Resistance characterization of ledipasvir and velpatasvir in hepatitis C virus genotype 4.

HCV genotype 4 (GT4) has often been overlooked in drug development, even though it infects ~20 million people worldwide. Ledipasvir/sofosbuvir and sofosbuvir/velpatasvir were highly efficacious in GT4 HCV-infected patients from GS-US-337-1119 and GS-US-342-1138. Here, we characterize the resistance profile of ledipasvir (LDV) and velpatasvir (VEL) in patients with GT4 HCV infection. NS5A deep-sequencing was performed for 454 patients infected with HCV GT4 at baseline, including 44 patients enrolled in GS-US-337-1119 and 116 patients enrolled in GS-US-342-1138, and at relapse for patients with virologic failure. LDV and VEL susceptibilities of 56 patient isolates were determined. In GS-US-337-1119, SVR12 rates were 100% for all subtypes except 4b and 4r. Phenotypic assessment of 56 HCV NS5A patient isolates from various GT4 subtypes indicated that LDV had high potency for the common subtypes 4a/d, and subtypes 4c/f/k/l/m/n/o/p/r/t despite the presence of resistance-associated substitutions (RASs). For the rare GT4b, LDV median EC50 was higher, but with a broad range of individual values. Importantly, all GT4b isolates tested had 2-4 NS5A RASs, some including Y93H. Similarly, the 2 GT4r infected patients who had virologic relapse had rare triple RASs. Reversion of these substitutions to the consensus residue significantly increased LDV susceptibility. In GS-US-342-1138, all patients achieved SVR12, regardless of their subtype or presence of RASs. In vitro data confirmed that VEL is potent against all GT4 isolates tested. LDV and VEL are potent antiviral drugs, estimated to be effective against >95% and >99%, respectively, of GT4 HCV isolates.

Sofosbuvir plus ribavirin in treatment-naïve patients with chronic hepatitis C virus genotype 1 or 3 infection in India.

Until 2014, pegylated interferon plus ribavirin was the recommended standard of care for the treatment of chronic hepatitis C virus (HCV) infection in India. This open-label phase 3b study, conducted across 14 sites in India between 31 March 2014 and 30 November 2015, evaluated the efficacy and safety of sofosbuvir plus ribavirin therapy among treatment-naïve patients with chronic genotype 1 or 3 HCV infection. A total of 117 patients with genotype 1 or 3 HCV infection were randomized 1:1 to receive sofosbuvir 400 mg and weight-based ribavirin (1000 or 1200 mg) daily for 16 or 24 weeks. Among those with genotype 1 infection, the primary efficacy endpoint of sustained virologic response at 12 weeks post-treatment (SVR12) was reported in 90% (95% confidence intervals [CI], 73-98) and 96% (95% CI, 82-100) of patients following 16 and 24 weeks of treatment, respectively. For patients with genotype 3 infection, SVR12 rates were 100% (95% CI, 88-100) and 93% (95% CI, 78-99) after 16 and 24 weeks of therapy, respectively. Adverse events, most of which were mild or moderate in severity, occurred in 69% and 57% of patients receiving 16 and 24 weeks of treatment, respectively. The most common treatment-emergent adverse events were asthenia, headache and cough. Only one patient in the 24-week group discontinued treatment with sofosbuvir during this study. Overall, sofosbuvir plus ribavirin therapy achieved SVR12 rates ≥90% and was well tolerated among treatment-naïve patients with chronic genotype 1 or 3 HCV infection in India.

Workplace screening for hand dermatitis: a pilot study.

BACKGROUND: Health care workers (HCWs) are at increased risk for developing occupational skin disease (OSD) such as dermatitis primarily due to exposure to wet work. Identification of risk factors and workplace screening can help early detection of OSD to avoid the condition becoming chronic. AIMS: To determine risk factors and clinical findings for hand dermatitis using a workplace screening tool. METHODS: Employees at a large teaching hospital in Toronto, Canada, were invited to complete a two-part hand dermatitis screening tool. Part 1 inquired about hand hygiene practices and Part 2 comprised a visual assessment of participants' hands by a health professional and classification as (i) normal, (ii) mild dermatitis or (iii) moderate/severe dermatitis. Risk factors were determined using chi-square and Cochran-Armitage analysis on a dichotomous variable, where Yes represented either a mild or moderate/severe disease classification. RESULTS: There were 183 participants out of 643 eligible employees; response rate 28%. Mild or moderate/severe dermatitis was present in 72% of participants. These employees were more likely to work directly with patients, have worked longer in a health care setting, wash hands and change gloves more frequently, wear gloves for more hours per day, have a history of eczema or dermatitis and report a current rash on the hands or rash in the past 12 months. CONCLUSIONS: There was a high percentage of HCWs with dermatitis and risk factors for dermatitis. These findings argue for increased attention to prevention and early identification of hand dermatitis and support further testing of the workplace screening tool.

An in vitro investigation of the cardiovascular effects of the 5-HT(4) receptor selective agonists, velusetrag and TD-8954.

The 5-HT(4) receptor agonists, and gastrointestinal (GI) prokinetic agents, cisapride and tegaserod, lack selectivity for the 5-HT(4) receptor. Cisapride is a potent human ether-à-go-go-related gene (hERG) potassium channel inhibitor while cisapride and tegaserod have significant affinity for 5-HT(1) and 5-HT(2) receptor subtypes. Marketing of both compounds was discontinued due to cardiovascular concerns (cardiac arrhythmias with cisapride and ischemic events with tegaserod). The reported association of tegaserod with ischemia has been postulated to involve coronary artery constriction or augmentation of platelet aggregation. This in vitro study investigated the effects of two of the new generation of highly selective 5-HT(4) receptor agonists, velusetrag and TD-8954, on canine, porcine and human coronary artery tone, human platelet aggregation and hERG potassium channel conductance. No significant off-target actions of velusetrag or TD-8954 were identified in these, and prior, studies. While cisapride inhibited potently the hERG channel currents, tegaserod failed to affect platelet aggregation, and had only a small contractile effect on the canine coronary artery at high concentrations. Tegaserod inhibited the 5-HT-induced contractile response in the porcine coronary artery. New generation 5-HT(4) receptor agonists hold promise for the treatment of patients suffering from GI motility disorders with a reduced cardiovascular risk.

Clinical trial: the efficacy and tolerability of velusetrag, a selective 5-HT4 agonist with high intrinsic activity, in chronic idiopathic constipation - a 4-week, randomized, double-blind, placebo-controlled, dose-response study.

BACKGROUND: Velusetrag is an orally active 5-HT(4) receptor agonist of potential benefit in treating chronic idiopathic constipation. AIM: To evaluate the efficacy, safety and tolerability of velusetrag in chronic idiopathic constipation. METHODS: After a 2-week baseline period, patients [<3 spontaneous bowel movements (SBM)/week] received placebo or velusetrag (15, 30 or 50 mg) daily for 4 weeks in a randomized, double-blind design, followed by a 1-week follow-up period. The primary endpoint was the change from baseline in weekly SBM frequency averaged over the 4-week treatment period. RESULTS: Patients receiving velusetrag (15, 30 and 50 mg) achieved statistically and clinically significant increases in weekly SBM frequency relative to those receiving placebo. Mean increases were 3.6, 3.3 and 3.5 SBM/week respectively, compared with 1.4 SBM/week for placebo (P < 0.0001). Statistically significant increases in the weekly frequency of complete SBM (CSBM) were also reported (mean increases of 2.3, 1.8 and 2.3 for 15, 30 and 50 mg velusetrag respectively, compared with 0.6 for placebo). Common adverse events associated with velusetrag were diarrhoea, headache, nausea and vomiting, generally occurring during the initial days of dosing. CONCLUSION: Velusetrag was efficacious and well tolerated in patients with chronic idiopathic constipation (ClinicalTrials.gov identifier NCT00391820).

Phase I study of the heat shock protein 90 inhibitor alvespimycin (KOS-1022, 17-DMAG) administered intravenously twice weekly to patients with acute myeloid leukemia.

Heat shock protein 90 (Hsp90) is a molecular chaperone with many oncogenic client proteins. The small-molecule Hsp90 inhibitor alvespimycin, a geldanamycin derivative, is being developed for various malignancies. This phase 1 study examined the maximum-tolerated dose (MTD), safety and pharmacokinetic/pharmacodynamic profiles of alvespimycin in patients with advanced acute myeloid leukemia (AML). Patients with advanced AML received escalating doses of intravenous alvespimycin (8-32 mg/m(2)), twice weekly, for 2 of 3 weeks. Dose-limiting toxicities (DLTs) were assessed during cycle 1. A total of 24 enrolled patients were evaluable for toxicity. Alvespimycin was well tolerated; the MTD was 24 mg/m(2) twice weekly. Common toxicities included neutropenic fever, fatigue, nausea and diarrhea. Cardiac DLTs occurred at 32 mg/m(2) (elevated troponin and myocardial infarction). Pharmacokinetics revealed linear increases in C(max) and area under the curve (AUC) from 8 to 32 mg/m(2) and minor accumulation upon repeated doses. Pharmacodynamic analyses on day 15 revealed increased apoptosis and Hsp70 levels when compared with baseline within marrow blasts. Antileukemia activity occurred in 3 of 17 evaluable patients (complete remission with incomplete blood count recovery). The twice-weekly administered alvespimycin was well tolerated in patients with advanced AML, showing linear pharmacokinetics, target inhibition and signs of clinical activity. We determined a recommended phase 2 dose of 24 mg/m(2).

The effects of the 5-HT3 antagonist, alosetron, on brain serotonin synthesis in patients with irritable bowel syndrome.

Serotonin (5-HT) plays an important role in the pathophysiology of irritable bowel syndrome (IBS). Using alpha-[(11)C]methyl-L-tryptophan-positron emission tomography (PET), it was demonstrated that brain 5-HT synthesis is increased in patients with IBS, in a gender-specific manner. The aims of the study were to evaluate the effects of alosetron on brain 5-HT synthesis in patients with IBS. Six male and five female non-constipation-predominant IBS patients were enrolled. The subjects received alosetron or a placebo for 14 days, separated by a 2-week washout period. On day 14, rectal distensions commenced just prior to the PET scan (which was performed for 80 min), and continued for 20-min periods. The functional images were analysed with SPM99. Alosetron vs placebo treatments, in a randomized, double-blinded, crossover manner, were studied. 5-HT synthesis was greater in several regions in the males than in the females during the alosetron treatment, whereas there was no region in which the females had greater synthesis. There were significant gender-treatment interactions of synthesis in the cingulate gyrus, caudate nucleus, globus pallidus, and cerebellum. The gender differences in the effect of alosetron on brain 5-HT synthesis may be related to the gender differences in the efficacy of alosetron.

Alosetron repeat dose pharmacokinetics, effects on enzyme activities, and influence of demographic factors.

AIM: To assess the pharmacokinetics of alosetron, its effect on in vivo enzyme activities, and influence of demographic factors during repeated dosing. METHODS: Thirty healthy men and women received 1 mg oral alosetron twice-daily for 29.5 days and a single oral dose of a metabolic probe cocktail before and on the last day of alosetron dosing. Serum alosetron concentrations were measured on days 1, 8, 15, 22 and 29. Probe-substrate and metabolite concentrations were measured after each cocktail dose. RESULTS: Alosetron accumulation in serum was negligible. Exposure to alosetron did not alter probe-metabolite/substrate ratios associated with CYP2C19, 2E1, 2C9, or 3A4 activity, but modestly decreased those associated with CYP1A2 and N-acetyltransferase activity. Systemic exposure to alosetron was higher in women, positively correlated with age and body mass index, and negatively correlated with CYP1A2 activity. Incidence of constipation was higher in women, but not associated with alosetron concentration. CONCLUSIONS: Single dose data can reliably predict the pharmacokinetics of alosetron after repeated doses. Alosetron exhibits limited potential for inhibition of cytochrome P450-mediated metabolism. Interindividual differences in alosetron pharmacokinetics associated with demographic factors may be related to strong dependence on metabolism by CYP1A2.

Effect of alosetron on theophylline pharmacokinetics.

AIMS: To examine the potential for alosetron to alter the pharmacokinetics of theophylline by inhibiting its metabolism, as suggested by in vitro and in vivo effects on CYP1A2 activity. METHODS: Ten healthy female volunteers received theophylline 200 mg twice daily alone for 8 days and with alosetron 1 mg twice daily for 15 days in this randomized, placebo-controlled, two-way-crossover study. RESULTS: Alosetron had no significant effect on theophylline plasma concentrations (Cmax approximately 9 microg ml(-1), AUC approximately 90 microg ml(-1) h) or oral formation clearance of three major metabolites produced via CYP1A2: 3-methylxanthine, 1-methylurate and 1,3-dimethylurate (5, 7 and 16 ml min(-1), respectively). Concomitant administration of alosetron and theophylline was well tolerated. CONCLUSIONS: The absence of a clinical drug interaction involving inhibition of theophylline metabolism by alosetron was not predicted by in vitro and in vivo metabolic probe data.

Effect of ranitidine on the pharmacokinetics of triazolam and alpha-hydroxytriazolam in both young (19-60 years) and older (61-78 years) people.

OBJECTIVE: This study evaluated the effect of oral ranitidine (75 mg and 150 mg) on the pharmacokinetics of triazolam (0.25 mg) and its major metabolite, alpha-hydroxytriazolam, in both young and older people. Metabolite data were used to distinguish the mechanism of this interaction. METHOD: This was a randomized, open-label, 3-way crossover study. Eighteen young (19-60 years) and 12 older (61-78 years) men and women were randomly assigned to receive evening doses of triazolam 0.25 mg (1) alone, (2) on the third day of dosing ranitidine 75 mg twice daily for 4 days, and (3) on the third day of dosing ranitidine 150 mg twice daily for 4 days. RESULTS: In the young group, mean triazolam area under the concentration-time curve from time zero to infinity [AUC(0-infinity)] was 10% and 28% higher after treatment with 75 mg and 150 mg ranitidine, respectively. In the older group, mean triazolam AUC(0-infinity) was 31% and 28% higher after treatment with 75 mg and 150 mg ranitidine, respectively. There was no change in the alpha-hydroxytriazolam/triazolam AUC(0-infinity) ratio in either age group, indicating that neither formation nor elimination of alpha-hydroxytriazolam was affected by ranitidine. There were no changes in the half-life of triazolam or alpha-hydroxytriazolam. CONCLUSION: Ranitidine increases oral absorption of triazolam in both young and older people. This effect is likely caused by elevation of gastrointestinal pH, allowing for greater absorption of acid-labile triazolam. The difference in this effect between age groups at the lower 75-mg dose of ranitidine suggests that older people may be more sensitive to the antisecretory effect of ranitidine.

Proteosomes, emulsomes, and cholera toxin B improve nasal immunogenicity of human immunodeficiency virus gp160 in mice: induction of serum, intestinal, vaginal, and lung IgA and IgG.

Intranasal immunization of mice with human immunodeficiency virus (HIV) rgp160 complexed to proteosomes improved anti-gp160 serum IgA and IgG titers, increased the number of gp160 peptides recognized, and stimulated anti-gp160 intestinal IgA compared with immunization with uncomplexed rgp160 in saline. These enhanced responses were especially evident when either a bioadhesive nanoemulsion (emulsomes) or cholera toxin B subunit (CTB) was added to the proteosome-rgp160 vaccine. Furthermore, anti-gp160 IgG and IgA in vaginal secretions and fecal extracts were induced after intranasal immunization with proteosome-rgp160 delivered either in saline or with emulsomes. Formulation of uncomplexed rgp160 with emulsomes or CTB also enhanced serum and selected mucosal IgA responses. Induction of serum, vaginal, bronchial, intestinal, and fecal IgA and IgG by intranasal proteosome-rgp160 vaccines delivered in saline or with emulsomes or CTB is encouraging for mucosal vaccine development to help control the spread of HIV transmission and AIDS.

Effervescent and standard formulations of ranitidine--a comparison of their pharmacokinetics and pharmacology.

BACKGROUND: An effervescent formulation of ranitidine may be absorbed faster and achieve a faster onset of action than conventional tablet form. The aim of this study was to compare the effects of effervescent formulations of ranitidine with equivalent dose standard tablets, in terms of intragastric pH and plasma pharmacokinetics in the initial 6 h following dosing. METHODS: Fifteen fasting healthy males, aged 18-31 (mean 29) years, were each randomly given, at weekly intervals, 150 mg standard and effervescent ranitidine and 300 mg standard and effervescent ranitidine. Ambulatory gastric pH was performed and plasma drug levels measured at regular intervals. RESULTS: Plasma ranitidine levels increased more rapidly with both effervescent formulations compared with standard tablets as indicated by mean area under curve (AUC) at 1 h (P < 0.001). However, the pH profiles produced by all four treatments were similar with a steep rise in pH at 40-60 min to give a sustained level of pH 7 for the following 5 h. The effervescent formulations produced a transient rise in pH immediately following dosing, and for 300 mg this rise was significantly different at 10-20 min compared with the standard tablet (median pH 4.75 vs. 2.3, P < 0.05). CONCLUSIONS: Plasma drug levels increase more rapidly following effervescent ranitidine. Effervescent and standard formulations of 150 and 300 mg are all equally effective in producing gastric pH 7 after 1 h. However, effervescent formulations produce an early transient rise in pH which may be of clinical benefit.

Humoral responses to linear epitopes on the HIV-1 envelope in seropositive volunteers after vaccine therapy with rgp160.

Humoral responses to the HIV-1 envelope were investigated in 30 human volunteers enrolled in a phase I vaccine therapy trial of rgp160 (LAI/LAV) using two techniques that emphasize detection of antibody response against linear (continuous) epitopes: immunoblotting and PEPSCAN. Seven fusion proteins containing large portions from constant regions 1, 2, 3, and 5, and variable region 3 of gp120 and two regions in the transmembrane protein, gp41, were employed in immunoblots to quantitatively measure immune response as a function of immunization. In addition, the entire gp160 (LAI/LAV) envelope protein was constructed in duplicate sets of 211 overlapping 12-mer peptides to fine-map the changes. Immunoblotting defined significant changes in reactivity to epitopes in constant regions; of 28 volunteers completing the trial, the percentage with reactivity against C1 changed from 62 to 100%; for C2, from 0 to 46%; for C3, from 0 to 82%; and for a constant region in gp41, from 25 to 68%. PEPSCAN on a subset (n = 8) of these volunteers identified new reactivity to epitopes throughout the envelope, concentrated in V1, C3, and C5 in gp120 and several peptides in gp41. Completely immunized patients responded to double the number of linear epitopes compared with two patients receiving alum alone. The results verify that the response to rgp160 is significantly broadened after immunization, providing additional evidence that HIV-1-infected volunteers can expand their antibody repertoire against a protein from a pathogen during chronic infection with that same pathogen. These results expand those previously obtained in this patient cohort, by defining explicitly the immunogenic regions recognized postvaccination and by providing methodology for quantitating those changes.

Effect of intravenous infusion of ranitidine on intragastric acidity in fasting subjects: comparison with bolus or Gastrojet (pH-stat-adjusted) infusion.

This study was undertaken in nine fasting healthy volunteers to compare the effect of intravenous continuous infusion versus bolus injection of ranitidine on 12-h intragastric pH, and to compare the efficacy of these two modes of administration of pH-stat-adjusted infusion of ranitidine using the Gastrojet. Each volunteer had three study sessions with 12-h pH measurements. In the ranitidine infusion treatment arm (RAN-INF), ranitidine was continually infused intravenously using an IVAC-pump at a dose of 0.125 mg mg.kg over a 12-h period. In the ranitidine bolus treatment arm (RAN-BOL), ranitidine bolus 50 mg was given over 10 min, every 6 h. When ranitidine infusion was given by the pH stat method using the Gastrojet (RAN-JET), sufficient ranitidine was given to maintain a present value of pH > or = 5. The study was analysed with a 3 x 3 Latin square cross-over design with multiple measurements of each phase of the cross-over. No difference was found between RAN-INF and RAN-BOL in 12-h or in daytime (10.00-18.00 h) mean pH, median pH, or percentage of pH > or = 5. Using RAN-JET, 89.5% of the pH values were > or = 5., compared with 39.7% and 40.0% with RAN-INF or RAN-BOL. RAN-JET also gave higher (P < 0.05) mean and median 12-h or daytime pH values, as compared with RAN-INF or RAN-BOL. The mean doses of ranitidine given in the 12-h infusion periods were 100 mg, 109 mg and 112 mg (RAN-BOL, RAN-INF and RAN-JET, respectively). Thus, this superior inhibition of acid inhibition achieved with Gastrojet does not require higher mean doses of ranitidine. These findings cannot necessarily be applied to persons with duodenal ulcer disease or to patients in an intensive-care unit setting. However, the data do raise the possibility that much greater inhibition of acid inhibition can be achieved by individualizing the dose of ranitidine using the Gastrojet.

Immunohistochemical demonstration of differential substance P-, met-enkephalin-, and glutamic-acid-decarboxylase-containing cell body and axon distributions in the corpus striatum of the cat.

The immunohistochemical localization of neuronal cell bodies and axons reactive for substance P (SP) and methionine-enkephalin (ME) was investigated in the corpus striatum of the adult cat brain and compared with that of glutamate decarboxylase (GAD), synthetic enzyme for gamma-aminobutyric acid. Striatal cell bodies reactive for ME could be identified only in colchicine treated cats, are medium size, ovoid striatal cells, and are found in large numbers in a more or less even distribution throughout the caudate nucleus, putamen, and nucleus accumbens. The striatal region most densely occupied by ME-immunoreactive cells is the ventral and central part of the caudate head. Modest numbers of larger ME-reactive neurons are dispersed throughout the entopeduncular nucleus and the pars reticulata of the substantia nigra. Striatal cells of medium size reactive for SP could be identified, with or without colchicine, in largest numbers in the medial half of the caudal three-fourths of the putamen and in clusters of irregular size and shape in the head of the caudate nucleus. Cells reactive for SP are also common in layer II and the islands of Calleja of the olfactory tubercle. We could not reliably visualize GAD-positive cell bodies in the striatum, even with colchicine treatment; however, they could be seen readily in all pallidal structures such as the globus pallidus, ventral pallidum, entopeduncular nucleus, and substantia nigra. Axons reactive for ME are found mainly in the globus pallidus where they form a dense and even network throughout the nucleus. The globus pallidus is almost devoid of SP reactivity except near its extreme caudal pole. Conversely, SP-immunoreactive axons form dense meshworks in the entopeduncular nucleus and substantia nigra where ME immunoreactivity is minimal. Fewer, but still ample numbers, of SP-reactive axons are present also in the ventral tegmental and retrorubral areas of the midbrain tegmentum and in the ventral pallidum of the basal forebrain, but only sparse ME-reactive axons are present in these areas. This differential distribution of SP- and ME-containing axons in the pallidal and nigral structures stands in contrast to the relatively homogeneous and dense distribution of GAD-containing axons throughout the dorsal and ventral pallidum, entopeduncular nucleus, and substantia nigra.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence that changes in tonic luteinizing hormone secretion determine the growth of preovulatory follicles in the rat.

Physiological concentrations of progesterone (20-100 ng/ml), maintained by the insertion of implants into 30-day-old rats, delayed first ovulation, and withdrawal of progesterone on day 47 of age synchronized first ovulation in rats. Inhibition of ovulation involved negative feedback regulation of tonic LH and FSH secretion, blockage of gonadotropin surges, and suppression of preovulatory, but not antral, follicular growth. Removal of implants resulted in a rapid decline in serum progestrone from 100 to 5 ng/ml within 0-12 h. Between 0-36 h there were progressive increases in serum concentrations of LH and FSH, enhanced accumulation of estradiol by individual follicles incubated in vitro with or without exogenous substrate, and marked progressive increases in the content of LH (but not FSH) receptors in both thecal and granulosa cells. These events were followed by gonadotropin surges at 48 h (1800 h on day 49), ovulation, and morphological and biochemical signs of luteinization, including decreases in follicular gonadotropin receptor content and estradiol accumulation, evident by 60 h. With the exception of changes in basal LH, this sequence of events is remarkably similar in time and pattern to that after the decline of progesterone on diestrous day 2 and ovulation on proestrus of a 5-day cycle. Although a direct effect of progesterone on ovarian follicular cell function cannot be excluded, the data suggest that subtle but sustained increases in LH (and possibly FSH) are required for the enhanced follicular accumulation of estradiol and LH-binding activity occurring between diestrus and proestrus of the rat estrous cycle. Thus, perhaps some of the mystery surrounding the endocrine events between diestrus and proestrus can be ascribed to changes in serum LH that have been too small and/or variable for current nonserial sampling methods and RIAs to detect reliably.