Evaluation of miR-122 as a Serum Biomarker for Hepatotoxicity in Investigative Rat Toxicology Studies.

MicroRNAs are short noncoding RNAs involved in regulation of gene expression. Certain microRNAs, including miR-122, seem to have ideal properties as biomarkers due to good stability, high tissue specificity, and ease of detection across multiple species. Recent reports have indicated that miR-122 is a highly liver-specific marker detectable in serum after liver injury. The purpose of the current study was to assess the performance of miR-122 as a serum biomarker for hepatotoxicity in short-term (5-28 days) repeat-dose rat toxicology studies when benchmarked against routine clinical chemistry and histopathology. A total of 23 studies with multiple dose levels of experimental compounds were examined, and they included animals with or without liver injury and with various hepatic histopathologic changes. Serum miR-122 levels were quantified by reverse transcription quantitative polymerase chain reaction. Increases in circulating miR-122 levels highly correlated with serum elevations of liver enzymes, such as alanine aminotransferase (ALT), aspartate aminotransferase (AST) and glutamate dehydrogenase (GLDH). Statistical analysis showed that miR-122 outperformed ALT as a biomarker for histopathologically confirmed liver toxicity and was equivalent in performance to AST and GLDH. Additionally, an increase of 4% in predictive accuracy was obtained using a multiparameter approach incorporating miR-122 with ALT, AST, and GLDH. In conclusion, serum miR-122 levels can be utilized as a biomarker of hepatotoxicity in acute and subacute rat toxicology studies, and its performance can rival or exceed those of standard enzyme biomarkers such as the liver transaminases.

Circulating ghrelin levels are decreased in human obesity.

Ghrelin is a novel endogenous natural ligand for the growth hormone (GH) secretagogue receptor that has recently been isolated from the rat stomach. Ghrelin administration stimulates GH secretion but also causes weight gain by increasing food intake and reducing fat utilization in rodents. To investigate the possible involvement of ghrelin in the pathogenesis of human obesity, we measured body composition (by dual X-ray absorption) as well as fasting plasma ghrelin concentrations (radioimmunoassay) in 15 Caucasians (8 men and 7 women, 31+/-9 years of age, 92+/-24 kg body wt, and 29+/-10% body fat, mean +/- SD) and 15 Pima Indians (8 men and 7 women, 33+/-5 years of age, 97+/-29 kg body wt, and 30+/-8% body fat). Fasting plasma ghrelin was negatively correlated with percent body fat (r = -0.45; P = 0.01), fasting insulin (r = -0.45; P = 0.01) and leptin (r = -0.38; P = 0.03) concentrations. Plasma ghrelin concentration was decreased in obese Caucasians as compared with lean Caucasians (P < 0.01). Also, fasting plasma ghrelin was lower in Pima Indians, a population with a very high prevalence of obesity, compared with Caucasians (87+/-28 vs. 129+/-34 fmol/ml; P < 0.01). This result did not change after adjustment for fasting plasma insulin concentration. There was no correlation between fasting plasma ghrelin and height. Prospective clinical studies are now needed to establish the role of ghrelin in the pathogenesis of human obesity.

Ritonavir and saquinavir combination therapy for the treatment of HIV infection.

OBJECTIVE: To evaluate the safety and antiretroviral activity of ritonavir (Norvir) and saquinavir (Invirase) combination therapy in patients with HIV infection. DESIGN: A multicenter, randomized, open-label clinical trial. SETTING: Seven HIV research units in the USA and Canada. PATIENTS: A group of 141 adults with HIV infection, CD4 T lymphocyte counts of 100-500 x 10(6) cells/l, whether treated previously or not with reverse transcriptase inhibitor therapy, but without previous HIV protease inhibitor drug therapy. INTERVENTIONS: After discontinuation of prior therapy for 2 weeks, group I patients were randomized to receive either combination (A) ritonavir 400 mg and saquinavir 400 mg twice daily or (B) ritonavir 600 mg and saquinavir 400 mg twice daily. After an initial safety assessment of group I patients, group II patients were randomized to receive either (C) ritonavir 400 mg and saquinavir 400 mg three times daily or (D) ritonavir 600 mg and saquinavir 600 mg twice daily. Investigators were allowed to add up to two reverse transcriptase inhibitors (including at least one with which the patient had not been previously treated) to a patient's regimen after week 12 for failure to achieve or maintain an HIV RNA level < or = 200 copies/ml documented on two consecutive occasions. MEASUREMENTS: Plasma HIV RNA levels and CD4+ T-lymphocyte counts were measured at baseline, every 2 weeks for 2 months, and monthly thereafter. Safety was assessed through the reporting of adverse events, physical examinations, and the monitoring of routine laboratory tests. RESULTS: The 48 weeks of study treatment was completed by 75% (106/141) of the patients. Over 80% of the patients on treatment at week 48 had an HIV RNA level < or = 200 copies/ml. In addition, intent-to-treat and on-treatment analyses revealed comparable results. Suppression of plasma HIV RNA levels was similar for all treatment arms (mean areas under the curve minus baseline through 48 weeks were-1.9, -2.0, -1.6, -1.8 log10 copies/ml in ritonavir-saquinavir 400-400 mg twice daily, 600-400 mg twice daily, 400-400 mg three times daily, and 600-600 mg twice daily, respectively). Median CD4 T-lymphocyte count rose by 128 x 10(6) cells/l from baseline, with an interquartile range (IQR) of 82-221 x 10(6) cells/l. The most common adverse events were diarrhea, circumoral paresthesia, asthenia, and nausea. Reversible elevation of serum transaminases (> 5 x upper limit of normal) occurred in 10% (14/141) of the patients enrolled in this study and was associated with baseline abnormalities in liver function tests, baseline hepatitis B surface antigen positivity, or hepatitis C antibody positivity (relative risk, 5.0; 95% confidence interval 1.5-16.9). Most moderate or severe elevations in liver function tests occurred in patients treated with ritonavir-saquinavir 600-600 mg twice daily. CONCLUSIONS: Ritonavir 400 mg combined with saquinavir 400 mg twice daily with the selective addition of reverse transcriptase inhibitors was the best-tolerated regimen of four dose-ranging regimens and was equally as active as the higher dose combinations in HIV-positive patients without previous protease inhibitor treatment.

In vivo comparison of activated protein-1 gene activation in response to human parathyroid hormone (hPTH)(1-34) and hPTH(1-84) in the distal femur metaphyses of young mice.

Intermittent parathyroid hormone (PTH) treatment increases bone mass in humans and animals. Although intact human PTH has 84 amino acids, the N-terminal 31 to 38 amino acids are sufficient for bone anabolic activity in vivo. Prior studies have evaluated hPTH(1-34) and hPTH(1-84) with respect to bone mass increase and quality, but there have been no in vivo comparisons of dose-dependent molecular responses. After confirming that young male BALB/c mice respond to daily PTH with increased bone mass, we profiled the steady-state mRNA levels of activating protein-1 (AP-1) genes regulated by hPTH(1-34) and hPTH(1-84) at doses ranging from 0 to 19.4 nmol/kg in the distal femur metaphyses. We selected AP-1 genes, which include jun and fos, as they play a fundamental role mediating signals for proliferation, differentiation, and apoptosis in cells of different origins, including bone, and are known to be regulated by PTH. Human PTH(1-34) and hPTH(1-84) increased steady-state mRNA expression of c-jun, junB, c-fos, and fra-2 in an equivalent dose- and time-dependent manner. Expression of fosB or fra-1 was not detected with either peptide. When averaged across dose and time, responses to hPTH(1-34) and hPTH(1-84) were not significantly different from each other. Expression of c-jun, junB, and c-fos peaked 30 minutes after the injection while fra-2 expression peaked 30 minutes later. All AP-1 genes stimulated by PTH returned to the levels of vehicle treated controls by 3 h after injection. The expression level of junD, which was abundant in the distal metaphysis, was not altered by either peptide. No change in magnitude was observed after 1, 3, or 7 days of once-daily subcutaneous treatment of either peptide. When individual comparisons for each dose between peptides were made, the minimum effective dose necessary to stimulate a significant increase in c-fos and junB expression was equivalent for both peptides. The minimum effective dose for hPTH(1-34) was at least tenfold lower than hPTH(1-84) in stimulating c-jun and fra-2 expression. Area under the curve for the highest dose (19.4 nmol/kg) of either peptide showed no significant differences in the expression of any of the genes. In conclusion, in young mice given once-daily subcutaneous injections up to 7 days, hPTH(1-34) and hPTH(1-84) induced equivalent responses by time and dose in the selected AP-1 genes. These data on molecular regulation in mouse bone confirm and extend prior data from rat studies showing equivalence on bone mass at equimolar doses.

Assessment and reporting of the clinical immunogenicity of therapeutic proteins and peptides-harmonized terminology and tactical recommendations.

Immunogenicity is a significant concern for biologic drugs as it can affect both safety and efficacy. To date, the descriptions of product immunogenicity have varied not only due to different degrees of understanding of product immunogenicity at the time of licensing but also due to an evolving lexicon that has generated some confusion in the field. In recent years, there has been growing consensus regarding the data needed to assess product immunogenicity. Harmonization of the strategy for the elucidation of product immunogenicity by drug developers, as well as the use of defined common terminology, can benefit medical practitioners, health regulatory agencies, and ultimately the patients. Clearly, understanding the incidence, kinetics and magnitude of anti-drug antibody (ADA), its neutralizing ability, cross-reactivity with endogenous molecules or other marketed biologic drugs, and related clinical impact may enhance clinical management of patients treated with biologic drugs. To that end, the authors present terms and definitions for describing and analyzing clinical immunogenicity data and suggest approaches to data presentation, emphasizing associations of ADA development with pharmacokinetics, efficacy, and safety that are necessary to assess the clinical relevance of immunogenicity.

A prototypical process for creating evidentiary standards for biomarkers and diagnostics.

A framework for developing evidentiary standards for qualification of biomarkers is a key need identified in the Food and Drug Administration's Critical Path Initiative. This article describes a systematic framework that was developed by Pharmaceutical Research and Manufacturers of America (PhRMA) committees and tested at a workshop in collaboration with the Food and Drug Administration and academia. With some necessary refinements, this could be applied to create an appropriately individualized evidentiary standard for any biomarker purpose.