Cardiovascular safety assessments in the conscious telemetered dog: utilisation of super-intervals to enhance statistical power.

INTRODUCTION: ICH S7A and S7B guidelines recommend the use of conscious animals for assessment of non-clinical cardiovascular safety of new chemical entities prior to testing in humans. Protocol design and data analysis techniques can affect the quality of the data produced and can therefore ultimately influence the clinical management of cardiovascular risk. It is therefore essential to have an understanding of the magnitude of changes detectable and the clinical relevance of these changes. This paper describes the utilisation of "super-intervals" to analyse and interpret data obtained from our conscious telemetered dog cardiovascular safety protocol and reports the statistical power achieved to detect changes in various cardiovascular parameters. METHODS: Cardiovascular data from 18 dog telemetry studies were used to calculate the statistical power to detect changes in cardiovascular parameters. Each study followed a test compound versus vehicle cross-over experimental design with 24h monitoring (n=4). 1 min mean raw data from each individual animal was compressed into 15 min mean data for each dose group for visualisation. Larger summary periods, or "super-intervals", were then selected to best represent any observed cardiovascular effects whilst taking into account the pharmacokinetic profile of the drug e.g. intervals of 1 to 6, 7 to 14 and 14 to 22h post-dose. RESULTS: With this methodology and study design we predict, using the median percentile that our studies have 80% power to detect the following changes: HR (+/-10bpm), LV +dP/dt max (+/-375mmHg/s), MBP (+/-5mmHg) and QTc (+/-4ms). DISCUSSION: Super-intervals are a simple way to handle the high degree of natural variability seen with any ambulatory cardiovascular assessment and, in our hands, result in highly statistically powered studies. The ability of this model to detect cardiovascular changes of small, but biologically relevant, magnitude enables confident decision making around the cardiovascular safety of new chemical entities.

The production of hydrogen sulfide limits myocardial ischemia and reperfusion injury and contributes to the cardioprotective effects of preconditioning with endotoxin, but not ischemia in the rat.

We investigated whether (endogenous) hydrogen sulfide (H2S) protects the heart against myocardial ischemia and reperfusion injury. Furthermore, we investigated whether endogenous H2S is involved in the protection afforded by (1) ischemic preconditioning and (2) the second window of protection caused by endotoxin. The involvement of one of the potential (end) effectors of the cardioprotection afforded by H2S was investigated using the mitochondrial KATP channel blocker, 5-hydroxydecanoate (5-HD; 5 mg/kg). Animals were subjected to 25 min regional myocardial ischemia followed by reperfusion (2 h) and were pretreated with the H2S donor, sodium hydrosulfide (3 mg/kg i.v.). Animals were also subjected to shorter periods of myocardial ischemia (15 min) and reperfusion (2 h) and pretreated with an irreversible inhibitor of cystathionine-gamma-lyase, dl-propargylglycine (PAG; 50 mg/kg i.v.). Animals were also pretreated with PAG (50 mg/kg) and subjected to either (1) ischemic preconditioning or (2) endotoxin (1 mg/kg i.p.) 16 h before myocardial ischemia. Myocardial infarct size was determined by p-nitroblue tetrazolium staining. Administration of sodium hydrosulfide significantly reduced myocardial infarct size, and this effect was abolished by 5-HD. Administration of PAG (50 mg/kg) or 5-HD significantly increased infarct size caused by 15 min of myocardial ischemia. The delayed cardioprotection afforded by endotoxin was abolished by 5-HD or PAG. In contrast, PAG (50 mg/kg) did not affect the cardioprotective effects of ischemic preconditioning. These findings suggest that (1) endogenous H2S is produced by myocardial ischemia in sufficient amounts to limit myocardial injury and (2) the synthesis or formation of H2S by cystathionine-gamma-lyase may contribute to the second window of protection caused by endotoxin.

Inhibition of papilloma progression by antisense oligonucleotides targeted to HPV11 E6/E7 RNA.

Human papillomaviruses (HPVs) are recognized as important human pathogens, causing a spectrum of hyperproliferative lesions from benign warts to cervical dysplasias/carcinomas. HPV-associated lesions require continued production of the oncogenic E6/E7 proteins, which are encoded by either bicistronic or overlapping mRNAs. Here we targeted the E6/E7 mRNA of HPV11, a type implicated in causation of genital warts, using molecular reagents. Accessible sites in the HPV11(E6/E7) RNA were identified using library selection protocols, and nucleic acids (DNAzymes, antisense oligonucleotides) targeted to these sites were constructed, and tested in cell culture and on human foreskin grafts. While DNAzymes were at least equally effective in cell culture, antisense oligonucleotides targeted to the region surrounding one of the library-selected sites (ASO(407)) proved most effective in blocking progression of HPV11-induced papillomas in human foreskin grafts on immunodeficient mice. In total, 11 papillomas were treated with ASO(407). Of these, four of seven small papillomas treated with ASO(407) showed loss of detectable virus by in situ hybridization (ISH), and in all four of these, papillomas were no longer evident grossly or histologically after treatment. When larger papillomas were treated, one of four showed loss of virus by ISH, associated with a minor decrease in papilloma size. Considering all 11 papillomas treated with ASO(407), loss of viral staining by ISH was significantly different from that observed in controls (P<0.016), as was true for the seven small treated papillomas (P<0.012). DNAzymes targeted to the same site (or other library selected sites) did not produce statistically significant differences in ISH staining (P<0.15). Our results with ASO(407) appear to represent the first specific molecular therapy against a bona fide HPV infection, and provide a rational proof-of-principle strategy for development of molecular therapeutics targeting other HPV-associated lesions.

The melanocortin 5 receptor is expressed in human sebaceous glands and rat preputial cells.

Melanocortins regulate pigmentation, adrenal hormone secretion, immune functions, lipid metabolism, and feeding behaviors in rodents. These peptides include adrenocorticotrophic hormone, melanocyte stimulating hormone, beta-lipotrophin, and the endorphins. Lipid metabolism in sebaceous glands and preputial glands of rodents is regulated by alpha-melanocyte stimulating hormone, the major agonist for melanocortin receptors. Five melanocortin receptor subtypes have been identified that differ in their tissue localization and affinities for melanocortin ligands. Targeted disruption of the melanocortin 5 receptor in transgenic mice results in widespread dysfunction of exocrine glands, including a marked decrease in sebum production. A role for melanocortins in the modulation of human sebum production has not been established. The goal of this study is to determine which melanocortin receptors are expressed in human sebaceous glands. Messenger RNA was isolated from human sebaceous glands and the reverse transcriptase polymerase chain reaction was performed using primers specific for each of the melanocortin receptor subtypes. Transcripts were detected for the melanocortin 5 receptor. A polyclonal chicken antihuman antibody to the melanocortin 5 receptor localized to sebaceous glands, eccrine glands, hair follicles, and epidermis in human skin, rat skin, cultured human sebocytes, and rat preputial cells. Presence of the melanocortin 5 receptor protein in human sebaceous glands and rat preputial glands was further verified by Western blotting. These data support further investigation of the role of melanocortins in the regulation of human sebum production and support the use of the rat preputial system as an experimental model in sebaceous gland physiology.

Modulation of HLA-DR expression in epithelial cells by interleukin 1 and estradiol-17 beta.

Both ultrapure human interleukin-1 (IL-1) and Escherichia coli derived recombinant IL-1 alpha and beta consistently induced the expression of major histocompatibility class II (HLA-DR) molecules in a human endometrial and a breast carcinoma cell line. [35S]Methionine incorporation into IL-1 induced, immunoprecipitable HLA-DR molecules demonstrated de novo synthesis of both light and heavy chains of the HLA-DR molecules. Lipopolysaccharide, recombinant interleukin-2 and recombinant interleukin-6 failed to induce HLA-DR expression in these epithelial cells. In contrast to the dramatic effect on HLA-DR expression, IL-1 had no effect on the epithelial cell proliferation. Pretreatment of T47D cells with estradiol-17 beta significantly decreased the IL-1 induced HLA-DR expression, and pretreatment of IL-1 with an IL-1 specific antibody, neutralized IL-1 action. These studies demonstrate that a cytokine (IL-1) and a sex steroid hormone estradiol-17 beta can interact to regulate the expression of HLA-DR molecules in epithelial cells.

Serum stimulation of lysyl hydroxylase activity in cultured human skin fibroblasts.

In the absence of ascorbic acid, confluent human skin fibroblasts incubated in 0.5% serum-supplemented medium had one-third of the level of lysyl hydroxylase activity of cells incubated in media containing high serum concentrations (5-20%). This difference appeared to be due to a decline in the enzyme activity following serum deficiency, and was largely abolished by addition of ascorbic acid to the medium. The effect of serum deficiency was slow, manifesting in 48 h at the earliest, and was completely reversed by replenishing the medium with serum. Prolyl hydroxylase activity was independent of serum concentration, both in the absence and in the presence of ascorbic acid in the culture medium.

Regulation of collagen synthesis by ascorbic acid.

After prolonged exposure to ascorbate, collagen synthesis in cultured human skin fibroblasts increased approximately 8-fold with no significant change in synthesis of noncollagen protein. This effect of ascorbate appears to be unrelated to its cofactor function in collagen hydroxylation. The collagenous protein secreted in the absence of added ascorbate was normal in hydroxylysine but was mildly deficient in hydroxyproline. In parallel experiments, lysine hydroxylase (peptidyllysine, 2-oxoglutarate:oxygen 5-oxidoreductase, EC 1.14.11.4) activity increased 3-fold in response to ascorbate administration whereas proline hydroxylase (prolyl-glycyl-peptide, 2-oxoglutarate:oxygen oxidoreductase, EC 1.14.11.2) activity decreased considerably. These results suggest that collage polypeptide synthesis, posttranslational hydroxylations, and activities of the two hydroxylases are independently regulated by ascorbate.

Prolyl and lysyl hydroxylase activities of human skin fibroblasts: effect of donor age and ascorbate.

Prolyl and lysyl hydroxylase activities in cultures of human skin fibroblasts from fetal to 94-yr-old donors were measured. In contrast to earlier studies with whole skin, neither prolyl nor lysyl hydroxylase activity was found related to donor age. Prolyl hydroxylase activity increased 3- to 6-fold when cell extracts were incubated with ascorbate and other hydroxylation cofactors before assay. A similar increase in prolyl hydroxylase activity occurred when cells were incubated with ascorbate. Lysyl hydroxylase activity remained unaltered under these conditions.

Procollagen I synthesis in human skin fibroblasts: effect on culture conditions on biosynthesis.

Human skin fibroblast culture promises to be a useful system for the investigation of the regulation of collagen biosynthesis and the study of abnormalities in collagen biosynthesis in connective tissue disorders. The effect of culture conditions on procollagen I biosynthesis has been determined. Optimal conditions for collagen biosynthesis were: 10% dialyzed heat-inactivated fetal calf serum, 0.15 mM ascorbate, and 0.078 mM beta-aminopropionitrile. Newly synthesized procollagen I accumulated in the medium at a linear rate for 18 hr. Preincubation of cells in labelling media for 4 hr before adding radioactive proline enhanced synthesis. Collagenase digestion was used to study overall collagen biosynthesis. 94% of all collagen synthesized was found in the medium, and 6% in the cell pellet. Under optimal conditions, collagen comprised 24% of all protein in the medium, and 14% of protein produced by the whole culture.