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Stearoyl‐coenzyme A desaturase 1 (SCD‐1) in sebaceous glands is a key enzyme in the synthesis of monounsaturated fatty acids essential for acne development. GSK1940029 gel, a novel SCD‐1 inhibitor, is being developed as a potential treatment for acne. To assess the irritation potential, pharmacokinetics (PK), and safety of topical GSK1940029 to the skin of healthy adults, two interdependent studies were conducted in parallel. Study 1 (n = 54) investigated the irritation potential of GSK1940029 (0.3% and 1%, occluded application) to allow for its application to larger surface areas in study 2 (n = 39), which investigated the safety, tolerability, and PK of GSK1940029 after single and repeat doses as occluded and nonoccluded applications. GSK1940029 was not a primary or cumulative irritant after 2 and 21 days of dosing in study 1. In study 2, single and repeat applications of GSK1940029 (0.1% to 1%) doses were well tolerated with little or no influence on AUC and Cmax under occluded or unoccluded conditions. Systemic exposure increased proportionally with surface area and was higher in occluded conditions. Design of these interdependent studies allowed for the assessment of the irritation potential for topical GSK1940029 in parallel with the investigation of PK and safety profiles.

Diacylglycerol acyltransferase (DGAT) enzymes are involved in triglyceride (TG) biosynthesis. GSK3008356 is a potent and selective DGAT1 inhibitor that was administered orally in a 2-part study as double-blind, randomized, placebo-controlled single doses (SDs) and repeat doses (RDs) in healthy subjects to investigate its pharmacokinetics, pharmacodynamics, and safety/tolerability. Gastrointestinal adverse events were considered drug related and increased with dose and when given as multiple doses. In the SD part (n = 80), GSK3008356 was dosed from 5 to 200 mg as single or multiple doses per day. In the RD part (n = 24), GSK3008356 was dosed twice daily at 1, 3, and 10 mg for 14 days. GSK3008356 was generally well tolerated in the SD and RD parts. With single doses, absorption was rapid (median tmax , 0.5-1.5 hours), whereas single-day divided dosing resulted in higher tmax . Following 14-day RD oral administration, GSK3008356 was also rapidly absorbed, with median tmax ranging from 0.5 to 0.75 hours on days 1 and 14. Estimated mean half-life ranged from 1.5 to 4.6 hours with SDs and 1.3 to 2.1 hours with RDs. Exposure of GSK3008356 was largely dose proportional after RDs. At higher doses, there was a trend toward lower absolute postprandial TG level in some subjects.

Non-alcoholic steatohepatitis (NASH) is a liver disease in which fatty infiltration is accompanied by liver inflammation. GSK3008356 is under development as a selective inhibitor of diacylglycerol acyltransferase 1 (DGAT1), a key enzyme involved in the formation of triglyceride (TG). Decreased DGAT1 activity can reduce circulating TG and liver TG, and therefore could potentially prevent or treat NASH. The aim of the current study was to develop a population pharmacokinetic-pharmacodynamic (PKPD) model that characterizes the PK disposition of GSK3008356 and its relation to the changes in blood TG. Drug concentrations were measured in 104 healthy adults receiving various single (SD) and repeat doses (RD) in a first time in human (FiH) study. A 30% fat meal was given at hour 2 postdose, and blood postprandial TG concentrations were measured at various time points. The population PKPD model consists of several parts including a PK model, drug effect model, meal effect model, and a turnover model. The pharmacokinetic data were described using a 3-compartment model. Drug effect was described by an inhibitory sigmoidal Emax model. Since TG levels change with the introduction of a meal, a bi-exponential meal effect model was utilized. The total change in TG was fitted using a turnover model with drug and meal effects on the TG production rate. The current analysis presents a PKPD modeling strategy of time-varying TG data coming from both endogenous and exogenous sources. In general, the presented model could be utilized in the model-based drug development of drugs that influence TG levels in blood.

On the basis of a mechanistic understanding of the toxicity of the 4-aminoquinoline amodiaquine (1b), three series of amodiaquine analogues have been prepared where the 4-aminophenol "metabolic alert" has been modified by replacement of the 4'-hydroxy group with a hydrogen, fluorine, or chlorine atom. Following antimalarial assessment and studies on mechanism of action, two candidates were selected for detailed ADME studies and in vitro and in vivo toxicological assessment. 4'-Fluoro-N-tert-butylamodiaquine (2k) was subsequently identified as a candidate for further development studies based on potent activity versus chloroquine-sensitive and resistant parasites, moderate to excellent oral bioavailability, low toxicity in in vitro studies, and an acceptable safety profile.

N-tert-Butyl isoquine (4) (GSK369796) is a 4-aminoquinoline drug candidate selected and developed as part of a public-private partnership between academics at Liverpool, MMV, and GSK pharmaceuticals. This molecule was rationally designed based on chemical, toxicological, pharmacokinetic, and pharmacodynamic considerations and was selected based on excellent activity against Plasmodium falciparum in vitro and rodent malaria parasites in vivo. The optimized chemistry delivered this novel synthetic quinoline in a two-step procedure from cheap and readily available starting materials. The molecule has a full industry standard preclinical development program allowing first into humans to proceed. Employing chloroquine (1) and amodiaquine (2) as comparator molecules in the preclinical plan, the first preclinical dossier of pharmacokinetic, toxicity, and safety pharmacology has also been established for the 4-aminoquinoline antimalarial class. These studies have revealed preclinical liabilities that have never translated into the human experience. This has resulted in the availability of critical information to other drug development teams interested in developing antimalarials within this class.

Biopharmaceuticals that target specific disease-mediating molecules have advanced our understanding of the pathogenesis of psoriasis. The traditional paradigm that psoriasis is primarily a disease of epidermal cells has been replaced with a model that now includes keratinocyte-derived factors, inflammatory mediators and angiogenic mechanisms. Recent studies have highlighted some of the key molecules involved in all of these pathogenic processes. Several have already been evaluated as putative targets in in vitro and in vivo studies, whereas other molecules are significantly upregulated in psoriasis and require further study to elucidate their role and contribution to disease. Although not all these molecules will eventually qualify as drug targets, data from similar experimental strategies are predicted to underpin the next generation of candidate targets and novel therapeutic approaches.

Background Anemia associated with chronic kidney disease (CKD) often requires treatment with recombinant human erythropoietin (EPO). Hypoxia-inducible factor−prolyl hydroxylase inhibitors (PHIs) stimulate endogenous EPO synthesis and induce effective erythropoiesis by non-EPO effects. GSK1278863 is an orally administered small-molecule PHI. Study Design Multicenter, single-blind, randomized, placebo-controlled, parallel-group study. Setting & Participants Anemic non–dialysis-dependent patients with CKD stages 3-5 (CKD-3/4/5 group; n=70) and anemic hemodialysis patients with CKD stage 5D (CKD-5D group; n=37). Interventions Patients with CKD-3/4/5 received placebo or GSK1278863 (10, 25, 50, or 100mg), and patients with CKD-5D received placebo or GSK1278863 (10 or 25mg) once daily for 28 days. Outcomes & Measurements Primary pharmacokinetic and pharmacodynamic (increase and response rates in achieving the target hemoglobin [Hb] concentration, plasma EPO concentrations, reticulocyte count, and others]) and safety and tolerability end points were obtained. Results Both CKD-3/4/5 and CKD-5D populations showed a dose-dependent increase in EPO concentrations and consequent increases in reticulocytes and Hb levels. Percentages of GSK1278863 participants with an Hb level increase > 1.0g/dL (CKD-3/4/5) and >0.5g/dL (CKD-5D) were 63% to 91% and 71% to 89%, respectively. Per-protocol–defined criteria, high rate of increase in Hb level, or high absolute Hb value was the main cause for withdrawal (CKD-3/4/5, 30%; CKD-5D, 22%). A dose-dependent decrease in hepcidin levels and increase in total and unsaturated iron binding were observed in all GSK1278863-treated patients. Limitations Sparse pharmacokinetic sampling may have limited covariate characterization. EPO concentrations at the last pharmacodynamic sample (5-6 hours) postdose may not represent peak concentrations, which occurred 8 to 10 hours postdose in previous studies. Patients were not stratified by diabetes status, potentially confounding vascular endothelial growth factor and glucose analyses. Conclusions GSK1278863 induced an effective EPO response and stimulated non-EPO mechanisms for erythropoiesis in anemic non–dialysis-dependent and dialysis-dependent patients with CKD.

The PI3K/AKT pathway is constitutively active in hematologic malignancies, providing proliferative and antiapoptotic signals and possibly contributing to drug resistance. We conducted an open-label phase 1 study to evaluate the maximum tolerated dose (MTD), safety, pharmacokinetics, and clinical activity of afuresertib-an oral AKT inhibitor-in patients with advanced hematologic malignancies. Seventy-three patients were treated at doses ranging from 25 to 150 mg per day. The MTD was established at 125 mg per day because of 2 dose-limiting toxicities in the 150-mg cohort (liver function test abnormalities). The most frequent adverse events were nausea (35.6%), diarrhea (32.9%), and dyspepsia (24.7%). Maximum plasma concentrations and area under the plasma concentration-time curves from time 0 to 24 hours were generally dose proportional at > 75-mg doses; the median time to peak plasma concentrations was 1.5 to 2.5 hours post dose, with a half-life of approximately 1.7 days. Three multiple myeloma patients attained partial responses; an additional 3 attained minimal responses. Clinical activity was also observed in non-Hodgkin lymphoma, Langerhan's cell histiocytosis, and Hodgkin disease. Single-agent afuresertib showed a favorable safety profile and demonstrated clinical activity against hematologic malignancies, including multiple myeloma.

Mice immunized against Strongyloides stercoralis L3 were shown to kill greater than 90% of challenge larvae contained within diffusion chambers. The objective of the present study was to identify the host components responsible for immunity. Serum from unprotected, control mice and protected, immune mice in doses of 25-500 microliters was transferred into naive mice at the same time and location as larval challenge. Transfer of as little as 50 microliters of immune serum was able to confer protective immunity. The serum-transferred immunity was ablated by excluding cells from the larval microenvironment or by depleting granulocytes through monoclonal antibody treatment in the recipient mice. Specific antibody isotypes were isolated using protein G and isotype-specific affinity columns. The resulting transfer experiments identified IgM as the isotype responsible for protective immunity to S. stercoralis L3. Antibody binding studies in vivo were performed and only IgM bound to the surface of infective L3 and host-derived L3 (L3+) in immune animals. Elevated levels of C3 were also found bound to the surface of L3/L3+ in immune mice. Cobra venom factor treatment of immunized mice to deplete complement completely eliminated C3 binding to the surface of L3/L3+ and ablated immunity. Therefore, IgM, complement, and granulocytes are necessary for immune elimination of S. stercoralis L3/L3+. Identification of antigens recognized by IgM may help select possible vaccine candidates.

Immunization of mice with irradiated Onchocerca volvulus infective stage larvae (L3) has been demonstrated to confer protection against challenge infections with these larvae. Additionally, cytokine level measurements and cytokine depletion studies have shown that both IL-4 and IL-5 are important in generating a protective immune response against O. volvulus challenge infections, thus suggesting a dependency of protective immunity on IgG1, IgE and/or eosinophils. In the present study, we examined the humoral responses of immunized mice to O. volvulus L3 antigens. ELISA measurements of total serum antibody levels indicated that IgE was the only antibody isotype elevated in mice immunized with O. volvulus L3. IgM from immunized mice was the only isotype that recognized surface antigens on intact O. volvulus L3. IgG1, IgG3, IgE and IgA recognized internal parasite antigens on O. volvulus L3 frozen sections. Western blot analysis of L3 proteins showed that in serum from mice immunized with O. volvulus L3 IgG1, IgG2a/2b, IgA, and IgE, as well as IgM, recognized unique L3 proteins. Antibodies in serum from L3 immunized mice were able to detect O. volvulus adult antigens in a pattern similar to the recognition found in O. volvulus L3. Some L3 antigens were shared by adults, while other antigens were L3 specific. The ELISA, immunohistochemistry and Western blot findings thus demonstrate a complex pattern of antigen recognition of parasite antigens by antibodies found in mice immune to the L3 of O. volvulus.

Challenge worm survival was significantly reduced when BALB/cByJ mice were vaccinated againstStrongyloides stercoralisinfective third stage larvae (L3) regardless of whether the challenge infections consisted of systemically migrating L3 or L3 implanted in diffusion chambers. The only cell type that increased in number in diffusion chambers in immunized mice, 1 week after booster immunizations, was the eosinophil, and maximal levels of eosinophils were coincident with parasite killing. Mice were treated with mAb to eliminate IL-5 or granulocytes to assess the role that eosinophils play in larval killing. Treated animals showed no decrease in immunity when challenge infections consisted of systemically migrating L3 administered 3 weeks after booster immunizations. Eosinophil numbers in immunized mice decreased to control levels when measured 3 weeks post-booster immunization, both in diffusion chambers and in the peripheral blood, whereas they were elevated at 1 week after booster immunizations. Direct contact between host cells and L3 was, however, still required for larval killing in immunized hosts 3 weeks after booster immunizations. Elimination of eosinophils by treatment with mAb to IL-5 or granulocytes significantly reduced protective immunity, when L3 were implanted in diffusion chambers at 1 and 3 weeks post-booster. However, as systemically migrating L3 were still killed in immunized, eosinophil-depleted animals, other cell types may play a role in larval destruction. Two human eosinophil granule products were found to be toxic for host-adapted L3+, but had no effect on infective L3, indicating that host-adapted larvae are possible targets for eosinophil-mediated destruction of third stage larvae. These findings suggest that inactivation of eosinophils by mAb treatment abolishes protective immunity to L3 contained within diffusion chambers and that small numbers of eosinophils are sufficient for immune-mediated killing ofS. stercoralisL3.

A murine model system was developed to study the induction and mechanism of protective immunity to L3 of Strongyloides stercoralis. L3 were implanted in BALB/cByJ mice in diffusion chambers constructed with 0.1- or 2.0-microns-pore-size membranes. Parasites survived equally well regardless or membrane type for 7 days, after which larval survival decreased in diffusion chambers constructed with 2.0-microns-pore-size membranes, which allowed host cells to enter. Survival of S. stercoralis L3 in diffusion chambers implanted in mice was assayed after immunization with live, heat-killed, and homogenized L3. Optimal immunization was achieved with 10,000 live L3, whereby immunized mice eliminated 97% of the larvae either contained within diffusion chambers or free within the tissues of the mouse by 24 hr postinfection. Sera from immunized mice had elevated levels of IgG1, IgM, and IgA parasitic-specific antibody; IgM was the only antibody isotype that recognized surface antigens of L3. Larvae were not killed in immunized mice if contact between host cells and the parasites was prevented. In the peripheral blood and diffusion chamber fluid of immunized mice, eosinophil levels were significantly higher when compared to the levels found in control mice. The rodent model developed in the present study has thus demonstrated that virtually complete immunity can be induced to the L3 of S. stercoralis and that larval killing was found to be associated with the presence of both specific antibody and eosinophils.

Abstract 1856 Background: Despite significant progress in treatment, multiple myeloma (MM) remains an incurable disease. There is a well recognized need for new agents with novel mechanisms of action, which would complement currently available drugs. The PI3K/AKT pathway is constitutively active in MM, providing proliferative and anti-apoptotic signals and possibly contributing to resistance to treatment. Therefore, AKT is a potential pharmacologic target in MM. GSK2110183 is a potent, orally available, ATP competitive inhibitor of all three isoforms of AKT. Methods: We conducted a phase I study to define the maximum tolerated dose (MTD) and to evaluate the PK, PD and clinical activity in patients with advanced hematologic malignancies. This two-part study consisted of dose escalation (Part1) followed by expansion in selected malignancies (Part2) to evaluate safety and clinical activity at the MTD. GSK2110183 was administered continuously once daily until unacceptable toxicity or disease progression. Results: Preliminary data are available for 73 patients who have received >1 dose of GSK2110183. Patients had the following malignancies: Non-Hodgkin's Lymphoma (NHL, 14), Hodgkin lymphoma (HL, 8), chronic lymphocytic leukemia (CLL 7), MM (34), acute leukemia (10). In Part1 GSK2110183 was administered at daily doses of 25mg, 75mg, 100mg, 125mg and 150mg. Dose limiting toxicities (DLTs) were observed in 3 of 6 patients at 150mg, thereby defining the MTD as 125mg daily. DLTs at 150mg were: short term memory loss (n=1) and elevation of AST/ALT accompanied by AP and bilirubin elevations (n=2). One of the two patients with liver toxicities also had serum lipase and amylase elevations. DLTs were reversible upon drug discontinuation in two patients. The third patient had diffuse large B cell lymphoma with hepatic involvement, which led to persistent liver enzyme elevations. The most common observed drug-related adverse events for all patients (>10%) were: nausea (20%), diarrhea (16%), dyspepsia (15%), fatigue (15%), anorexia (12%) and gastrointestinal reflux disease (11%). Observed grade 3 drug related hematologic toxicities were: neutropenia (n=4), and thrombocytopenia (n=1). No grade 4 hematologic toxicities were observed. In general, therapy was well-tolerated, with subjects continuing on therapy for up to 21 months. Mean AUC(0–24) and Cmax values increased with increasing doses; however, there was variability among subjects. Median Tmax across doses was 2 hrs, and the mean t 1/2 was approximately 1.7 days. GSK2110183 accumulated 1.4 to 5.1-fold with repeat daily dosing. Median (range) duration of treatment for different types of malignancies was as follows: NHL 88.5 days (12–597), HL 266 days (38–478), CLL 64 days (1–546), MM 87 days (8–323) and acute leukemia 37days (22–85). Subjects with MM were the main focus of the expansion cohort to evaluate for preliminary signals of efficacy. A total of 32 heavily pretreated, relapsed/refractory MM patients were treated at the 125mg dose, achieving an overall response rate of 19% (3 PR, 3 MR). All but one responder had prior treatment with proteasome inhibitor (PI), immunomodulatory agents (IMID) and alkylating agents. Responders were as equally heavily pretreated as the non-responders, with a median of 5 prior lines of treatment (range 2–8). Most significant reductions in M protein occurred very early in the treatment course (4/6 within 21 days). The respective mean (range) duration on study treatment for those who achieved MR or PR was 216 days (105–315), for subjects achieving stable disease 117 days (84–147), and for subjects with progressive disease 52 days (15–105). Conclusions: The AKT inhibitor GSK2110183 is well tolerated and demonstrates clinical activity as monotherapy in heavily pretreated MM patients. Further work is ongoing to identify the patients who are most likely to respond to AKT inhibition, as well as to define the most rational combination of the AKT inhibitor with other agents in order to maximize the clinical benefit for MM patients. Disclosures: Spencer: GSK: Honoraria, Research Funding. Yoon:GSK: Honoraria. Harrison:GSK: Honoraria, Research Funding. Morris:GSK: Employment. Smith:GSK: Employment. Freedman:GSK: Employment. Brigandi:GSK: Employment. Oliff:GSK: Employment. Opalinska:GlaxoSmithKline: Employment. Chen:GSK: Research Funding.

We have prepared a strain of Escherichia coli that expresses both the HIV protease and a Tet protein which has been modified to contain the HIV protease recognition sequence. When the protease is expressed, the bacteria will not grow in the presence of tetracycline. However, when the protease is inhibited the bacteria can grow in tetracycline containing media (Block and Grafstrom 1990). We have selected spontaneously arising Tet resistant mutants and have screened them for those that could be producing an inhibitor of HIV protease. The problems in the construction of this strain and the characterization of the various Teti mutants are discussed.

Host-adapted, transformed, Strongyloides stercoralis third-stage larvae (L3+) were previously found to be antigenically different from free-living, infective, third-stage larvae (L3). These antigenic differences were reproduced by transformation of free-living larvae in tissue culture medium at 37 C over 24 hr. Transformed L3 of both derivations were given as challenge infections in diffusion chambers to naive mice and mice immunized with S. stercoralis L3. Within 12 hr, the challenge infections were killed regardless of whether the L3+ were generated in vitro or in vivo. Eosinophils, previously found to be important in the immune response to S. stercoralis larvae, were recruited into the L3+ microenvironment within 12 hr of challenge infection in immune mice, which supports the previously proposed mechanisms of S. stercoralis larval killing. Thus, S. stercoralis L3+ appear to be targets of the immune response in mice instead of being involved in immune evasion.