Long-term lamivudine therapy in chronic hepatitis B.

BACKGROUND: One to 5 years of therapy of chronic hepatitis B with oral nucleoside analogues result in significant clinical improvements, but effects of more prolonged therapy are not well defined. AIM: To describe outcomes of chronic hepatitis B with long-term lamivudine therapy. METHODS: Forty-two patients with chronic hepatitis B treated with lamivudine were followed for 3.2-19.5 (median = 16.1) years. Therapy was switched to other agents (n = 16) if patients developed lamivudine resistance and relapse of disease. RESULTS: Among 22 HBeAg-positive patients, 17 (77%) became HBeAg negative, of whom 5 (23%) subsequently cleared HBsAg. Among 20 HBeAg-negative patients, 10 (50%) cleared HBsAg. The time to HBsAg clearance ranged from 0.9 to 16.8 (median = 9.3) years. Lamivudine resistance arose in 24 patients (57%) of whom 6 (25%) lost HBsAg. HBsAg clearance was not always accompanied by seroconversion; anti-HBs appearing concurrently in only five patients (33%). Nevertheless, HBsAg loss allowed for stopping therapy in all patients, none re-developing HBsAg or suffering relapse; all having normal alanine aminotransferase levels and no (n = 13) or unquantifiable HBV DNA levels (n = 2) when last seen. In contrast, seven of 27 patients (26%) who remained HBsAg-positive died of liver disease or liver cancer or underwent liver transplantation, all of whom had cirrhosis. CONCLUSIONS: Long-term viral suppression with nucleoside analogues leads to HBsAg loss in a substantial proportion of patients, particularly if HBeAg-negative. Serious outcomes during the first 10-20 years of treatment occur largely among patients with pre-existing cirrhosis who do not clear HBsAg with therapy.

Drug resistance of a viral population and its individual intrahost variants during the first 48 hours of therapy.

Using hepatitis C virus (HCV) and interferon (IFN) resistance as a proof of concept, we have devised a new method for calculating the effect of a drug on a viral population, as well as the resistance of the population's individual intrahost variants. By means of next-generation sequencing, HCV variants were obtained from sera collected at nine time points from 16 patients during the first 48 h after injection of IFN-α. IFN-resistance coefficients were calculated for individual variants using changes in their relative frequencies, and for the entire intrahost viral population using changes in viral titer. Population-wide resistance and presence of IFN-resistant variants were highly associated with pegylated IFN-α2a/ribavirin treatment outcome at week 12 (P = 3.78 × 10(-5) and 0.0114, respectively). This new method allows an accurate measurement of resistance based solely on changes in viral titer or the relative frequency of intrahost viral variants during a short observation time.

HLA-G polymorphisms, genetic susceptibility, and clinical outcome in childhood neuroblastoma.

Neuroblastoma is the most common solid tumor in children less than 5 years of age. The early onset of neuroblastoma suggests that genes involved in fetal development and pregnancy may have a putative role in the etiology of neuroblastoma. The human leukocyte antigen subtype G (HLA-G) molecule plays an important role in immune response regulation and appears to regulate immune tolerance during early pregnancy as well as tumor immunosurveillance. Elevated levels of soluble HLA-G (sHLA-G) have been detected in a number of malignancies including serum samples from neuroblastoma and have been reported to be predictive of tumor relapse in neuroblastoma. In light of previous investigations suggesting that single nucleotide polymorphisms in the HLA-G gene may impact on protein expression levels and isoform production, we examined the influence of HLA-G polymorphisms on the susceptibility and clinical outcome of neuroblastoma in 163 neuroblastoma patients and 404 healthy controls. The distribution of HLA-G polymorphisms, alleles, or allelic groups did not differ between children diagnosed with neuroblastoma and healthy controls. Our analyses did not detect an association between common HLA-G polymorphisms and clinical outcome in patients treated for neuroblastoma.

Contribution of gut bacteria to the metabolism of the spleen tyrosine kinase (Syk) inhibitor R406 in cynomolgus monkey.

The spleen tyrosine kinase (Syk) inhibitor R406 is orally administered as the prodrug R788. Following administration of R788 (12.5 mg kg(-1), 20 microCi kg(-1 14)C-R788) to intact and bile duct-cannulated cynomolgus monkeys, drug-related radioactivity was rapidly observed in plasma. No R788 was observed in plasma, while R406 was the major radioactive peak observed at all time points. Only low levels of metabolites were observed in plasma. The half-life for plasma radioactivity was 2.0-2.8 h. The majority (68.9%) of drug-related radioactivity was eliminated into bile. No intact R406 was observed in excreta. Biliary and urinary metabolites consisted of glucuronide and sulfate conjugates of the para-O-demethylated metabolite of R406 (R529), and a direct N-glucuronide of R406. The major metabolite in faeces from intact and bile duct-cannulated monkeys was a unique 3,5-benzene diol metabolite of R406. This metabolite was formed following the sequential O-demethylation and para-dehydroxylation of R529 by anaerobic gut bacteria.

The multidrug transporter ABCG2 (BCRP) is inhibited by plant-derived cannabinoids.

BACKGROUND AND PURPOSE: Cannabinoids are used therapeutically for the palliation of the adverse side effects associated with cancer chemotherapy. However, cannabinoids also inhibit both the activity and expression of the multidrug transporter, P-glycoprotein in vitro. Here we address the interaction of cannabinol (CBN), cannabidiol (CBD) and delta 9-tetrahydrocannabinol (THC) with the related multidrug transporter, ABCG2. EXPERIMENTAL APPROACH: Cannabinoid inhibition of Abcg2/ABCG2 was assessed using flow cytometric analysis of substrate accumulation and ATPase activity assays. The cytotoxicity and chemosensitization by cannabinoids was determined with cell viability assays. Expression of cannabinoid and vanilloid receptors was assessed using reverse transcriptase polymerase chain reaction, and cannabinoid modulation of ABCG2 expression was examined using immunoblotting. KEY RESULTS: CBN, CBD and THC increased the intracellular accumulation of the Abcg2/ABCG2 substrate, mitoxantrone, in an over-expressing cell line. The THC metabolite, (-)-11-nor-9-carboxy-delta 9-THC was much less potent. The plant cannabinoids inhibited both basal and substrate stimulated ATPase activity of human ABCG2. Cannabinoid cytotoxicity occurred in the absence of known cannabinoid cell surface receptors, and only at concentrations higher than those required for Abcg2/ABCG2 inhibition. Sub-toxic concentrations of the cannabinoids resensitized the overexpressing cell line to the cytotoxic effect of Abcg2/ABCG2 substrates, mitoxantrone and topotecan. This occurred in the absence of any effect on ABCG2 expression. CONCLUSIONS AND IMPLICATIONS: Cannabinoids are novel Abcg2/ABCG2 inhibitors, reversing the Abcg2-mediated multidrug-resistant phenotype in vitro. This finding may have implications for the co-administration of cannabinoids with pharmaceuticals that are ABCG2 substrates.

A rapid immunochromatographic assay for hepatitis B virus screening.

Simple, rapid and accurate assays for hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) are helpful for clinical diagnosis and field epidemiological surveys. A commercially developed, rapid immunochromatographic test for simultaneous detection of HBsAg and HBeAg was evaluated using a total of 2463 selected samples (827 frozen sera, 1011 fresh sera, and 625 whole blood samples). Results of the rapid test were compared with standard enzyme immunoassay (EIA) methods for HBsAg and HBeAg detection. The accuracy of the rapid test was excellent and was similar for frozen sera, fresh sera and whole blood. The overall sensitivity and specificity for the detection of HBsAg were 95 and 100%, and the corresponding positive and negative predictive values were 100 and 99.7%, respectively. The sensitivity and specificity for the detection of HBeAg were slightly less than that for HBsAg, and were 80 and 98%, with positive and negative predictive values of 91 and 94%, respectively. Thus, compared with the EIA method, the rapid test was highly sensitive and accurate for the detection of HBsAg although somewhat less sensitive and specific for detection of HBeAg. Because of its speed, simplicity and flexibility, the rapid test is ideally suited for HBsAg and HBeAg screening in population-based epidemiological studies and in low risk populations, particularly in regions of the world where hepatitis B is endemic.

Pharmacokinetics and its role in small molecule drug discovery research.

Pharmacokinetics (PK), which describes the disposition of a drug in the body, should be a primary consideration in the selection of a drug candidate, ultimately contributing to its eventual clinical success or failure. Accordingly, a sound understanding of PK concepts and an appreciation of the judicious use of PK and related (e.g., metabolism, transporter) data in drug discovery can be beneficial to those involved in the process. This review defines important PK parameters (e.g., clearance, volume of distribution, half-life), describes methods of PK data analysis (noncompartmental vs. compartmental) and provides an overview of additional concepts such as allometric scaling, PK/pharmacodynamic modeling, and nonlinear PK. Furthermore, the role and strategic use of PK screens in drug discovery are discussed.

Authentication of medicinal Dendrobium species by the internal transcribed spacer of ribosomal DNA.

Herba Dendrobii (Shihu) is a commonly used Chinese medicine derived from the stem of several orchid species belonging to the genus Dendrobium. It is rather expensive and adulteration is frequent. Proper authentication of the medicinal species is necessary to protect consumers and support conservation measures. DNA sequences of the internal transcribed spacer 2 (ITS 2) of 16 Dendrobium species were shown to be significantly different from one another by an average of 12.4% and from non-orchids and Pholidota (an adulterant of Shihu) by 29.8% and 18.8%, respectively. The intra-specific variation among the Dendrobium species studied was only about 1%. Therefore, ITS 2 regions could be adopted as a molecular marker for differentiating medicinal Dendrobium species from one another and also from non-orchids and adulterants.

Long-term therapy of chronic hepatitis B with lamivudine.

Lamivudine therapy induces improvements in chronic hepatitis B in a high proportion of patients, but prolonged therapy is limited by the development of viral resistance. We analyzed clinical responses and virologic resistance in 27 patients treated continuously with lamivudine for 2 to 4 years. Serum transaminases, hepatitis B virus (HBV) DNA by both branched DNA (bDNA) signal amplification and quantitative polymerase chain reaction were monitored at 4- to 8-week intervals. Virologic resistance to lamivudine was confirmed by the presence of mutations in the YMDD motif of the polymerase gene by restriction fragment-length polymorphism analysis. Serum HBV-DNA levels decreased rapidly in all treated patients, falling by 4 to 5 logs within 1 year. Transaminase levels also decreased and were normal in 70% of patients at 1 year, at which point liver histology had improved in 81% of patients. Viral resistance began to emerge after 8 months of therapy, eventually developing in 14 patients, including 76% of hepatitis B e antigen (HBeAg)-positive patients but only 10% of HBeAg-negative patients. Lamivudine withdrawal led to reappearance of wild-type HBV species, but retreatment led to more rapid reappearance of the mutant virus. Clinical, serum biochemical, and histologic improvements were maintained in the 13 patients who did not develop resistance. Thus, long-term therapy with lamivudine resulted in maintained improvements in virologic, biochemical, and histologic features of disease in most patients with HBeAg-negative chronic hepatitis B and in the subgroup of HBeAg-positive patients with high serum transaminase levels. A high rate of resistance limited efficacy, particularly in patients who remained HBeAg positive on therapy.

Resolution of chronic delta hepatitis after 12 years of interferon alfa therapy.

Chronic delta hepatitis is an uncommon but severe form of chronic viral hepatitis for which there is currently no satisfactory therapy. A patient with chronic delta hepatitis was treated with interferon alfa, 5 million units daily for 12 years. Serial serum samples were tested for routine liver tests and selected samples for quantitative levels of hepatitis B surface antigen (HBsAg) and hepatitis delta virus RNA. Liver biopsies were performed before, during, and after an initial 1-year course of therapy and again after 3 and 10 years of continuous therapy. With initiation of interferon therapy, serum aminotransferase levels decreased to normal range, became abnormal again when the dose was reduced, and increased to pretreatment levels when therapy was stopped. With reinstitution and prolonged therapy, aminotransferase levels became persistently normal; after several years, both hepatitis delta virus RNA and serum HBsAg became undetectable. Liver biopsy, which initially revealed cirrhosis, showed gradual improvement in inflammatory and fibrosis scores and, after 10 years, no abnormalities or fibrosis. Therapy was stopped, and the patient remained free of evidence of infection. In conclusion, long-term therapy with interferon alfa in high doses led to resolution of chronic delta hepatitis, disappearance of hepatitis delta and B virus markers, and improvement in fibrosis.

Hepatitis G virus and human immunodeficiency virus coinfection: response to interferon-alpha therapy.

The prevalence and consequences of hepatitis G virus (HGV) infection were determined in 180 patients with human immunodeficiency virus (HIV) infection (predominantly male homosexuals) who participated in a trial that compared treatment with zidovudine versus interferon (IFN)-alpha versus the combination. HGV RNA levels were measured by branched DNA signal amplification assay. Initially, 66 (37%) had HGV RNA. Sexual transmission was the sole risk factor for infection in all but 4 subjects. Pretreatment clinical features were similar between HGV RNA-positive and -negative patients. After 6 months, only 5% treated with zidovudine became HGV RNA negative, compared with 95% who received IFN-alpha alone and 66% on combination therapy with low-dose IFN-alpha. After therapy, HGV RNA levels returned to baseline in most subjects. Thus, HGV infection is common among HIV-infected homosexual males but does not appear to influence clinical features in early HIV infection. HGV RNA levels are suppressed by IFN but not by zidovudine.

Lamivudine for chronic delta hepatitis.

Chronic delta hepatitis is a severe form of chronic liver disease caused by hepatitis delta virus (HDV) infection superimposed on chronic hepatitis B or the hepatitis B surface antigen (HBsAg) carrier state. Therapy of delta hepatitis is currently unsatisfactory. We have evaluated lamivudine (3-thiacytidine), an oral nucleoside analogue with marked effects against hepatitis B, as therapy in 5 patients with chronic hepatitis D. Five men, ages 38 to 65 years, were treated. All had HBsAg, antibody to HDV, and HDV RNA in serum, as well as persistent elevations in alanine aminotransferase (ALT) levels and liver histology showing severe chronic hepatitis with fibrosis or cirrhosis. Lamivudine was given in a dose of 100 mg orally daily for 12 months. Patients were monitored carefully and tested for HBsAg, HBV-DNA and HDV-RNA levels serially during the year of treatment and for 6 months thereafter. Liver biopsies were performed before therapy and repeated after 1 year. Serum levels of HBV DNA fell rapidly in all 5 patients, becoming undetectable even by polymerase chain reaction (PCR) in 4. However, all 5 patients remained HBsAg- and HDV-RNA-positive, and serum ALT levels and liver histology did not improve. All patients tolerated therapy well. When lamivudine was stopped, HBV-DNA levels returned to pretreatment values without a change in disease activity. Lamivudine is a potent inhibitor of HBV-DNA replication, but does not improve disease activity or lower HDV-RNA levels in patients with chronic delta hepatitis.

10-Year follow-up after interferon-alpha therapy for chronic hepatitis C.

Sustained responses to interferon-alpha occur in 10% to 25% of patients with chronic hepatitis C, but the long-term outcome is not well defined. We evaluated the long-term clinical, histological, and virological outcomes of 10 patients with chronic hepatitis C who were treated between 1984 and 1987 with interferon-alpha-2b for 52 +/- 6 weeks (total doses of 492 +/- 116 MU). Before therapy, all 10 had hepatitis C virus (HCV) RNA, elevations of serum aminotransferases, and chronic hepatitis with fibrosis on liver biopsy. Clinical follow up was 6 to 13 years, and liver biopsies were done 5 to 11 years after initiation of therapy. HCV RNA was assayed by qualitative and quantitative reverse transcriptase-polymerase chain reaction assays. Among 5 patients who had a 6-month sustained response after therapy, all remained HCV RNA negative, and at last follow-up, 4 had normal and 1 minimally elevated serum aminotransferase levels. Liver biopsy specimens were nonreactive for HCV RNA, and all the patients showed improvements in both inflammation and fibrosis and were either normal or had mild, nonspecific inflammatory changes. Among 5 patients without a sustained response, all continued to have HCV RNA in serum and persistent or intermittent aminotransferase elevations. Liver biopsy specimens showed little or no change in necrosis and inflammation; all except 1 patient had progression of fibrosis scores or cirrhosis. All 5 patients had symptoms of chronic hepatitis, 1 underwent liver transplantation, and another had progressive hepatic decompensation. In conclusion, patients with a 6-month posttreatment virological response have a favorable long-term clinical and histological outcome.

Combination therapy with famciclovir and interferon-alpha for the treatment of chronic hepatitis B.

Interferon-alpha (IFN-alpha) treatment results in long-term remissions in only 25%-40% of patients with chronic hepatitis B virus (HBV) infection. Famciclovir, the oral prodrug of penciclovir, inhibits HBV DNA replication. Five adults with chronic HBV infection in whom previous IFN-alpha therapy had failed were treated in a pilot study of overlapping IFN-alpha and famciclovir therapy totaling 20 weeks. HBV DNA levels decreased by 0.9 log units during the initial 4-week period of famciclovir alone, followed by a further decrease of 1.8 logs during the middle 12-week period of combination therapy. HBV DNA rose by 0.9 log during the final 4-week period of IFN-alpha alone. Two patients cleared HBV DNA, and their liver disease improved by clinical and histologic criteria. The combination of famciclovir and IFN-alpha appeared to be at least additive in suppressing HBV DNA. Efficacy trials of combination therapy with famciclovir and IFN-alpha are warranted.

Statistical models for predicting a beneficial response to interferon-alpha in patients with chronic hepatitis B.

Therapy with interferon-alpha has been reported to induce remissions in 35% of patients with chronic hepatitis B. The ability to identify patients likely to respond would be helpful in making recommendations for treatment. In this statistical analysis we included 82 patients with chronic hepatitis B who received interferon-alpha in clinical trials at the National Institutes of Health between 1984 and 1991. A response was defined as the loss of hepatitis B virus (HBV) DNA and hepatitis B e antigen (HBeAg) within 1 year of therapy. Multiple clinical parameters measured at pretreatment (month 0) and after the first month (month 1) of therapy were selected by stepwise regression to support the development of the prognostic models: the two-stage logistic regression model and a neural network that utilized higher-order non-linear interactions between variables. Among the 82 patients, 24 (29%) were responders. The two-stage logistic model using pretreatment variables: sex, hepatic fibrosis and alanine aminotransferase (ALT) levels correctly identified 61% of responders and 76% of non-responders. When HBV DNA at month 1 along with sex, initial ALT and fibrosis was included, the resultant model correctly identified 69% of responders and 77% of non-responders. The neural network, by incorporating interactions between variables, correctly identified 77% and 86% of responders, and 87% and 92% of non-responders, using pretreatment factors alone and the combination of pretreatment and month 1 factors respectively. Hence, the neural network was more accurate than the simple logistic regression model in predicting a response to interferon-alpha in chronic hepatitis B. The universality of these models needs to be further verified.

Long-term follow-up of patients with chronic hepatitis B treated with interferon alfa.

BACKGROUND & AIMS: Therapy with interferon alfa (IFN-alpha) leads to remission of disease in one third of patients with chronic hepatitis B. The aim of this study was to better define the long-term prognosis of this outcome. METHODS: One hundred three patients with chronic hepatitis B who underwent IFN-alpha therapy in three clinical trials between 1984 and 1991 were followed up for serological status, biochemical evidence of liver disease, and liver complications or mortality through 1994. RESULTS: Among 103 patients, 31 (30%) responded to therapy with loss of hepatitis B e antigen and viral DNA from serum. Responders were more likely than nonresponders to be women, black, and to have more severe liver disease including cirrhosis (P < 0.05). Up to 11 years (mean, 6.2 years) after therapy, a higher percentage of responders than nonresponders were still negative for hepatitis B e antigen (94% vs. 40%; P < 0.001) and hepatitis B surface antigen (71% vs 8.3%; P < 0.001). Overall, the rate of liver-related complications and death did not differ by IFN-alpha response, but with adjustment for cirrhosis, nonresponders had higher rates of liver-related complications and mortality (hazard ratio, 13.7; 95% confidence interval, 3.0-63.5). CONCLUSIONS: The response to IFN-alpha therapy in chronic hepatitis B is usually a sustained improvement in disease markers and, when cirrhosis is considered, patient outcome.

The effect of food on the absorption of 14C-SDZ FOX 988, an antidiabetic agent, in healthy human volunteers.

The objective of this study is to examine the effect of food on oral absorption of SDZ FOX 988 (FOX 988), an antidiabetic agent, and circulating levels of its active metabolite, SDZ 53-450 (53-450). Sixteen normal volunteers received a single 10 mg dose of 14C-FOX 988, either as gelatin capsules or in a suspension (0.5% CMC). For subjects receiving each formulation, four subjects received a meal, consisting of 50% fat by calories, immediately following dosing, while the other four received the same meal at 2 h post-dose. Serial blood, urine, and fecal samples were collected for 120 h and analyzed for total radioactivity. Blood concentrations of 53-450 were analyzed using an HPLC-UV method. Concomitant administration with food increased the extent of FOX 988 absorption from either suspension or capsule, as shown by an increase in AUC and in urinary recovery of radioactivity. Blood concentrations of 53-450 were only detected in subjects receiving food at dosing. No difference in absorption was observed between the capsule and the suspension. Results from this study showed that oral absorption of FOX 988 is enhanced by co-administration of food in normal volunteers.

The effect of the fat content of food on the pharmacokinetics and pharmacodynamics of SDZ FOX 988, an antidiabetic agent, in the dog.

SDZ FOX 988 (FOX 988) is being developed for the treatment of type II diabetes. The objective of this study was to examine the effect of the fat content of food on the pharmacokinetics and pharmacodynamics of FOX 988 following oral administration in the dog. In a randomized, cross-over design, four dogs received a single 10 mg kg-1 dose of 14C-FOX 988 suspension concomitantly with food containing 10% fat or 40% fat, or with the 10% fat food at 4 h post-dose. Serial blood, urine, and fecal samples were collected for 96 h and analyzed for total radioactivity. Blood concentrations of 53-450, the active metabolite of FOX 988, were also determined. Serum concentrations of beta-hydroxybutyrate and glucose, pharmacological markers for the antidiabetic effects, were measured serially for 24 h after dosing. The animals receiving the low-fat meal at dosing and at 4 h post-dose exhibited similar extents of absorption, as shown by similar AUC values and urinary radioactivity recovery. Administration of the high-fat meal at dosing significantly enhanced the absorption of FOX 988 and resulted in high blood concentrations of 53-450. However, no significant differences in the pharmacological activity of the drug were observed among the three treatments.

Applying Bailer's method for AUC confidence intervals to sparse sampling.

Bailer developed a method for constructing confidence intervals for areas under the concentration-vs-time curve (AUC's) with only one sample per subject but with multiple subjects sampled at each of several time points post dose. We have modified this method to account for estimation of the variances. How the need to estimate variances affects study design is discussed. An extension of Bailer's method is proposed where variances are modeled as a function of the means, in order to get more precise estimates of variances. The modified and extended methods are applied to a rat toxicokinetic study with only two rats per time point per treatment group.