Interleukin (IL) 31 induces in cynomolgus monkeys a rapid and intense itch response that can be inhibited by an IL-31 neutralizing antibody.

BACKGROUND: Overexpression or administration of interleukin 31 (IL-31) has been shown to induce a profound itch response in mice and dogs. The chronic pruritus observed in mouse IL-31 transgenic mice results in the development of skin lesions and alopecia through excoriation from excessive scratching, a condition similar to that observed in patients with atopic dermatitis (AD). OBJECTIVE: To test whether IL-31 induces pruritus in non-human primates and, if so, whether treatment with an anti-IL-31 neutralizing monoclonal antibody (mAb) can block the response. METHODS: A series of studies was conducted in cynomolgus monkeys to evaluate the itch response to recombinant cynomolgus IL-31 (cIL-31) administration. Three routes of cIL-31 administration (intravenous, intradermal, and subcutaneous) were evaluated. Subcutaneous treatment with a humanized anti-human IL-31 mAb cross-reactive to cIL-31 was subsequently tested for its ability to block the response to intradermal cIL-31 administration. RESULTS: Each route of cIL-31 delivery elicited a scratching response immediately after cIL-31 administration and lasted at least 3 h. Treatment with the IL-31 mAb inhibited the cIL-31-mediated scratching response in a dose-dependent manner. CONCLUSION: These results demonstrate that an IL-31 mAb can inhibit IL-31-mediated pruritus in vivo, and could be an effective therapy for pruritic skin conditions like AD where IL-31 upregulation may play a role.

Bioavailability and relative tissue distribution of [125I]-recombinant human thrombin following intravenous or subcutaneous administration to non-human primates.

BACKGROUND: Recombinant human thrombin (rhThrombin) is being developed as a general adjunct to hemostasis. Endogenous thrombin is rapidly inactivated by complex formation with antithrombin III and other inhibitors. It follows that these inhibitors will also inactivate any rhThrombin that reaches the systemic circulation. OBJECTIVES: Study goals were to determine the pharmacokinetic characteristics of [(125)I]-rhThrombin and [(125)I]-rhThrombin complexed to endogenous inhibitors, and the tissue distribution of rhThrombin-associated radioactivity in non-human primates. Hematology, serum chemistry and coagulation status were also monitored. METHODS: [(125)I]-rhThrombin was administered intravenously (i.v.; 3.5 U kg(-1)) or subcutaneously (s.c.; 350 U kg(-1)) to male cynomolgus monkeys. Plasma was analyzed for rhThrombin-associated radioactivity and non-compartmental analysis was used to determine the corresponding pharmacokinetic parameters. A size exclusion-high pressure liquid chromatography (SE-HPLC) method was used to quantitate rhThrombin complexes, non-complexed rhThrombin, and free [(125)I]. Whole-body gamma scintigraphy was used to follow radioactivity localization up to 72 h postdose. RESULTS: No adverse events were observed following [(125)I]-rhThrombin administration. The pharmacokinetic profile of rhThrombin-associated radioactivity following i.v. injection was multi-exponential with an initial half-life of approximately 10 min. Following both i.v. and s.c. dosing, the terminal half-life was approximately 15 h. SE-HPLC analysis revealed that rhThrombin was rapidly complexed to antithrombin III and other inhibitors in the systemic circulation following i.v. administration. Thus, rhThrombin-associated radioactivity in the blood was complexed and presumed inactive. [(125)I]-rhThrombin inhibitor complexes accumulated and were eliminated in the liver following both routes of administration. CONCLUSIONS: These data suggest that rhThrombin rapidly binds to endogenous inhibitors following either i.v. or s.c. administration.

Isolation and expression of an isoform of human CDP-diacylglycerol synthase cDNA.

Phosphatidic acid (PA) is a phospholipid involved in signal transduction and in glycerolipid biosynthesis. CDP-diacylglycerol synthase (CDS) or CTP:phosphatidate cytidylyltransferase (EC 2.7.7.41) catalyzes the conversion of PA to CDP-diacylglycerol (CDP-DAG), an important precursor for the synthesis of phosphatidylinositol, phosphatidylglycerol, and cardiolipin. We describe in this study the isolation and characterization of a human cDNA clone that encodes amino acid sequences homologous to Escherichia coli, yeast, and Drosophila CDS sequences. Expression of this human cDNA under the control of a GAL1 promoter in a null cds1 mutant yeast strain complements its growth defect and produces CDS activity when induced with galactose. Transfection of this cDNA into mammalian cells leads to increased CDS activity in cell-free extracts using an in vitro assay that measures the conversion of [alpha-32P]CTP to [32P]CDP-DAG. This increase in CDS activity also leads to increased secretion of tumor necrosis factor-alpha and interleukin-6 from endothelial ECV304 cells upon stimulation with interleukin-1beta, suggesting that CDS overexpression may amplify cellular signaling responses from cytokines.

Cloning and expression of two human lysophosphatidic acid acyltransferase cDNAs that enhance cytokine-induced signaling responses in cells.

Lysophosphatidic acid (LPA) and phosphatidic acid (PA) are two phospholipids involved in signal transduction and in lipid biosynthesis in cells. LPA acyltransferase (LPAAT), also known as 1-acyl sn-glycerol-3-phosphate acetyltransferase (EC 2.3.1.51), catalyzes the conversion of LPA to PA. In this study, we describe the isolation and characterization of two human cDNAs that encode proteins possessing LPAAT activities. These two proteins, designated here as LPAAT-alpha and LPAAT-beta, contain extensive sequence sequence similarities to microbial or plant LPAAT sequences. LPAAT-alpha mRNA was detected in all tissues with highest expression in skeletal muscle whereas LPAAT-beta was expressed predominantly in heart and liver tissues. Expression of these two cDNAs in an Escherichia coli strain with a mutated LPAAT gene (plsC) complements its growth defect and shifts the equilibrium of cellular lipid content from LPA to PA and other lipids. Overexpression of these two cDNAs in mammalian cells leads to increased LPAAT activity in cell-free extracts using an in vitro assay that measures the conversion of fluorescently labeled LPA to PA. This increase in LPAAT activity correlates with enhancement of transcription and synthesis of tumor necrosis factor-alpha and interleukin-6 from cells upon stimulation with interleukin-1beta, suggesting LPAAT overexpression may amplify cellular signaling responses from cytokines.

Lisofylline causes rapid and prolonged suppression of serum levels of free fatty acids.

Lisofylline (LSF), a novel anti-inflammatory compound that modulates stress-associated changes in lipid metabolism, is under development to modify toxicity for patients undergoing dose-intensive cytotoxic therapy for neoplasia and to prevent multiorgan failure and acute respiratory distress syndrome after oxidative injury. The present investigation, a component of a pharmacokinetics study, was performed to assess the effect of LSF on serum-free fatty acids (FFA). LSF was administered at doses of either 1, 2 or 3 mg/kg every 24 hr for 3 days by 10 min intravenous infusion to 12 healthy volunteers, followed 24 hr later by a single oral dose of 6 mg/kg, which was determined not to be bioavailable. Total serum FFA were quantitated after separation from other lipids by thin-layer chromatography in samples from 10 of 12 subjects, and serum levels of individual fatty acids were measured by high-performance liquid chromatography in samples from 11 of 12 subjects. Six hours after the first LSF dose of 1, 2 or 3 mg/kg, FFA levels decreased from the time zero levels by a mean (+/- S.D.) of 64.7 +/- 7.4% (range, 37-80%; P < .001 vs. time zero levels). Six hours after the third i.v. LSF dose, the FFA reached a nadir of 71.5 +/- 5.5% below the time zero levels (range, 55-88%; P < .001 vs. time zero). Equivalent effects were observed after the first LSF dose regardless of whether patients received LSF at 1, 2 or 3 mg/kg. The decrease in serum FFA was still present 48 hr after the final i.v. dose and 24 hr after the oral dose, with a mean decrease of 34 +/- 9.8% (P < .01 vs. time zero). Serum triglycerides began to increase after the first i.v. LSF dose and were at the highest measured level 6 hr after the third dose, increasing by 74.5 +/- 19.7% from the time zero levels (range, 36-146%; P = .02 vs. time zero). The increase in serum triglycerides also persisted for 36 hr after the final i.v. LSF dose. LSF and its two principal metabolites had plasma clearance t 1/2 values of 0.75 hr, 0.78 hr and 1.17 hr, respectively. Therefore the effects of LSF on lipid metabolism were present for a prolonged period compared with measurable persistence in plasma; this points to unique functions or unknown metabolites of LSF. These alterations in serum lipids may be relevant to the anti-inflammatory activity of LSF and may serve as surrogate markers for the pharmacodynamics of LSF.