Manu Tandon,¹ Anuradha,¹ Pralhad S. Patki, MD²

¹Department of Gastroenterology, C. Dayakar Reddy Hospital, Narayanguda, Hyderabad, India

²Medical Services and Clinical Trials, Research and Development Center, Himalaya Drug Company, Makali, Bangalore, India

CORRESPONDENCE TO:

Pralhad S. Patki, MD

Medical Services and Clinical Trials

Research and Development Center

Himalaya Drug Company

Makali, Bangalore 562 123, India

email: dr.patki@himalayahealthcare.com

Running title:

HD-03/ES IN HBeAg-negative chronic hepatitis B

Abstract

Background: Hepatitis B e antigen (HBeAg)–negative chronic hepatitis B (CHB) refers to an atypical presentation characterized by the absence of HBeAg but with clinical and histologic evidence of chronic hepatitis and the presence of ongoing viral replication. Individuals with this condition are at higher risk for progression to end-stage liver disease and for development of hepatocellular carcinoma.

Aims of the Study: To study the safety and efficacy of polyherbal extract HD-03/ES in the treatment of HBeAg-negative CHB.

Materials and Methods: This study was a phase 2 prospective open-label clinical trial among 42 patients attending the outpatient department of C. Dayakar Reddy Hospital, Narayanguda, Hyderabad, India. Patients with HBeAg-negative CHB who fulfilled the inclusion criteria comprised the study cohort. Clearance of hepatitis B surface antigen (HBsAg) and HBeAg and normalization of alanine aminotransferase (ALT) level were assessed after administration of HD-03/ES (1 capsule twice daily for 24 weeks). Biochemical variables (serum ALT and total bilirubin levels) were statistically analyzed using paired t-test. Variables such as symptomatic relief, hepatitis B virus (HBV) DNA loss, and HBsAg and HBeAg clearance were assessed using the exact binomial test. Missing values for 5 patients who were lost to follow-up were evaluated using the last-observation-carry-forward method.

Results: Statistically significant effects were observed with HD-03/ES treatment based on symptomatic improvement in appetite, jaundice, nausea, vomiting, fatigue, and normalization or reduction of ALT and bilirubin levels. Significant clearance was found in HBsAg; however, HBeAg clearance and HBV DNA loss were not significant. Improved rates of HBV infection were also observed. HD-03/ES was well tolerated in this study.

Conclusions: Clearance of HBsAg may be achieved in HBeAg-negative patients, supporting the use of HD-03/ES for the treatment of HBeAg-negative CHB. Although the initial results of this study are promising, large head-to-head randomized comparative studies with conventional drugs are needed to determine whether virological response can be sustained during chronic dosing and whether relapse occurs after stopping HD-03/ES.

Keywords:

HD-03/ES, chronic hepatitis B, HBsAg, HBeAg

Introduction

Hepatitis B e antigen (HBeAg)–negative chronic hepatitis B (CHB) refers to an atypical presentation characterized by the absence of HBeAg but with clinical and histologic evidence of chronic hepatitis and the presence of ongoing viral replication. It is caused by various mutations affecting transcription and translation of the precore-core RNA, the most common being the precore and double-core promoter mutations. Hepatitis B virus (HBV) has a worldwide distribution, and development of the precore variant is genotype dependent. Individuals with these variants may initially be seen with fulminant hepatitis but more commonly are seen with asymptomatic hepatitis during the natural course of chronic infection. The most characteristic pattern is severe flares, followed by remissions.¹ Patients with HBeAg-negative chronic hepatitis (active carriers) retain HBV DNA, have persistent alanine aminotransferase (ALT) elevation and active necroinflammation on liver biopsy, and are at higher risk for progression to end-stage liver disease and for development of hepatocellular carcinoma (HCC). A primary malignant neoplasm of the liver, HCC often occurs secondary to viral hepatitis infection (hepatitis B or C) or to alcoholic cirrhosis.

Most active carriers of HBeAg-negative HCC are from southern Europe or Asia, but the prevalence has increased worldwide. The precore stop codon variant was detected among a median of 60% (range, 0%-100%) of HBeAg-negative patients overall, among 92% in the Mediterranean, among 50% in the Asia-Pacific region, and among 24% in the United States and northern Europe; there are few data on the prevalence of core promoter variants outside of Asia, where the median prevalence is 77% among HBeAg-negative patients.²

The treatment of patients with HBeAg-negative hepatitis has been discouraging; only 15% to 27% have sustained virological responses to standard interferon therapy, and although antiviral agents can suppress the virus and lead to undetectable HBV DNA levels, indefinite treatment is required, with relapse and resistance to antiviral agents being common. Two studies evaluated the treatment of patients with HBeAg-negative hepatitis B. In one study,³ an international group of researchers randomized 537 HBeAg-negative patients to receive 48 weeks of peginterferon alfa-2a (180 µg weekly) plus placebo, peginterferon alfa-2a plus lamivudine (100 mg daily), or lamivudine alone. In the other study,⁴ researchers in Italy performed a retrospective analysis of 656 patients with HBeAg-negative chronic hepatitis for 22 months. Drugs to treat these patients have to be continued indefinitely, a strategy associated with increased risk of resistance and with unknown long-term safety complications.⁴ Lamivudine therapy is associated with drug resistance, which increases with extended use.^5,6

Ayurveda, an indigenous system of medicine, has a tradition of using herbals to manage liver diseases. HD-03/ES is an indigenous capsule formulation consisting of hydroalcoholic extracts of the herbs Cyperus rotundus and Cyperus scariosus (25 mg of each). Acute and subacute toxicity studies conducted in rats indicated that HD-03/ES is devoid of significant toxic effects following acute and successive administration (unpublished data). Surface antigen suppression and HBV virus elimination activities were examined using 2 hepatitis B surface antigens (HBsAgs) expressing human HCC cell lines, namely, PLC/PRF/5 and HepG2.2.215. Also assessed to evaluate the efficacy of the plant extract were polymerase chain reaction to study amplification of DNA specific to HBV, reverse transcriptase inhibition assay, immunomodulatory effects, and hepatoprotective ability against oxidative damage. The efficacy of the plant extract to eliminate HBV was studied in experimentally infected Pekin ducks. Results of our investigations indicated that the plant extracts could reversibly inhibit cell growth and suppress HBsAg expression in both human HCC cell line models.

A preliminary case study⁷ found significant clearance of HBsAg and disappearance of viral DNA in a patient treated with HD-03/ES (2 capsules twice daily) for 24 weeks. Studies have demonstrated antioxidant,^8-10 free radical scavenging,¹⁰ antiproliferative,⁸ antibacterial,⁹ cytotoxic,⁹ and apoptotic⁹ activities of C rotundus. A clinical study¹¹ of HD-03/ES treatment in patients with CHB virus infection showed reductions in ALT, HBsAg, HBeAg, and HBV DNA. The present study was performed to investigate the safety and efficacy of polyherbal extract HD-03/ES in the management of HBeAg-negative CHB.

Materials and Methods

Study Design

A prospective open-label clinical trial was performed in the Department of Gastroenterology, C. Dayakar Reddy Hospital, Narayanguda, Hyderabad, India, between February 2007 and December 2008 to evaluate the effect of HD-03/ES in the management of HBeAg-negative hepatitis B. Informed written consent was obtained from all study participants, and the protocol of the study was approved by the institutional ethics committee. The study was conducted in accord with the Declaration of Helsinki and with good clinical practice guidelines issued by the Ministry of Health Government of India.

Inclusion Criteria

Patients included in the study were aged 18 to 60 years and were required to have had positive test results for HBsAg for at least 6 months. Participants also had to have negative test results for HBeAg and an alanine transaminase (ALT) level that was 5 to 10 times the upper limit of normal at the screening visit.

Exclusion Criteria

Excluded were the following: patients older than 60 years or younger than 18 years; patients who were pregnant or breast-feeding; patients who had hepatitis C or other hepatic viral infection, autoimmune hepatitis, or drug-induced or alcoholic hepatitis; patients with severe complications of the cardiovascular, renal, or hematopoietic system; and patients with mental diseases. Patients were excluded from the study if they had decompensated liver disease (defined as serum albumin level £36 g/dL, bilirubin level ³15 g/dL, prolonged prothrombin time ³2 seconds, or a history of ascites, variceal hemorrhage, or hepatic encephalopathy) or pancytopenia (defined as hemoglobin level <11 g/dL, white blood cell count <4000/mm³, or platelet count <105/mm³ (to convert albumin level to grams per liter, multiply by 10; bilirubin level to micromoles per liter, multiply by 17.104; hemoglobin level to grams per liter, multiply by 10; white blood cell count to ×10⁹/L, multiply by 0.001; and platelet count to ×10⁹/L, multiply by 1.0). Patients with a history of using interferon, antiviral agents, corticosteroids, or immunosuppressive drugs were also excluded.

Study Procedures

Forty-two patients visiting the outpatient department of C. Dayakar Reddy Hospital with HBeAg-negative CHB were included in the study and were asked to take HD-03/ES for 24 weeks (1 capsule in the morning after breakfast and 1 capsule at bedtime). The signs and symptoms of patients were recorded in detail at baseline and monthly during treatment.

Interventional Treatment

Patients were provided HD-03/ES capsules (each containing C rotundus [150 mg] and C scariosus [100 mg]) to be taken twice daily for 24 weeks. The purity and standardization of the drug were evaluated using high-performance thin-layer chromatography.

Follow-up and Assessment

Serum samples obtained from patients were stored at −20°C until analysis. Serum was assayed for HBsAg and HBV DNA at baseline and after 24 weeks of therapy using commercially available enzyme-linked immunosorbent assay kits (Roche, Welwyn Garden City, England). The patients underwent monthly liver function tests (total bilirubin and ALT) during the study period.

Primary and Secondary Outcome Measures

The primary end point was symptomatic relief, HBsAg clearance, and the safety profile of the drug. Secondary end points included HBV DNA level, HBeAg clearance, and ALT normalization to 40 U/L at the end of treatment (to convert ALT level to microkatals per liter, multiply by 0.0167).

Statistical Analysis

All values are expressed as the mean (SD) or as the incidence of symptoms. Biochemical variables (ALT and total bilirubin levels) were statistically analyzed using paired t-test. Variables such as HBsAg and HBeAg clearance and HBV DNA loss and symptomatic relief were assessed using exact binomial test. Missing values for 5 patients who were lost to follow-up were evaluated using the last-observation-carry-forward (LOCF) method. The minimum level of significance was fixed at P < .05. Statistical analysis was performed using commercially available software (GraphPad Prism, version 4.01; GraphPad Software, San Diego, CA).

RESULTS

Included in the phase 2 prospective open-label clinical trial were 35 male and 7 female patients (mean age, 37.1 [11.2] years). Of 42 patients enrolled in the study, 37 completed the study per protocol. Missing data for 5 patients who were lost to follow-up were evaluated using the LOCF method.

Improvement in appetite was statistically significant compared with baseline values (P < .001) and with values at week 8 (P < .01) (Table 1). Nausea and vomiting also decreased compared with baseline values, showing statistical significance at week 16 (P < .03) and at week 24 (P < .02). Of 28 patients who were seen with fatigue at baseline, only 4 continued to have fatigue at the end of the study period. These findings were significant at week 8 (P < .002), week 16 (P < .001), and week 24 (P < .001) compared with baseline values. Fatigue was reduced at week 24 compared with week 8 values (P < .03). Jaundice, present in 15 patients at the beginning of treatment, remained in only 1 patient at the end of the study period (P < .001).

Serum ALT and total bilirubin levels were compared before and after 24 weeks of treatment using paired t-test (Table 2 and the Figure). After 6 months of treatment, ALT (P < .03) and total bilirubin (P < .04) levels had decreased significantly. Serological variables, including HBsAg and HBeAg clearance and HBV DNA loss, were analyzed using exact binomial test (Table 3). Following 24 weeks of treatment with HD-03/ES, there was significant improvement in HBsAg clearance; of 42 patients who were positive for HBsAg at baseline, 12 achieved HBsAg clearance (P < .001). Of 12 patients who had HBV DNA at baseline, only 5 had HBV DNA at the end of the study. Of 12 patients who were HBeAg positive at baseline, only 8 demonstrated HBeAg at the end of the study. However, the HBV DNA loss and HBeAg clearance were not significant.

HD-03/ES was well tolerated in this study. No patient was withdrawn from therapy because of adverse events. Most observed adverse effects were mild and included abdominal discomfort (4 patients), headache (1 patient), and insomnia (1 patient). Mild ascites (1 patient) and exacerbation of preexisting psoriasis (1 patient) were also noted, but these were thought to be unrelated to the investigational drug. No serious clinical or biochemical abnormalities were experienced by any patient.

DISCUSSION

The results of this preliminary study indicate that short-term therapy with HD-03/ES may be effective in the management of HBeAg-negative CHB. The goal of therapy in HBeAg-negative CHB is to abolish or efficiently suppress viral replication, which is the main cause of underlying liver necroinflammation and fibrosis.¹² In this study, HBsAg clearance was observed in 12 of 42 patients after 24 weeks of therapy. These findings are promising and indicate that short-term therapy with HD-03/ES may be effective in the management of HBeAg-negative CHB.

The treatment options available for HBeAg-negative CHB are limited. In the present study, HD-03/ES showed beneficial therapeutic activity in patients having HBeAg-negative CHB, with minimal adverse effects. This study was an open investigation; a randomized controlled trial with larger sample size and with longer duration is warranted to evaluate relapse rates after cessation of HD-03/ES therapy.

Although HBsAg clearance occurs in patients with HBeAg-negative CHB after interferon therapy, it tends to occur later than 24 weeks after the start of therapy.¹³ Clearance of HBsAg was not reported in some clinical trials of lamivudine or adefovir among patients with HBeAg-negative CHB.^6,14

Findings in the present study, including ALT normalization, HBsAg clearance, and HBV DNA loss during short-term treatment with HD-03/ES, indicate that patients treated with HD-03/ES may lose their infectivity sooner than with other treatments, leading to potentially lower relapse rates. To be useful for treatment, an agent should have no serious adverse events associated with use. With its good profile during short-term application, HD-03/ES may be ideal if safety persists during long-term therapy.

HD-03/ES contains hydroalcoholic extracts of the 2 herbs C rotundus and C scariosus. Using the HBsAg-expressing human HCC cell lines PLC/PRF/5 and HepG2.2.215, two possible mechanisms of action have been studied.⁷ Although requiring further elucidation, preliminary results indicate that HD03/ES suppresses HBsAg, probably by binding to the antigen, and eliminates HBV by reverse transcriptase inhibition. In vitro studies have demonstrated that C rotundus has antioxidant,^8-10 free radical scavenging,¹⁰ antiproliferative,⁸ antibacterial,⁹ cytotoxic,⁹ and apoptosis-inducing⁹ activities.

CONCLUSION

Our data demonstrate the possibility of achieving HBsAg clearance in patients with HBeAg-negative CHB and support the use of HD-03/ES for the treatment of HBeAg-negative CHB. Results demonstrating ALT normalization and HBsAg clearance after short-term treatment indicate that HD-03/ES therapy may offer quick resolution of infection and low rates of relapse. Our conclusions are uncertain given the uncontrolled nature of the trial; however, with the limited treatment options available, HD-03/ES warrants further investigation in a large randomized controlled clinical trial. Outcomes to consider in a larger controlled study should include sustained virological response during chronic dosing and relapse rates after terminating HD-03/ES therapy.

References

1.          Ghany MG, Liang TJ. Natural history of HBsAg-negative chronic hepatitis B. Curr Hepat Rep. 2006;5(1):27-32.

2.          Funk ML, Rosenberg DM, Lok ASF. World-wide epidemiology of HBeAg-negative chronic hepatitis B and associated precore and core promoter variants. J Viral Hepat. 2002;9(1):52-61.

3.          Baltayiannis G, Katsanos K, Karayiannis P, Tsianos EV. Interferon-alpha therapy in HBeAg-negative chronic hepatitis B: a long-term prospective study from north-western Greece. Aliment Pharmacol Ther. 2006;24:525-533.

4.          Marcellin P, Lau GK, Bonino F, et al; Peginterferon Alfa-2a HBeAg-Negative Chronic Hepatitis B Study Group. Peginterferon alfa-2a alone, lamivudine alone, and the two in combination in patients with HBeAg-negative chronic hepatitis B. N Engl J Med. 2004;351:1206-1217.

5.          Liaw YF, Chien RN, Yeh CT, Tsai SL, Chu CM. Acute exacerbation and hepatitis B virus clearance after emergence of YMDD motif mutation during lamivudine therapy. Hepatology. 1999;30:567-572.

6.          Hadziyannis SJ, Papatheodoridis GV, Dimou E, Laras A, Papaioannou C. Efficacy of long-term lamivudine monotherapy in patients with hepatitis B e antigen–negative chronic hepatitis B. Hepatology. 2000;32:847-851.

7.          Kulkarni KS, Dixit MN, Bhagwat VG, et al. Reduction of HBsAg with decrease in viral load with herbal formulation, HD-03/ES: a case study. Med Update. 2002;7:61-63.

8.          Kilani-Jaziri S, Neffati A, Limem I, et al. Relationship correlation of antioxidant and antiproliferative capacity of Cyperus rotundus products towards K562 erythroleukemia cells. Chem Biol Interact. 2009;181(1):85-94.

9.          Kilani S, Ben Sghaier M, Limem I, et al. In vitro evaluation of antibacterial, antioxidant, cytotoxic and apoptotic activities of the tubers infusion and extracts of Cyperus rotundus. Bioresour Technol. 2008;99(18):9004-9008.

10.          Yazdanparast R, Ardestani A. In vitro antioxidant and free radical scavenging activity of Cyperus rotundus. J Med Food. 2007;10(4):667-674.

11.          Rajkumar JS, Sekar MG, Mitra SK. Safety and efficacy of oral HD-03/ES given for six months in patients with chronic hepatitis B virus infection. World J Gastroenterol. 2007;13(30):4103-4107.

12.          Hadziyannis SJ, Papatheodoridis GV, Vassilopoulos D. Treatment of HBeAg-negative chronic hepatitis B. Semin Liver Dis. 2003;23:81-88.

13.          Brunetto MR, Oliveri F, Coco B, Leandro G, Colombatto P, Gorin JM. Outcome of anti-HBe positive chronic hepatitis B in alpha-interferon treated and untreated patients: a long term cohort study. J Hepatol. 2002;36:263-270.

14.          Santantonio T, Mazzola M, Iacovazzi T, Miglietta A, Guastadisegni A, Pastore G. Long-term follow-up of patients with anti-HBe/HBV DNA–positive chronic hepatitis B treated for 12 months with lamivudine. Hepatology. 2000;32:300-306.

Table 1. Effect of Drug Therapy on Clinical Symptoms

Variable

No. of Patients

Baseline

At 8 wk

At 16 wk

At 24 wk

Loss of Appetite

Present

19

12

2

2

Absent

23

30

40

40

Nausea and Vomiting

Present

13

6

4

3

Absent

29

36

38

39

Fatigue

Present

28

13

7

4

Absent

14

29

35

38

Jaundice

Present

15

9

1

1

Absent

27

33

41

41

^a

P < .001 compared with baseline and P < .01 compared with at 8 weeks.

^b

P < .03 compared with baseline.

^c

P < .02 compared with baseline.

^d

P < .002 compared with baseline.

^e

P < .001 compared with baseline.

^f

P < .001 compared with baseline and P < .03 compared with at 8 weeks.

Table 2. Biochemical Variables Before and After 24 Weeks of HD-03/ES Therapy

SI conversion factors: To convert alanine aminotransferase level to microkatals per liter, multiply by 0.0167; bilirubin level to micromoles per liter, multiply by 17.104.

^aP = .03 compared with baseline.

^bP = .04 compared with baseline.

Table 3. Serological Response After 24 Weeks of HD-03/ES Therapy

^a

Compared with baseline.

Figure. Alanine aminotransferase (ALT) levels in response to 24 weeks of HD-03/ES therapy. To convert ALT level to microkatals per liter, multiply by 0.0167.