet is granular, light brown with a characteristic taste and smell.
2.2 Differentiation and verification:
(1) To 1.0 g of the ground tablets add 2 mol/L solution of hydrochloric acid, then dissolve both. Next add ninhydrin 2 mg, slowly heating so the solution changes from deep blue to light blue.
(2) To 1.0 g of the ground tablets add 20ml water to dissolve. To 5ml of this solution add silver nitrate 2.5ml and a black deposition of silver appears.
2.3 This product should measure up to all the medicine rules in the first addendum of “The Codex in the People’s Republic of China” (Edition 1).
2.4 Shelf life experiment: Divide the tablets into 3 groups and store them under normal temperature. Periodically perform character differentiation, assay the solubility in water, and differentiate with computer aided analytical equipment GC, FTIR and HPLC spectroscopy to assure a minimum of 90% conformance with datum as-manufactured. Confirm sterility with SPC method for total CFU count, yeast and mold and e-coli in January, February, March, June, and December. All the datum accords with the related rules in the first addendum of “The Codex in the People’s Republic of China” (Edition 1).

Toxicity experiment (presided over by Li Songhua MD at Daogen Medical College in Japan )
3.1 Acute toxicity experiment: 20 baby mice (20±1g each, half male half female), administer P.O. a solution of the polysaccharide tablet 3 times / 24 hours (500 mg for each mouse every time), total dose is 75g/kg/day. Maintain this dosage for 7 days and otherwise feed according to normal. At the end of the seven days all the mice are healthy and none show any signs of toxicity or abnormality. This short-term overdosage is approximately 835 times the normal recommended human adult dosage on a mg/kg basis.
3.2 Long-term toxicity experiment: 80 healthy adult mice were administered the solution P.O. at 10g/kg/day for 90 days. At 90 days, the mice are sacrificed and all organs assayed for signs of toxicity. The mice showed no abnormal characteristics and the tissues show no toxicity changes. This confirms that this product has little toxicity for long-term administration.

Clinical observation
4.1 The trial group consists of 60 subjects who have complete datum agreement for hepatitis B since February 2000. These cases are all in accordance with the chronic hepatitis B diagnostic standard which was made at the Beijing conference in 1995. That is to say, HbsAg and HbeAG are both positive. The patients are divided into two groups: the experimental group and the control group. The experimental group consists of 32 subjects: 19 males and 13 females, whose ages range from 16 y to 55 y with the average age of 32 y. Among these cases, the lightly sick number 10 individuals and the moderately sick are 22 in number. The comparison group consisted of 28 cases: 16 males and 12 females, with ages from 14 y to 53 y and an average age of 31 y. Among these cases, the lightly sick are 9 in number and the moderately sick number 19 individuals. As far as sex, age and degree of disease considerations, there is no apparent differentiation between the groups. (P>0.05).

Treatment Methods
These two groups were all treated by accepted Western medicine protocol. lamivudine was administered 100mg per day for a period of 9 months. Fufangyiganling or Ganlixinpian was also given 3 times a day, 4 tablets in each dose and continued for 6 months. The experimental group was treated the same, except for the addition of the Immune Assist™ mushroom polysaccharide tablets, 18 tablets per day divided into 3 doses, for a total daily dosage of 9 grams, continued for 9 months. During the treatment period, the two groups were checked for hepatic function every other month as well as checking the hepatic antigen and antibody system.

Treatment Standards Rating
4.2 Treatment efficacy standards:

Marked effect: The main symptoms disappear, swelling and inflammation of the liver and spleen subsides, hepatic function becomes normal, and one or more of either the HbsAL or HBeAg converts to negative.

Moderate Effect: The symptom improve, swelling and inflammation of the liver and spleen subsides or becomes steady, the hepatic function approaches normal or becomes normal, but neither the HbsAL or the HBeAg converts to negative.

Ineffective: None of the above datum is approached.

Treatment result
4.3 In the experimental group, there are 20 cases of marked effect, 7 cases of Moderate Effect, and 5 cases considered ineffective, and the totally efficiency is 84.4%. In the comparison group, there are 8 cases of marked effect, 15 cases of Moderate Effect, 5 cases considered ineffective and the totally efficiency is 82.1%. The totally efficiency difference of these two groups has no apparent meaning (P>0.05). See Table 1 below:

Table 1 – Effectivness comparison of lamivudine alone compared to lamivudine & Immune-Assist

Comparison of rate of hepatitis antibodies converting from positive to negative
4.4 After 9 months of treatment, 20 cases of HBeAg in the experimental group convert to negative (62.5%), while only 8 cases of HbeAg in comparison group convert to negative (28.6%). The sero-conversion rate of these two groups is significant (P<0.05). See Table 2 below:

Patients whose HBeAg convert to negative in these two groups continue to take lamivudine for another 3 months. There are 3 cases whose HBeAg returns to positive after stopping the medication in the experimental group, and 1 case in the comparison group. There is no apparent meaning.

Discussion
There are two treatment methods to cure hepatitis B in the world today: one is to effect a cure through antiviral activity, the other is by assisting the immune system to fight the disease. When inflammation is promoted by the reproduction of the virus, the main treatment method for chronic hepatitis is through the use of an antiviral drug. Up until now the efficiency of antiviral therapy is the decrease of HBV-DNA (<0.1pg/ml) and the elimination of HBeAg, since these results are related to changes of the imflammatory necrosis (or damage) and the reduction of communicability. After antiviral therapy, HBeAg and HBsAg convert to negative. However, HBV-DNA can still be confirmed in serum and liver by PCR analysis, which means that the disease still exists. Because of this it is clinically significant that we choose the sero-conversion of HBeAg as the main guideline for curative effect.

The antiviral drugs, which are certified in international medical science protocols are interferon and lamivudine. But since interferon only can be used under the rigorous observation of veteran doctors, there are only a small portion of patients which can be treated in this manor out of the millions of cases worldwide. lamivudine is similar to a nucleotide and causes a type of pyrimidine ramification. It is synthetically manufactured and is an L-structured artificial enantiomorph. It is a strong and safe inhibitor for the replication of HBV. It can play a great role in wide treatment usage, but it is not fit for all chronic hepatic infections. The medicine for all chronic infections should restrain the synthesis of HBV-DNA and eliminate CCC-DNA, but does not rely on the immunological activity of the patients themselves. It has proven very difficult to produce any kind of medicine with both antiviral and immunomodulatory activity to date.

The HbsAL / HBeAg indicators has one or more advantages. First, it can accurately choose medicines for treatment and let these medicines exert their advantages singly; and secondly, it can improve the curative effect concurrently with other medicines. However lamivudine takes a long time to display its disease resistance role when used as a singular drug therapy (The sero-conversion rate from positive to negative of HBeAg is about 3-4 years to achieve 60% efficacy in all patients), and it is likely to induce virus genetic variation if taken for a long period of time. So we used the mixed medicine approach of direct acting antiviral and the immune modulation effect of the mushroom polysaccharides together to cure hepatitis in a shorter timeframe than usual, and use Chinese medicine alone for long-term follow-up. According to the experience of these authors (more than eighteen months involved in this study), when we use the mushroom polysaccharides and lamivudine together, the sensitivity to the lamivudine can be increased through the pretreatment with Chinese medicine. The multi-targeted antiviral activity of Chinese medicine, mushroom polysaccharide immune modulation and the concurrent action of lamivudine makes up for the short comings of lamivudine alone, for example by allowing shorter periods of treatment, greater reduction in swelling and inflammation, and decreased toxic side effects, leading to greater patient compliance.

According to the references cited below [2-4], edible fungi have many good biological functions. They can prolong the patient’s dormancy period, increase appetite, ameliorate fatigue and enhance immunity. They can strengthen the body’s nonspecific immunity, improve the secretory product of mucosa’s secretor type IgA, increase the function of mononuclear-phagocyte and the activity of NK cell, maintain immune balance, ameleorate alcoholic damage to liver cell efficiency and accelerate the restoration and regeneration of liver tissue cells. Edible fungi also have great attenuation of virulence, improve the body’s overall health, protect hemopoietic action of bone marrow, and lighten the damage of the esophagus. Edible fungi have certain curative effect for chemical liver damage and healthy blood lipid regulation, and can play an effective function in the clinic. From this trial we find that the mushroom polysaccharides can play an important roll in curing hepatitis B, and this mushroom polysaccharide mixture (Immune-Assist) should be considered as an adjunct to conventional hepatitis treatment.

Reference:
1.Wang Jiyao The Curing of Modern Hepatitis, Shanghai Medicial College Publishing House, 1999,26
2.Hileino H, Yoshxaha M, Suzuk Y, et al. Isoation and hypoglycemic activity of trichosons A.B.C.D. and E, glycans of trichosanthes kirilowii root, planta med, 1989; 55(4); 349
3.EA Boxle et al. Pharma pharmacol 1982; 34; 563
4.Li Guangzhou Research for Increasing Antineoplastic Function of lentinan, The Application Medicine in China 2000, 17(5):354-355

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