Atherosclerotic cardiovascular disease risk profile of patients with chronic hepatitis B treated with tenofovir alafenamide or tenofovir disoproxil fumarate for 96 weeks.

BACKGROUND: Patients with chronic hepatitis B (CHB) who switch from tenofovir disoproxil fumarate (TDF) to tenofovir alafenamide (TAF) show changes in lipid profiles. AIM: To evaluate how these changes affect cardiovascular risk. METHODS: This pooled analysis, based on two large prospective studies, evaluated fasting lipid profiles of patients with CHB who were treated with TAF 25 mg/day or TDF 300 mg/day for 96 weeks. Patients who fulfilled the American College of Cardiology criteria (age 40-79 years, high-density lipoprotein [HDL] 20-100 mg/dL, total cholesterol [TC] 130-320 mg/dL and systolic blood pressure 90-200 mmHg) required to assess 10-year atherosclerotic cardiovascular disease (ASCVD) risk with baseline lipid data and at least one post-baseline measurement were included in the ASCVD-risk population. The 10-year ASCVD risk was calculated for patients in this population, and changes from baseline to Week 96 were assessed using intermediate- (≥7.5%) and high-risk (≥20%) cut-offs. RESULTS: Among 1632 patients, 620 (38%) met the criteria for the ASCVD-risk population. At Week 96, fasting levels of all lipids, except TC:HDL ratio, were lower with TDF than TAF. No significant increase was observed in overall ASCVD risk or in any ASCVD-risk categories during the 96-week treatment period compared with baseline. A similar proportion of patients in the TAF and TDF treatment groups (1.3% and 2.3%, respectively; p = 0.34) reported cardiovascular events. CONCLUSION: Despite on-treatment differences in lipid profiles with TAF and TDF, predicted cardiovascular risk and clinical events were similar for both groups after 96 weeks.

Safety and efficacy of vebicorvir administered with entecavir in treatment-naïve patients with chronic hepatitis B virus infection.

BACKGROUND & AIMS: Nucleos(t)ide reverse transcriptase inhibitors do not completely suppress HBV DNA in chronic HBV infection (cHBV). Vebicorvir (VBR) is an investigational core inhibitor that interferes with multiple aspects of HBV replication. This phase II trial evaluated the safety and efficacy of VBR in combination with entecavir (ETV) in treatment-naïve patients with cHBV. METHODS: HBeAg-positive, treatment-naïve patients without cirrhosis were randomised 1:1 in a double-blind manner to once-daily VBR 300 mg+ETV 0.5 mg or placebo (PBO)+ETV 0.5 mg for 24 weeks. The primary endpoint was change in mean log10 HBV DNA from Baseline to Week 12 and 24. RESULTS: All patients in both treatment groups (PBO+ETV: 12/12; VBR+ETV: 13/13) completed the study. At Week 12, VBR+ETV led to a greater mean (SD) reduction from Baseline in log10 IU/ml HBV DNA (-4.45 [1.03]) vs. PBO+ETV (-3.30 [1.18]; p = 0.0077). At Week 24, VBR+ETV led to a greater reduction from Baseline in log10 IU/ml HBV DNA (-5.33 [1.59]) vs. PBO+ETV (-4.20 [0.98]; p = 0.0084). Greater mean reductions in pregenomic RNA were observed at Week 12 and 24 in patients receiving VBR+ETV vs. PBO+ETV (p <0.0001 and p <0.0001). Changes in viral antigens were similar in both groups. No drug interaction between VBR and ETV was observed. Two patients experienced HBV DNA rebound during treatment, with no resistance breakthrough detected. The safety of VBR+ETV was similar to PBO+ETV. All treatment-emergent adverse events and laboratory abnormalities were Grade 1/2. There were no deaths, serious adverse events, or evidence of drug-induced liver injury. CONCLUSIONS: In this 24-week study, VBR+ETV provided additive antiviral activity over PBO+ETV in treatment-naïve patients with cHBV, with a favourable safety and tolerability profile. CLINICAL TRIAL NUMBER: NCT03577171 LAY SUMMARY: Hepatitis B is a long-lasting viral infection of the liver. Current treatments can suppress hepatitis B virus but do not offer the opportunity of cure, hence, new treatment approaches are required. Herein, we show that the combination of the novel core inhibitor vebicorvir with an existing antiviral (entecavir) in treatment-naïve patients chronically infected with hepatitis B virus demonstrated greater antiviral activity than entecavir alone. Additionally, vebicorvir was safe and well tolerated. Thus, further studies evaluating its potential role in the treatment of chronic hepatitis B are warranted.

Safety and efficacy of vebicorvir in virologically suppressed patients with chronic hepatitis B virus infection.

BACKGROUND & AIMS: HBV nucleos(t)ide reverse transcriptase inhibitors (NrtIs) do not completely suppress HBV replication. Previous reports indicate persistent viremia during NrtI treatment despite HBV DNA being undetectable. HBV core inhibitors may enhance viral suppression when combined with NrtIs. This phase II trial (NCT03576066) evaluated the efficacy and safety of the investigational core inhibitor, vebicorvir (VBR), in virologically- suppressed patients on NrtIs. METHODS: Non-cirrhotic, NrtI-suppressed patients with chronic HBV were randomised to VBR 300 mg once daily or matching placebo (PBO) for 24 weeks. Treatment was stratified by hepatitis B e antigen (HBeAg) status. The primary endpoint was change from Baseline in serum HBeAg or hepatitis B surface antigen (HBsAg) after 24 weeks. RESULTS: Of 73 patients enrolled, 47 were HBeAg positive and 26 were HBeAg negative. In HBeAg-positive and -negative patients, there were no differences in the change from Baseline at Week 24 for HBsAg or HBeAg. Using a novel, high-sensitivity assay to detect HBV DNA, a greater proportion of patients with detectable HBV DNA at Baseline achieved undetectable HBV DNA at Week 24 in the VBR+NrtI vs. PBO+NrtI group. In HBeAg-positive patients, a greater change from Baseline in HBV pregenomic (pg)RNA was observed at Week 24 with VBR+NrtI vs. PBO+NrtI. Treatment-emergent adverse events (TEAEs) in VBR+NrtI patients included upper respiratory tract infection, nausea, and pruritus. No serious adverse events, Grade 4 TEAEs, or deaths were reported. CONCLUSIONS: In this 24-week study, VBR+NrtI demonstrated a favourable safety and tolerability profile. While there were no significant changes in viral antigen levels, enhanced viral suppression was demonstrated by greater changes in DNA and pgRNA with the addition of VBR compared to NrtI alone. CLINICAL TRIALS NUMBER: NCT03576066. LAY SUMMARY: Core inhibitors represent a novel approach for the treatment of chronic hepatitis B virus (HBV) infection, with mechanisms of action distinct from existing treatments. In this study, vebicorvir added to existing therapy reduced HBV replication to a greater extent than existing treatment and was generally safe and well tolerated.

Lower Observed Hepatocellular Carcinoma Incidence in Chronic Hepatitis B Patients Treated With Entecavir: Results of the ENUMERATE Study.

OBJECTIVES: Data from the United States are lacking regarding the impact of entecavir (ETV) on the risk of hepatocellular carcinoma (HCC). Our aim is to determine whether treatment with ETV is associated with a reduced HCC risk by calculating the expected HCC incidence based on the Risk Estimation for Hepatocellular Carcinoma in Chronic Hepatitis B (REACH-B) model and comparing it with the observed HCC incidence. METHODS: The incidence of HCC in US patients treated with ETV between 2005 and 2013 in a retrospective cohort was obtained. The predicted HCC incidence was calculated using the REACH-B model. The standardized incidence ratios (SIRs) were calculated as a ratio of observed over predicted HCC cases. RESULTS: Of 841 patients, 646 (65% male, 84% Asian, median age 47 years, 36% hepatitis B e antigen positive, 9.4% with cirrhosis) met the inclusion criteria. Over a median follow-up of 4 years, 17 (2.6%) cases of HCC were diagnosed, including 8 out of 61 (13.1%) patients with cirrhosis and 9 out of 585 (1.5%) without cirrhosis. Compared with those without HCC, the 17 patients with HCC were older at 53 years vs. 47 years and more likely to have cirrhosis at 47.1% vs. 8.4%. Among patients without cirrhosis, the observed HCC incidence was significantly lower than predicted by the fourth year (SIR, 0.37; 95% confidence interval: 0.166-0.82). A sensitivity analysis that comprised all patients, including those with cirrhosis, showed that at the maximum follow-up time of 8.2 years, a significantly lower than predicted HCC incidence was noted with an SIR of 0.56 (95% confidence interval: 0.35-0.905). CONCLUSIONS: Based on the REACH-B model, long-term ETV therapy was associated with a lower than predicted HCC incidence. However, the risk of HCC persisted, and careful HCC surveillance remains warranted despite the anti-viral treatment.

Question of ALT flare during switch to adefovir from lamivudine: A single center open-label, randomized, safety study (June 17, 2005 to February 5, 2009).

Earlier clinical studies have reported an ALT flare greater than 10 times the upper limit of normal in some patients with chronic hepatitis B when their lamivudine (LAM) treatment was switched to adefovir (ADV) therapy. The current study compared the safety of switching directly to ADV versus overlapping LAM and ADV for 3 months followed by ADV monotherapy. Patients with chronic hepatitis B receiving LAM therapy for > or = 6 months were eligible for the study regardless of the presence of LAM resistance, HBeAg status or serum ALT levels. Eighteen patients (13 males) were randomized to direct switch to ADV and 17 patients (10 males) to overlap. HBV-DNA, ALT, albumin, and total bilirubin were assayed at baseline, 3, 6, 9, and 12 months. Study drugs were discontinued at the end of 12 months with the follow up at 3 and 6 months. The decision to continue antiviral therapy was made at the discretion of the investigator. Baseline ALT levels were similar between the direct switch and overlap group: median ALT (U/L) was 44.0 (16-266) and 33.0 (19-367) for direct switch for overlap group, respectively (P = 0.42). No ALT flare was noted at 3 months in either group: median ALT decreased from 44.0 to 34.5 U/L in the direct switch group, and from 33.0 to 23.0 in the overlap group. Furthermore, no patient in either group exhibited ALT flare throughout the 12 months. This study did not show an ALT flare during switch to ADV at 3 months or at any time later.

Hepatocellular carcinoma with indeterminate or false-negative findings at initial MR imaging: effect on eligibility for curative treatment initial observations.

PURPOSE: To retrospectively evaluate the effect of indeterminate or false-negative findings at magnetic resonance (MR) imaging on eligibility for curative treatment of hepatocellular carcinoma (HCC). MATERIALS AND METHODS: This HIPAA-compliant retrospective study was approved by the institutional review board; the need for informed consent was waived. Of 166 patients with cirrhosis in whom HCC was detected with MR imaging, 21 (13 men, eight women; mean age, 60 years) had 33 proved HCCs that were not detected on previous MR images obtained 6-24 months earlier. MR imaging included T1-weighted, T2-weighted, and dynamic contrast material-enhanced T1-weighted imaging. Serial MR images and treatment records were reviewed to evaluate nodule growth and the effect of delayed diagnosis on treatment eligibility. RESULTS: Of 33 HCCs in 21 patients, 24 corresponding nodules (73%) were described on previous MR images as benign or indeterminate. Five additional nodules were visible at retrospective evaluation, but only on arterial phase images. The diameters of these 29 visible but indeterminate nodules were initially 0.6-1.9 cm (mean, 1.1 cm) and increased to 0.9-4.5 cm (mean, 1.9 cm) at HCC diagnosis (mean follow-up, 378 days). The mean doubling time was 856 days for diameter and 285 days for volume. All nine HCCs with a delayed diagnosis of less than 1 year were smaller than 3 cm at diagnosis, and the patients had undergone liver transplantation (n=3) or technically successful ablation or embolization (n=6). All 10 subcentimeter indeterminate nodules were smaller than 2 cm at HCC diagnosis, and none progressed to untreatable HCC. CONCLUSION: Indeterminate nodules smaller than 2 cm did not become untreatable HCC with delayed HCC diagnosis of 6-12 months.

Hepatitis B virus infection in 6,130 unvaccinated Korean-Americans surveyed between 1988 and 1990.

OBJECTIVES: During the past decades, the influx of immigrants from hepatitis B virus (HBV) endemic regions has brought significant changes in the prevalence of HBV-associated liver diseases and hepatocellular carcinoma (HCC) in the United States. Our program, which was intended to identify those in need of hepatitis B vaccination, helped us to learn of the natural history of HBV infection in Korean Americans. METHODS: Between November of 1988 and May 1990, we screened 6,130 Korean Americans in the eastern United States for HBV infection. RESULTS: The overall hepatitis B surface antigen (HBsAg) (+) rate was 6.1%, with 8.0% for males and 4.4% for females. The carrier rate peaked in subjects between the ages of 21 and 40 yr. The HBsAg (+) rate for 452 U.S.-born children was lower (2.7%) than that of 623 Korean-born (5.5%). None received hepatitis B immune-globulin or HBV vaccination. The vertical transmission rate was 30.3% in children born to HBsAg (+) mothers and 100% in those born to hepatitis B e antigen (HBeAg) positive mothers. In contrast, the paternal transmission rate was low; 10.3% in children with HBsAg (+) fathers and 19.2% in those with HBeAg (+) fathers. Another significant observation was the unexpected finding of ongoing liver diseases in incidentally identified carriers. Evaluation of 139 asymptomatic adult carriers revealed that 42% had elevated liver enzymes and 11% had already developed liver cirrhosis. CONCLUSION: These findings strongly suggest the need for active HBV screening of immigrants from endemic regions and, most importantly, the need for careful monitoring of the carriers.

Proteomic analysis of serum associated fucosylated glycoproteins in the development of primary hepatocellular carcinoma.

Changes in N-linked glycosylation are known to occur during the development of cancer. For example, increased branching of oligosaccharides has been associated with metastasis and has been correlated to tumor progression in human cancers of the breast, colon and melanomas. Increases in core fucosylation have also been associated with the development of hepatocellular carcinoma (HCC). Chronic infection with the hepatitis B virus is associated with more than 55% of all cases of hepatocellular carcinoma. We show here that increased levels of core fucosylation can be observed via glycan analysis of total serum and are associated with the development of HCC. In a blinded study, the serum glycoproteins derived from people diagnosed with HBV induced liver cancer were found to possess a dramatically higher level of fucosylation. This change occurs on both immunoglobulin molecules and on other serum glycoproteins. Targeted glycoproteomic analysis was used to identify those glycoproteins that are hyperfucosylated in cancer. In total, 19 proteins were found to be hyperfucosylated in cancer. The potential of these proteins as biomarkers of cancer is discussed.

MR Images of Bowel Wall Thickening in Patients with Minimal to Moderate Cirrhosis : Comparison with Clinical Findings

PURPOSE : To evaluate the MR imaging findings of bowel wall thickening in patients with minimal to moderate cirrhosis, and analyze their clinical significances comparing with laboratory findings. MATERIALS AND METHODS : We assessed retrospectively the MRI findings of 123 patients with minimal to moderate cirrhosis, and compared these with the clinical laboratory findings. We evaluated the involved sites and MR image findings of thickened bowel wall, as well as the presence of collateral vessels, ascites, and splenic size. These were compared with serum albumin and bilirubin levels, and prothrombin time. RESULTS : Gastrointestinal wall thickening was detected at 37 sites in 25 patients (20%), and more frequently detected in moderate cirrhosis (29%) than in minimal cirrhosis (17%). Jejunum and ascending colon were the most common sites of bowel wall thickening; each was involved at 22 and 9 sites, respectively. Ascending colonic wall thickening was more commonly detected in moderate cirrhosis than in minimal cirrhosis. The thickened bowel wall showed symmetric contour, high signal intensity on T2-weighted images, mixed iso- and low signal intensity on T1-weighted images, and homogeneous or target-like enhancement. Serum albumin level was significantly lower in patients with bowel wall thickening (3.3+/-0.9 g/dl vs. 3.9+/-0.7 g/dl;p=0.0024). Serum bilirubin level was significantly higher in patients with bowel wall thickening (1.7+/-1.0 mg/dl vs. 1.4+/-1.2 mg/dl; p=0.0160). Bowel wall thickening did not significantly correlate with the presence of collateral vessels, ascites, splenic size, and prolongation of prothrombin time. CONCLUSION : In minimal to moderate cirrhosis, the MR imaging evaluation of bowel wall thickening was useful for estimating the severity of cirrhosis and laboratory findings.

Factors associated with response to lamivudine: Retrospective study in a tertiary care clinic serving patients with chronic hepatitis B.

BACKGROUND AND AIMS: Chronic hepatitis B (CHB) is an important cause of end stage liver disease and hepatocellular carcinoma. Controlled clinical trials indicate treatment with lamivudine results in positive clinical responses. The study goal was to determine if the response to lamivudine treatment (HBeAg loss, HBV DNA loss and alanine aminotransferase [ALT] reduction) differs according to pretherapy (pre-tx) ALT levels. METHODS: This was a retrospective review of medical record data. All CHB patients at all stages of disease (including cirrhotic) with more than two visits to the clinic were included in the study (n = 719). Kaplan-Meier survival and Cox proportional hazards were estimated. RESULTS: Of the total 719 HBsAg (+) patients, 317 were treated with lamivudine 150 mg or 100 mg daily. Among HBeAg positive patients, at 3 years, Kaplan-Meier estimates of the loss of HBeAg were 40%, 57% and 61% for pre-tx ALT < upper limit of normal (ULN), 1-2 x ULN and >2 x ULN, respectively. Similar results of HBV-DNA loss were seen in HBeAg negative patients. CONCLUSIONS: In this setting, we observed that pre-tx ALT levels were not associated with response to lamivudine, but that lower platelet count and female sex in HBeAg (+) patients were important predictive factors of a favorable response to lamivudine therapy.

Preneoplastic markers of hepatitis B virus-associated hepatocellular carcinoma.

Hepatitis B virus (HBV) carriers are at high risk for the development of hepatocellular carcinoma (HCC), but there are no reliable markers that will identify such high-risk carriers. The objective of this work is to identify serologic markers that may indicate the early presence of HCC. Since HBV-encoded X antigen (HBxAg) likely contributes to HCC by up- or down-regulation of host gene expression, X positive and negative HepG2 cells were made and subjected to cDNA subtraction. When specific ELISAs were constructed measuring differentially expressed antigens and corresponding antibodies, antibodies to several differentially expressed genes were detected. In cross-sectional and longitudinal studies, antibodies were predominantly present in patients with HBV-associated cirrhosis and HCC, but not in most carriers with hepatic inflammation alone or without active liver disease. Antibodies were also present in patients with hepatitis C virus (HCV)-related HCC, but rarely detected in sera from uninfected individuals, those with tumors other than HCC, or those with drug-induced hepatitis. Statistical analysis showed that HCC patients with four or more antibodies detectable before the appearance of HCC had decreased survival, suggesting that these markers may reflect stepwise hepatocarcinogenesis. Hence, these antibodies may serve as preneoplastic markers for HCC in HBV carriers with chronic liver disease, and may be identified by a simple blood test.

A United States compassionate use study of lamivudine treatment in nontransplantation candidates with decompensated hepatitis B virus-related cirrhosis.

Patients with hepatitis B-related decompensated cirrhosis have limited treatment options. This prospective, multicenter study assessed lamivudine in 75 patients with decompensated cirrhosis, the majority of whom (93%) were not candidates for liver transplantation. At baseline, all 75 patients tested positive for hepatitis B surface antigen [HBsAg (+)] and 62% tested positive for hepatitis B e antigen [HBeAg (+)]. Hepatitis B virus (HBV) DNA levels were detectable in 64% of patients by the branched chain DNA (bDNA) assay. Patients received lamivudine 100 mg once daily (median duration, 12.7 months; range, 0.5 to 33 months). In patients with detectable HBV DNA pretreatment, the virus became undetectable by the bDNA assay in 69% of patients with > or = 6 months treatment and in 64% overall. Alanine aminotransferase (ALT) level improved in 90% and normalized in 55% of patients with > or = 6 months treatment and in 48% overall. Improvements in bilirubin and albumin levels occurred throughout treatment. The median Child-Pugh score improved from a baseline of 10 to 8 at last visit, with 31% (23/75) having an improved score of > or = 2 points, 57% (43/75) unchanged (< 2 points), and 12% (9/75) worsened (> or = 2 points). A virologic breakthrough developed in eight of 41 patients (18%) after a median of 13.1 months of treatment. Tyrosine-methionine-aspartate-aspartate (YMDD) variant HBV was detected in three of four patients tested. Nevertheless, at last visit, ALT, albumin, and bilirubin levels were similar for patients with and without breakthrough. Lamivudine treatment can lead to significant improvements in liver disease severity in nontransplantation candidates with advanced disease. Additional studies of lamivudine in combination with other antivirals are indicated for the large population of patients worldwide with advanced HBV-related cirrhosis and inadequate access to liver transplantation.

Determinants of early mortality in patients with decompensated chronic hepatitis B treated with antiviral therapy.

BACKGROUND & AIMS: Chronic hepatitis B is a leading cause of death worldwide. To identify patients who might require urgent liver transplantation despite antiviral therapy, we investigated the determinants of early mortality in a large cohort of patients with decompensated chronic hepatitis B treated with lamivudine. METHODS: One hundred fifty-four North American patients with decompensated chronic hepatitis B received lamivudine for a median of 16 months. Univariate and multivariate Cox regression modeling was used to develop a model of 6-month mortality. RESULTS: A biphasic survival pattern was observed, with most deaths occurring within the first 6 months of treatment (25 of 32, 78%) because of complications of liver failure. The estimated actuarial 3-year survival of patients who survived at least 6 months was 88% on continued treatment. In multivariate modeling, elevated pretreatment serum bilirubin and creatinine levels as well as the presence of detectable hepatitis B virus (HBV) DNA (by the bDNA assay) pretreatment were significantly associated with 6-month mortality. An equation approximating the probability of early mortality was developed from these variables. CONCLUSIONS: Our data demonstrate a distinct alteration in the slope of the survival curve after 6 months of lamivudine treatment for decompensated chronic hepatitis B. An equation consisting of 3 widely available pretreatment laboratory parameters was developed that can be used to predict the likelihood of early death in patients receiving lamivudine for decompensated chronic hepatitis B. These observations may help identify patients who can be stabilized with suppressive antiviral therapy vs. those who require urgent liver transplantation.

Elevation of fetal dopamine following exposure to methamphetamine in utero.

The effect of methamphetamine on fetal dopaminergic function was examined following treatment of pregnant mice twice daily with 40 mg/kg methamphetamine from either gestational day (GD) 7-13 or from GD 7-15. Dopamine levels were elevated in fetal GD 16 corpus striatum and rostral mesencephalon following both treatment regimens. This increase in fetal dopamine is consistent with our findings that exposure to methamphetamine in utero results in adult dopaminergic neurons which are more responsive in terms of methamphetamine induced release of the neurotransmitter and more sensitive to the neurotoxic effects of the drug.

Methamphetamine concentrations in fetal and maternal brain following prenatal exposure.

Levels of methamphetamine in maternal striatum and whole fetal mouse brain were assessed at 0.5, 1, 2, and 4 h postinjection on gestational day 14 (GD14) following a single, subcutaneous injection of 40 mg/kg (+)-methamphetamine hydrochloride to pregnant mice. In the dams, striatal concentrations of methamphetamine peaked at 1 h postinjection, reaching levels of approximately 510 ng/mg protein. Amphetamine, the primary metabolite of methamphetamine, increased to 77 ng/mg protein at 2 h and remained elevated by 4 h postinjection. In the fetal brain, peak methamphetamine concentrations of approximately 122 ng/mg protein were attained at 1 h. Amphetamine was only detectable in fetal brain at 2 and 4 h postinjection. Regional analysis of methamphetamine levels in fetal striatum, cortex, and brainstem revealed that the drug was not uniformly distributed. Maternal administration of methamphetamine results in fetal brain drug concentrations, which approximate those reported in human infants whose mother abused methamphetamine. This dosage regimen, therefore, serves as an appropriate animal model for assessing the potential risks to human offspring exposed to methamphetamine in utero.

Gender-dependent enhanced adult neurotoxic response to methamphetamine following fetal exposure to the drug.

Methamphetamine use by females of child-bearing age has become a major public health concern in terms of the long-term risk to the exposed fetus. We examined the possibility of enhanced adult neurotoxic potential of the drug in offspring that had been exposed to methamphetamine in utero during gestational days 7 to 18. While basal levels of monoamines were not affected by prenatal exposure to methamphetamine, we observed an enhanced neurotoxicity in adult male offspring following drug challenge with effects localized primarily to the dopaminergic nigrostriatal projection. This was evidenced by greater methamphetamine-induced reductions of dopaminergic markers in the striatum [dopamine (DA), dihydroxyphenylacetic acid, homovanillic acid (HVA), and 3-methoxytyramine (3-MT)] and ventral brainstem (DA) of prenatal methamphetamine-treated males compared with saline-treated animals. Some effects of prenatal methamphetamine exposure were observed in female offspring, but these were limited to striatal levels of 3-MT and HVA. Differential gender sensitivity to the neurotoxic effect of methamphetamine was shown to be correlated with hyperthermic response. Hyperthermic effects, however, do not account for the increased susceptibility of prenatal methamphetamine-treated males to drug-induced striatal DA neurotoxicity since methamphetamine challenge did not evoke a significantly greater hyperthermic response in these animals compared with prenatal saline-treated males. The findings raise the concern that male methamphetamine abusers may be at risk for an enhanced neurotoxic risk if they were exposed to the drug in utero.

Cellular dopamine is increased following exposure to a factor derived from immortalized striatal neurons [corrected].

A factor obtained from an immortalized hybrid monoclonal cell line (X61) of striatal origin is capable of increasing the dopamine content of hybrid, monoclonal cells expressing a dopaminergic phenotype (MN9D) and of aggregate cultures containing primary dopaminergic neurons. The factor is a protein smaller than 100 kDa and appears to be different than a number of other trophic agents with effects on the dopaminergic neuron. The effect on dopamine content appears to be specific to centrally-derived neuronal elements; the protein having no effect on the dopamine content of PC12 cells. Given that parkinsonian symptoms are only apparent following degeneration of a substantial portion of the mesencephalic dopaminergic cell population, activity capable of increasing dopamine content of the surviving cells may represent an interesting candidate therapeutic agent.

Preferential resistance of dopaminergic neurons to glutathione depletion in a reconstituted nigrostriatal system.

Depletion of glutathione in the substantia nigra is one of the earliest changes observed in Parkinson's disease (PD), and could initiate dopaminergic neuronal degeneration. Nevertheless, we have previously demonstrated that mesencephalic dopaminergic neurons in primary monolayer cultures are more resistant to the toxicity of glutathione depletion than nondopaminergic neurons. To extend this finding to a system that more closely resembles the in vivo situation, we characterized the effects of glutathione depletion on reaggregate cultures derived from ventral mesencephalic and their striatal target neurons, as well as supporting elements including glia. Dopaminergic neurons were found to be more resistant to the toxicity of buthionine-(S,R)-sulfoximine, an inhibitor of glutathione synthesis, than other nigrostriatal neurons, while striatal target cells exhibited an intermediate susceptibility when examined after 48 h. Glutathione depletion, however, decreased the intracellular content of catecholamines after 48 h and eventually led to the loss of dopaminergic neurons after 7 days. Our data indicate that the intrinsic resistance of dopaminergic neurons to the toxicity of glutathione depletion occurs in a variety of experimental paradigms, and suggest that global glutathione depletion alone is unlikely to account for the selective loss of dopaminergic neurons in PD. Rather, it is more likely that either the selective loss of glutathione from dopaminergic neurons, or the combination of glutathione loss with other insults contributes to the preferential death of dopaminergic neurons in PD.

Evidence of elevated intracellular calcium levels in weaver homozygote mice.

1. A mutation in the G-protein-linked, inwardly rectifying K+ channel GIRK2 leads to the loss of cerebellar and dopaminergic mesencephalic neurons in weaver mice. The steps leading to cell death are not well understood but may involve constitutive influx of Na+ and Ca2+ into the neurons. 2. We found that resting [Ca2+]i was dramatically higher in cerebellar neurons from weaver mice compared to wild-type neurons. 3. High-K+ stimuli elicited much smaller changes in [Ca2+]i in weaver cerebellar neurons compared to wild-type neurons. 4. weaver cerebellar granule cells could be rescued from cell death by the GIRK2wv cationic channel blocker, QX-314. 5. QX-314 lowered resting intracellular Ca2+ levels in weaver cerebellar granule cells. 6. These results suggest that changes in resting [Ca2+]i levels and alterations in K+ channel function are most likely to contribute to the developmental abnormalities and increased cerebellar cell death observed in weaver mice.

Forskolin and dopamine D1 receptor activation increase huntingtin's association with endosomes in immortalized neuronal cells of striatal origin.

Huntingtin is a cytoplasmic protein of unknown function that associates with vesicle membranes and microtubules. Its protein interactions suggest that huntingtin has a role in endocytosis and organelle transport. In this study we sought to identify factors that regulate the transport of huntingtin in striatal neurons, which are the cells most affected in Huntington's disease. In clonal striatal cells derived from fusions of neuroblastoma and embryonic striatal neurons, huntingtin localization is diffuse and slightly punctate in the cytoplasm. When these neurons were differentiated by treatment with forskolin, huntingtin redistributed to perinuclear regions, discrete puncta along plasma membranes, and branch points and terminal growth cones in neurites. Huntingtin staining overlapped with clathrin, a coat protein involved in endocytosis. Immunoblot analysis of subcellular membrane fractions separated by differential centrifugation confirmed that huntingtin immunoreactivity in differentiated neurons markedly increased in membrane fractions enriched with clathrin and with huntingtin-interacting protein 1. Dopamine treatment altered the subcellular localization of huntingtin and increased its expression in clathrin-enriched membrane fractions. The dopamine-induced changes were blocked by the D1 antagonist SCH 23390 and were absent in a clonal cell line lacking D1 receptors. Results suggest that the transport of huntingtin and its co-expression in clathrin and huntingtin-interacting protein 1-enriched membranes is influenced by activation of adenylyl cyclase and stimulation of dopamine D1 receptors.