Safety, tolerability and pharmacokinetics of rilpivirine following administration of a long-acting formulation in healthy volunteers.

OBJECTIVES: This phase I healthy volunteer study (NCT01031589) was carried out to investigate the safety/tolerability and pharmacokinetics of a rilpivirine (RPV; TMC278) long-acting (LA) formulation after single and multiple intramuscular (i.m.) injections. METHODS: In the first part of the study, which had an open-label design, a single RPV LA i.m. injection (300 mg/mL) of 300 (n = 6) or 600 (n = 5) mg was given to the volunteers. In the second part of the study, which had a double-blind, randomized, placebo-controlled design, three RPV LA i.m. injections (one every 4 weeks) at 1200/600/600 mg (n = 6) or placebo (n = 2) were given. Safety and local tolerability were monitored. RPV plasma concentrations were analysed up to 28 days after injection or until they were < 20 ng/mL. RESULTS: Grade 1/2 RPV-related adverse events in the 300, 600 and 1200/600/600 mg groups were: rash (zero, one and one subject, respectively, the last of whom discontinued participation in the study); musculoskeletal stiffness (three, zero and zero subjects, respectively); injection site reactions (one, two and two subjects, respectively). After one injection of 300, 600 or 1200 mg RPV LA, the mean (standard deviation) maximum plasma concentration was 39 (25), 48 (13) and 140 (16) ng/mL, and the mean (standard deviation) area under the concentration-time curve (28 days) was 17,090 (8907), 25,240 (8184) and 55,350 (13,550) ng h/mL, respectively. RPV pharmacokinetics were largely comparable after the 1200 mg loading dose and both 600 mg injections of RPV LA. The mean (standard deviation) RPV plasma concentration across the 28-day dosing interval after the last injection in the 1200/600/600 mg group was 79 (19) ng/mL. CONCLUSIONS: Single and multiple i.m. injections of RPV LA demonstrated favourable local/systemic tolerability in healthy volunteers. RPV pharmacokinetics suggested that clinically relevant plasma concentrations can be achieved with this LA formulation.

The effects of JNJ-26481585, a novel hydroxamate-based histone deacetylase inhibitor, on the development of multiple myeloma in the 5T2MM and 5T33MM murine models.

Multiple myeloma (MM) is a B-cell malignancy, which often remains incurable because of the development of drug resistance governed by the bone marrow (BM) microenvironment. Novel treatment strategies are therefore urgently needed. In this study, we evaluated the anti-MM activity of JNJ-26481585, a novel 'second-generation' pyrimidyl-hydroxamic acid-based histone deacetylase inhibitor, using the syngeneic murine 5TMM model of MM. In vitro, JNJ-26481585 induced caspase cascade activation and upregulation of p21, resulting in apoptosis and cell cycle arrest in the myeloma cells at low nanomolar concentrations. Similar results could be observed in BM endothelial cells using higher concentrations, indicating the selectivity of JNJ-26481585 toward cancer cells. In a prophylactic and therapeutic setting, treatment with JNJ-26481585 resulted in an almost complete reduction of the tumor load and a significant decrease in angiogenesis. 5T2MM-bearing mice also developed a MM-related bone disease, characterized by increased osteoclast number, development of osteolytic lesions and a reduction in cancellous bone. Treatment of these mice with JNJ-264815 significantly reduced the development of bone disease. These data suggest that JNJ-26481585 has a potent anti-MM activity that can overcome the stimulatory effect of the BM microenvironment in vivo making this drug a promising new anti-MM agent.

Human cystic fibrosis embryonic stem cell lines derived on placental mesenchymal stromal cells.

This study describes the production of two new human embryonic stem cell (hESC) lines affected by cystic fibrosis. These cell lines are heterozygous compounds, each a carrier of the DF508 mutations associated either with E585X or with 3849+10 kb C-->T. The derivation process was performed on irradiated human placental mesenchymal stromal cells and designed to minimize contact with xeno-components. This new source of feeder cells is easy to obtain and devoid of ethical concerns. The cells have a great capacity to proliferate which reduces the need for continuous preparation of new feeder cell lines. In addition, three normal hESC lines were obtained in the same conditions. The five stem cell lines retained hESC-specific features, including an unlimited and undifferentiated proliferation capacity, marker expression and the maintenance of stable karyotype. They also demonstrated pluripotency in vitro, forming cell lineages of the three germ layers, as indicated by immunolocalization of beta-tubulin, alpha-fetoprotein and actin. These new genetic cell lines represent an important in-vitro tool to study the physiological processes underlying this genetic disease, drug screening, and tissue engineering.

Study of gene expression in thyrotropin-stimulated thyroid cells by cDNA expression array: ID3 transcription modulating factor as an early response protein and tumor marker in thyroid carcinomas.

Induction of cell proliferation by mitogen or growth factor stimulation leads to the specific induction or repression of a large number of genes. To identify genes differentially regulated by the cAMP-dependent transduction pathway, which is poorly characterized so far, we used the cDNA expression array technology. Hybridizations of Atlas human cDNA expression arrays with (32)P-labeled cDNA probes derived from control or thyrotropin (TSH)-stimulated dog thyrocytes in primary culture generated expression profiles of hundreds of genes simultaneously. Among the genes that displayed modified expression, we selected the transcription factor ID3, whose expression was increased by a cAMP-dependent stimulus. ID3 overexpression after TSH stimulation was first verified by Northern blotting analysis, and its mRNA regulation was then investigated in response to a variety of agents acting on thyrocyte proliferation and/or differentiation. We show that: (1) ID3 mRNA induction was stronger after stimulation of the cAMP cascade, but was not restricted to this signaling pathway, as phorbol myristate ester (TPA) and insulin also stimulated mRNA accumulation; (2) in contrast, powerful mitogens for thyroid cells, epidermal growth factor and hepatocyte growth factor, did not significantly modify ID3 mRNA levels; (3) ID3 protein levels closely parallelled mRNA levels, as revealed by immunofluorescence experiments showing a nuclear signal regulated by TSH; (4) in papillary thyroid carcinomas, ID3 mRNA was downregulated. Our results suggest that ID3 expression might be more related to the differentiating process induced by TSH than to the proliferative action of this hormone.

The role of cyclic AMP and its effect on protein kinase A in the mitogenic action of thyrotropin on the thyroid cell.

Cyclic AMP has been shown to inhibit cell proliferation in many cell types and to activate it in some. The latter has been recognized only lately, thanks in large part to studies on the regulation of thyroid cell proliferation in dog thyroid cells. The steps that led to this conclusion are outlined. Thyrotropin activates cyclic accumulation in thyroid cells of all the studied species and also phospholipase C in human cells. It activates directly cell proliferation in rat cell lines, dog, and human thyroid cells but not in bovine or pig cells. The action of cyclic AMP is responsible for the proliferative effect of TSH. It accounts for several human diseases: congenital hyperthyroidism, autonomous adenomas, and Graves' disease; and, by default, for hypothyroidism by TSH receptor defect. Cyclic AMP proliferative action requires the activation of protein kinase A, but this effect is not sufficient to explain it. Cyclic AMP action also requires the permissive effect of IGF-1 or insulin through their receptors, mostly as a consequence of PI3 kinase activation. The mechanism of these effects at the level of cyclin and cyclin-dependent protein kinases involves an induction of cyclin D3 by IGF-1 and the cyclic AMP-elicited generation and activation of the cyclin D3-CDK4 complex.

Respective roles of carbamylcholine and cyclic adenosine monophosphate in their synergistic regulation of cell cycle in thyroid primary cultures.

The stimulation of thyroid cell proliferation by TSH through cAMP depends on permissive comitogenic factors, generally the insulin-like growth factors and insulin. In dog thyroid primary cultures, the use of the phosphodiesterase-resistant analog of cAMP (Bu)(2)cAMP instead of TSH allowed to unveil a potent comitogenic activity of carbamylcholine, which can substitute for insulin and was shown to mimic insulin action on cell cycle regulatory proteins. Like insulin, carbamylcholine induced the accumulation of cyclin D3 and overcame the repression by cAMP of this protein, which was shown 1) to be essential for cell cycle progression by means of microinjections of a neutralizing antibody; and 2) to be rate limiting for the cAMP-dependent assembly of cyclin D3-cdk4 complexes, their nuclear translocation and the phosphorylation of pRb. Relative to insulin, carbamylcholine offers the significant experimental advantage that its signaling cascades can be immediately deactivated by the muscarinic antagonist atropine. In the presence of carbamylcholine, the elimination of (Bu)(2)cAMP blocked within 2 h the entry of cells into DNA synthesis phase, but the addition of atropine still permitted the entry of cells in S phase. These data support our view that the progression in G1 phase stimulated by cAMP consists of at least two essential actions that are clearly dissociated: in a first stage, depending on the supportive activity of an agent that stimulates the required cyclin D3 accumulation, cAMP induces the assembly and nuclear translocation of cyclin D3-cdk4 complexes, and then cAMP can exert alone the last crucial control that determines the cell commitment toward DNA replication.

[Veterinarians:'Watch your affairs!']. / Dierenarts: 'let op uw saeck!'

The most relevant results of a written questionnaire among veterinarians in the Netherlands are presented and discussed. The inquiry was performed by MarketResponse Nederland BV. The objective was to get an overview of the current perception and the future view of the profession. The response was 37%. Most respondents (66%) were practitioners, 7% were active in research or teaching institutions, 5% were governmental employees, 5% were employed in industry, and 17% did not belong to any of these categories (retired, unemployed etc.). Forty-seven per cent of the veterinarians practised mainly small animal medicine, 24% large animal medicine (cattle 15%, swine 8%, and poultry 1%), and 4% equine medicine; 24% worked in mixed practices. This division reflects the real-life situation. The percentage of female respondents was considerably higher in the group of recent graduates than in the other groups of graduates (increasing from 27% in the graduation period 1980-1989 to 56% in the period 1990-1999). Sixty per cent of the veterinarians worked more than 40 hours a week. Veterinarians considered themselves reliable, honest, professional, client-minded, and animal-friendly. According to them, the public perceived veterinarians as being animal-friendly, professional, and reliable. Veterinarians were less satisfied in their current position than other professional groups, particularly with regard to their income. Their current market position was considered indifferent or good. This position was influenced negatively by the decrease in the number of animals and competition from non-veterinarians and others. The situation could be improved by collaboration and practice fusions, specialization, and differentiation. More demanding and price-conscious clients and governmental regulations were considered important trends. Social, management, and marketing skills, increased knowledge and cooperation, and a vision of future developments were considered essential in order to be able react to developments on the market. About 60% of the practitioners and 50% of the other veterinarians felt that they lacked the right skills, and especially management and marketing skills, to react to these developments adequately. Most veterinarians (77%) considered that their training did not provide them with the skills needed for their current position. During training, more time should be spent on management, communication, marketing, and social skills. Based on the results, veterinary medicine in the Netherlands seems to be an introspective but good profession; however, the interesting aspects of the profession appear to be over-shadowed by less appreciated aspects and the income is moderate. Entrepreneuship is poorly developed, in part because this is considered taboo. The importance of skills that are not an integral part of veterinary medicine, such as management, marketing, and communication, is recognized, as is the lack of these skills. Despite this, emphasis is put on continuing professional education, species specialization, modernization, accreditation, and expansion of facilities and treatment possibilities as ways to respond to the market situation.