Cellular Uptake and Transport Characteristics of FL118 Derivatives in Caco-2 Cell Monolayers.

In the evaluation of the druggability of candidate compounds, it was vital to predict the oral bioavailability of compounds from apparent permeability (Papp) across Caco-2 cell-culture model of intestinal epithelium cultured on commercial transwell plate inserts. The study was to investigate the transport characteristics and permeability of FL118 (10, 11-Methylenedioxy-20(S)-camptothecin) derivatives 7-Q6 (7-(4-Ethylphenyl)-10, 11-methylenedioxy-20(S)-camptothecin) and 7-Q20 (7-(4-Trifluoromethylphenyl)-10, 11-methylenedioxy-20(S)-camptothecin). Transport characteristics and permeability of the tested compounds to the small intestine were assessed at different concentrations (0.5, 1 µM) via Caco-2 cell monolayers model in vitro. Uptake studies based on Caco-2 cells, including temperatures, concentrations, and the influence of efflux transporters, were combined to confirm the transport characteristics of the tested compounds. Furthermore, cytotoxicity results showed that the concentrations used in the experiments were non-toxic and harmless to cells. In addition, The Papp of 7-Q6 was (3.69 ± 1.07) × 10-6 cm/s with efflux ratio (ER) 0.98, while the Papp of 7-Q20 was (7.78 ± 0.89) × 10-6 cm/s with ER 1.05 for apical-to-basolateral (AP→BL) at 0.5 µM, suggesting that 7-Q20 might possess higher oral bioavailability in vivo. Furthermore, P-glycoprotein (P-gp) was proved to slightly affect the accumulations of 7-Q20, while the absorption of 7-Q6 was irrelevant with P-gp and breast cancer resistant protein (BCRP) based on the cellular uptake assays. Accordingly, 7-Q6 was completely absorbed by passive diffusion, and 7-Q20 was mainly dependent on passive diffusion with being effluxed by P-gp slightly. Meanwhile, both 7-Q6 and 7-Q20 were potential antitumor drugs that might exhibit high oral bioavailability in the body.

Targeting transcription of MCL-1 sensitizes HER2-amplified breast cancers to HER2 inhibitors.

Human epidermal growth factor receptor 2 gene (HER2) is focally amplified in approximately 20% of breast cancers. HER2 inhibitors alone are not effective, and sensitizing agents will be necessary to move away from a reliance on heavily toxic chemotherapeutics. We recently demonstrated that the efficacy of HER2 inhibitors is mitigated by uniformly low levels of the myeloid cell leukemia 1 (MCL-1) endogenous inhibitor, NOXA. Emerging clinical data have demonstrated that clinically advanced cyclin-dependent kinase (CDK) inhibitors are effective MCL-1 inhibitors in patients, and, importantly, well tolerated. We, therefore, tested whether the CDK inhibitor, dinaciclib, could block MCL-1 in preclinical HER2-amplified breast cancer models and therefore sensitize these cancers to dual HER2/EGFR inhibitors neratinib and lapatinib, as well as to the novel selective HER2 inhibitor tucatinib. Indeed, we found dinaciclib suppresses MCL-1 RNA and is highly effective at sensitizing HER2 inhibitors both in vitro and in vivo. This combination was tolerable in vivo. Mechanistically, liberating the effector BCL-2 protein, BAK, from MCL-1 results in robust apoptosis. Thus, clinically advanced CDK inhibitors may effectively combine with HER2 inhibitors and present a chemotherapy-free therapeutic strategy in HER2-amplified breast cancer, which can be tested immediately in the clinic.

A Phase I Study of Dinaciclib in Combination With MK-2206 in Patients With Advanced Pancreatic Cancer.

The combination of drugs targeting Ral and PI3K/AKT signaling has antitumor efficacy in preclinical models of pancreatic cancer. We combined dinaciclib (small molecule cyclin dependent kinase inhibitor with MK-2206 (Akt inhibitor) in patients with previously treated/metastatic pancreatic cancer. Patients were treated with dinaciclib (6-12 mg/m2 i.v.) and MK-2206 (60-135 mg p.o.) weekly. Tumor biopsies were performed to measure pAKT, pERK, and Ki67 at baseline and after one completed cycle (dose level 2 and beyond). Thirty-nine patients participated in the study. The maximum tolerated doses were dinaciclib 9 mg/m2 and MK-2206 135 mg. Treatment-related grade 3 and 4 toxicities included neutropenia, lymphopenia, anemia, hyperglycemia, hyponatremia, and leukopenia. No objectives responses were observed. Four patients (10%) had stable disease as their best response. At the recommended dose, median survival was 2.2 months. Survival rates at 6 and 12 months were 11% and 5%, respectively. There was a nonsignificant reduction in pAKT composite scores between pretreatment and post-treatment biopsies (mean 0.76 vs. 0.63; P = 0.635). The combination of dinaciclib and MK-2206 was a safe regimen in patients with metastatic pancreatic cancer, although without clinical benefit, possibly due to not attaining biologically effective doses. Given the strong preclinical evidence of Ral and AKT inhibition, further studies with better tolerated agents should be considered.

Tylophorine reduces protein biosynthesis and rapidly decreases cyclin D1, inhibiting vascular smooth muscle cell proliferation in vitro and in organ culture.

BACKGROUND: Tylophorine (TYL) is an alkaloid with antiproliferative action in cancer cells. Vascular smooth muscle cell (VSMC) proliferation and neointima formation contribute to restenosis after percutaneous coronary interventions. HYPOTHESIS/PURPOSE: Our goal was to examine the potential of TYL to inhibit VSMC proliferation and migration, and to dissect underlying signaling pathways. STUDY DESIGN AND METHODS: TYL was administered to platelet-derived growth factor (PDGF-BB)-stimulated, serum-stimulated, quiescent and unsynchronized VSMC of rat and human origin. BrdU incorporation and resazurin conversion were used to assess cell proliferation. Cell cycle progression was analyzed by flow cytometry of propidium iodide-stained nuclei. Expression profiles of proteins and mRNAs were determined using western blot analysis and RT-qPCR. The Click-iT OPP Alexa Fluor 488 assay was used to monitor protein biosynthesis. RESULTS: TYL inhibited PDGF-BB-induced proliferation of rat aortic VSMCs by arresting cells in G1 phase of the cell cycle with an IC50 of 0.13 µmol/l. The lack of retinoblastoma protein phosphorylation and cyclin D1 downregulation corroborated a G1 arrest. Inhibition of proliferation and cyclin D1 downregulation were species- and stimulus-independent. TYL also decreased levels of p21 and p27 proteins, although at later time points than observed for cyclin D1. Co-treatment of VSMC with TYL and MG132 or cycloheximide (CHX) excluded proteasome activation by TYL as the mechanism of action. Comparable time-dependent downregulation of cyclin D1, p21 and p27 in TYL- or CHX-treated cells, together with decreased protein synthesis observed in the Click-iT assay, suggests that TYL is a protein synthesis inhibitor. Besides proliferation, TYL also suppressed migration of PDGF-activated VSMC. In a human saphenous vein organ culture model for graft disease, TYL potently inhibited intimal hyperplasia. CONCLUSION: This unique activity profile renders TYL an interesting lead for the treatment of vasculo-proliferative disorders, such as restenosis.

Novel indolizine derivatives lowers blood glucose levels in streptozotocin-induced diabetic rats: A histopathological approach.

BACKGROUND: Diabetes mellitus is a deadly disorder in human which induce chronic complications. The streptozotocin (STZ)-induced diabetes in rat is the most common animal model of human diabetes. The present study investigated the effects of novel indolizine derivatives (1-16) on plasma blood glucose concentrations in STZ-diabetic rats. METHODS: In vitro experiments were performed on 1,1-diphenyl-2-picrylhydrazyl (DPPH), superoxide free radicals, α-glucosidase enzyme and in vivo studies on normal, oral glucose loaded and STZ-induced diabetic rats. RESULTS: Among all synthetic derivatives, compound 12 showed good inhibitory profile against DPPH, superoxide free radicals and α-glucosidase enzyme with half maximal inhibitory concentration (IC50) values of 56.2, 33.5 and 26.5 µg/mL, respectively. The lethal dosage of indolizine derivatives was found to be above 1000 mg/kg body weight (b.w.). From the in vivo studies, it can be determined that the compound 12 depicted pronounced protective hypoglycemic effects in normal, glucose-loaded and STZ-induced diabetic rats with respect to the standard. Furthermore, 21 days of successive treatment with compound 12 in diabetic rats exhibited better recovery of body weight and considerable variations in biochemical parameters as that of the standard drug. Moreover, the histopathological section of pancreas and testes justifies the rehabilitation and regeneration of islets, acini and Sertoli cells in animals treated with compound 12. CONCLUSION: Our data suggest that the indolizine derivatives can be a benchmarks for designing potent oral antidiabetic agents.

Mitotic kinase PBK/TOPK as a therapeutic target for adult T­cell leukemia/lymphoma.

Adult T­cell leukemia/lymphoma (ATLL) is a disorder involving human T-cell leukemia virus type 1 (HTLV­1)-infected T­cells characterized by increased clonal neoplastic proliferation. PDZ-binding kinase (PBK) [also known as T­lymphokine-activated killer cell-originated protein kinase (TOPK)] is a serine/threonine kinase expressed in proliferative cells and is phosphorylated during mitosis. In this study, the expression and phosphorylation of PBK/TOPK were examined by western blot analysis and RT­PCR. We found that PBK/TOPK was upregulated and phosphorylated in HTLV­1-transformed T­cell lines and ATLL­derived T­cell lines. Notably, CDK1/cyclin B1, which phosphorylates PBK/TOPK, was overexpressed in these cells. HTLV­1 infection upregulated PBK/TOPK expression in peripheral blood mononuclear cells (PBMCs) in co-culture assays. The potent PBK/TOPK inhibitors, HI­TOPK­032, and fucoidan from brown algae, decreased the proliferation and viability of these cell lines in a dose­dependent manner. By contrast, the effect of HI­TOPK­032 on PBMCs was less pronounced. Treatment with HI­TOPK­032 resulted in G1 cell cycle arrest, and decreased CDK6 expression and pRb phosphorylation, which are critical determinants of progression through the G1 phase. In addition, HI­TOPK­032 induced apoptosis, as evidenced by morphological changes, the cleavage of poly(ADP-ribose) polymerase with the activation of caspase­3, -8 and -9, and an increase in the sub­G1 cell population and APO2.7-positive cells. Moreover, HI­TOPK­032 inhibited the expression of cellular inhibitor of apoptosis 2 (c-IAP2), X-linked inhibitor of apoptosis protein (XIAP), survivin and myeloid cell leukemia­1 (Mcl­1), and induced the expression of Bak and interferon-induced protein with tetratricopeptide repeats (IFIT)1, 2 and 3. It is noteworthy that the use of this inhibitor led to the inhibition of the phosphorylation of IκB kinase (IKK)α, IKKß, IκBα, phosphatase and tensin homolog (PTEN) and Akt, and to the decreased protein expression of JunB and JunD, suggesting that PBK/TOPK affects the nuclear factor-κB, Akt and activator protein­1 signaling pathways. In vivo, the administration of HI­TOPK­032 suppressed tumor growth in an ATLL xenograft model. Thus, on the whole, this study on the identification and functional analysis of PBK/TOPK suggests that this kinase is a promising molecular target for ATLL treatment.

Extended Evaluation of Virological, Immunological and Pharmacokinetic Endpoints of CELADEN: A Randomized, Placebo-Controlled Trial of Celgosivir in Dengue Fever Patients.

UNLABELLED: CELADEN was a randomized placebo-controlled trial of 50 patients with confirmed dengue fever to evaluate the efficacy and safety of celgosivir (A study registered at ClinicalTrials.gov, number NCT01619969). Celgosivir was given as a 400 mg loading dose and 200 mg bid (twice a day) over 5 days. Replication competent virus was measured by plaque assay and compared to reverse transcription quantitative PCR (qPCR) of viral RNA. Pharmacokinetics (PK) correlations with viremia, immunological profiling, next generation sequence (NGS) analysis and hematological data were evaluated as exploratory endpoints here to identify possible signals of pharmacological activity. Viremia by plaque assay strongly correlated with qPCR during the first four days. Immunological profiling demonstrated a qualitative shift in T helper cell profile during the course of infection. NGS analysis did not reveal any prominent signature that could be associated with drug treatment; however the phylogenetic spread of patients' isolates underlines the importance of strain variability that may potentially confound interpretation of dengue drug trials conducted during different outbreaks and in different countries. Celgosivir rapidly converted to castanospermine (Cast) with mean peak and trough concentrations of 5727 ng/mL (30.2 µM) and 430 ng/mL (2.3 µM), respectively and cleared with a half-life of 2.5 (± 0.6) hr. Mean viral log reduction between day 2 and 4 (VLR2-4) was significantly greater in secondary dengue than primary dengue (p = 0.002). VLR2-4 did not correlate with drug AUC but showed a trend of greater response with increasing Cmin. PK modeling identified dosing regimens predicted to achieve 2.4 to 4.5 times higher Cmin. than in the CELADEN trial for only 13% to 33% increase in overall dose. A small, non-statistical trend towards better outcome on platelet nadir and difference between maximum and minimum hematocrit was observed in celgosivir-treated patients with secondary dengue infection. Optimization of the dosing regimen and patient stratification may enhance the ability of a clinical trial to demonstrate celgosivir activity in treating dengue fever based on hematological endpoints. A new clinical trial with a revised dosing regimen is slated to start in 2016 (NCT02569827). Furthermore celgosivir's potential value for treatment of other flaviruses such as Zika virus should be investigated urgently. TRIAL REGISTRATION: ClinicalTrials.gov NCT01619969.

Optimizing celgosivir therapy in mouse models of dengue virus infection of serotypes 1 and 2: The search for a window for potential therapeutic efficacy.

Although the antiviral drug celgosivir, an α-glucosidase I inhibitor, is highly protective when given twice daily to AG129 mice infected with dengue virus, a similar regimen of twice daily dosing did not significantly reduce serum viral loads in patients in a recent clinical trial. This failure presumably might reflect the initiation of treatment when patients were already viremic. To better mimic the clinical setting, we used viruses isolated from patients to develop new mouse models of DENV1 and DENV2 infection and employed the models to test the twice daily treatment, begun either on the day of infection or on the third day post-infection, when the mice had peak of viremia. We found that, although the treatment started on day 0 was effective on viral load reduction, it provided no benefit when begun on day 3, indicating that in vivo antiviral efficacy becomes less prominent once viremia reaches the peak level. To determine if the therapeutic regimen in humans could be improved, we tested regimen of four-times daily treatment and found that the treatment significantly reduced viremia, suggesting that a similar regimen may be effective in a human clinical trial. A new clinical trial to investigate an altered dosing regimen has been approved (NCT02569827).

The novel anti-neuroblastoma agent PF403, inhibits proliferation and invasion in vitro and in brain xenografts.

Neuroblastoma is the most common cancer in infants and the fourth most common cancer in children. Our previous study showed that PF403 had a potent antitumor ability. In the present study, we evaluated the anti-neuroblastoma property of PF403 and investigated the underlying mechanisms. MTT assay, colony formation assay and flow cytometry assay were used to assess cytotoxicity of PF403 on SH-SY5Y cells. Transwell assay was chosen to estimate the anti-invasion ability of PF403 on neuroblastoma cells. The protein expression was detected by western blot analysis. The SH-SY5Y brain xenograft model was used to assess in vivo antitumor activity of PF403. PF403-mediated SH-SY5Y cell death was found to be dose- and time-dependent, and PF403 was able to limit invasion and metastasis of neuroblastoma cells. MRI and pathology analysis proved that the pro-drug of PF403, CAT3, inhibited SH-SY5Y cells in vivo. PF403 decreased expression of phosphorylated FAK, MMP-2 and MMP-9 proteins, and downregulated the activity of PI3K/AKT and Raf/ERK pathways, followed by regulation of the proteins expression of Bcl-2 family, activated caspase-3, -9 and PARP and initiation of apoptosis of neuroblastoma cells. PF403 exerted cytotoxicity against SH-SY5Y neuroblastoma cell both in vitro and in vivo, and inhibited its invasion ability, suggesting PF403 has potential as a new anticancer drug for the treatment of neuroblastoma.

FL118 induces p53-dependent senescence in colorectal cancer cells by promoting degradation of MdmX.

Anticancer agent FL118 was recently identified in screening of small-molecule inhibitors of human survivin expression. Although FL118 is a camptothecin analogue, its antitumor potency is much superior to other FDA-approved camptothecin analogues (irinotecan and topotecan). The mechanism of action (MOA) underlying the antitumor effects of FL118 remains to be fully elucidated. Here, we report that FL118 activates tumor suppressor p53 as a novel MOA in p53 wild-type cancer cells. Our studies show that this MOA involves an induction of proteasomal degradation of MdmX, a critical negative regulator of p53, in a manner largely independent of ATM-dependent DNA damage signaling pathway but dependent on E3-competent Mdm2. FL118 inhibits p53 polyubiquitination and monoubiquitination by Mdm2-MdmX E3 complex in cells and in cell-free systems. In contrast, FL118 stimulates Mdm2-mediated MdmX ubiquitination. Coimmunoprecipitation revealed that FL118 slightly decreases Mdm2-p53 interactions and moderately increases Mdm2-MdmX interactions, suggesting a change of targeting specificity of Mdm2-MdmX E3 complex from p53 to MdmX, resulting in accelerated MdmX degradation. As a result, p53 ubiquitination by Mdm2-MdmX E3 complex is reduced, which in turn activates p53 signaling. Activation of the p53 pathway by FL118 induces p53-dependent senescence in colorectal cancer cells. However, in the absence of p53 or in the presence of MdmX overexpression, FL118 promotes p53-independent apoptosis. These two distinct cellular consequences collectively contribute to the potent effects of FL118 to inhibit clonogenic potential of colon cancer cells. This study identifies a potential application of FL118 as an MdmX inhibitor for targeted therapies.

Tylophorine, a phenanthraindolizidine alkaloid isolated from Tylophora indica exerts antiangiogenic and antitumor activity by targeting vascular endothelial growth factor receptor 2-mediated angiogenesis.

BACKGROUND: Anti-angiogenesis targeting VEGFR2 has been considered as an important strategy for cancer therapy. Tylophorine is known to possess anti-inflammatory and antitumor activity, but its roles in tumor angiogenesis, the key step involved in tumor growth and metastasis, and the involved molecular mechanism is still unknown. Therefore, we examined its anti-angiogenic effects and mechanisms in vitro and in vivo. METHODS: We used tylophorine and analyzed its inhibitory effects on human umbilical vein endothelial cells (HUVEC) in vitro and Ehrlich ascites carcinoma (EAC) tumor in vivo. RESULTS: Tylophorine significantly inhibited a series of VEGF-induced angiogenesis processes including proliferation, migration, and tube formation of endothelial cells. Besides, it directly inhibited VEGFR2 tyrosine kinase activity and its downstream signaling pathways including Akt, Erk and ROS in endothelial cells. Using HUVECs we demonstrated that tylophorine inhibited VEGF-stimulated inflammatory responses including IL-6, IL-8, TNF-α, IFN-γ, MMP-2 and NO secretion. Tylophorine significantly inhibited neovascularization in sponge implant angiogenesis assay and also inhibited tumor angiogenesis and tumor growth in vivo. Molecular docking simulation indicated that tylophorine could form hydrogen bonds and aromatic interactions within the ATP-binding region of the VEGFR2 kinase unit. CONCLUSION: Tylophorine exerts anti-angiogenesis effects via VEGFR2 signaling pathway thus, may be a viable drug candidate in anti-angiogenesis and anti-cancer therapies.

Antibacterial activity of the alkaloid-enriched extract from Prosopis juliflora pods and its influence on in vitro ruminal digestion.

The purpose of this study was to assess the in vitro antimicrobial activity of alkaloid-enriched extracts from Prosopis juliflora (Fabaceae) pods in order to evaluate them as feed additives for ruminants. As only the basic chloroformic extract (BCE), whose main constituents were juliprosopine (juliflorine), prosoflorine and juliprosine, showed Gram-positive antibacterial activity against Micrococcus luteus (MIC = 25 µg/mL), Staphylococcus aureus (MIC = 50 µg/mL) and Streptococcus mutans (MIC = 50 µg/mL), its influence on ruminal digestion was evaluated using a semi-automated in vitro gas production technique, with monensin as the positive control. Results showed that BCE has decreased gas production as efficiently as monensin after 36 h of fermentation, revealing its positive influence on gas production during ruminal digestion. Since P. juliflora is a very affordable plant, this study points out this alkaloid enriched extract from the pods of Prosopis juliflora as a potential feed additive to decrease gas production during ruminal digestion.

Synthesis and biological evaluation of tylophorine-derived dibenzoquinolines as orally active agents: exploration of the role of tylophorine e ring on biological activity.

A series of novel tylophorine-derived dibenzoquinolines has been synthesized and their biological activity evaluated. Three assays were conducted: inhibition of cancer cell proliferation, inhibition of TGEV replication for anticoronavirus activity, and suppression of nitric oxide production in RAW264.7 cells (a measure of anti-inflammation). The most potent compound from these assays, dibenzoquinoline 33b, showed improved solubility compared to tylophorine 9a, in vivo efficacies in a lung A549 xenografted tumor mouse model and a murine paw edema model, good bioavailability, and no significant neurotoxicity (as tested by a rota-rod test for motor coordination). This is the first study to explore in detail the role of the tylophorine E ring on biological activity and very strongly suggests that tylophorine-derived dibenzoquinolines merit further development into orally active agents.

Dose- and schedule-dependent protective efficacy of celgosivir in a lethal mouse model for dengue virus infection informs dosing regimen for a proof of concept clinical trial.

Celgosivir (6-O-butanoyl castanospermine), a pro-drug of the naturally occurring castanospermine, is an inhibitor of α-glucosidase I and II that is found to be a potent inhibitor of several enveloped viruses including all four serotypes of dengue virus. We showed previously that the compound fully protected AG129 mice from lethal infection with a mouse adapted dengue virus at a dose of 50mg/kg twice daily (BID) for 5days and was effective even after 48h delayed treatment. Here we show that the protection by celgosivir is dose- and schedule-dependent and that a twice-a-day regimen of 50, 25 or 10mg/kg is more protective than a single daily dose of 100mg/kg. Treatment with 50mg/kg BID castanospermine had comparable efficacy as 25mg/kg BID celgosivir, suggesting that celgosivir is approximately twice as potent as castanospermine with respect to in vivo antiviral efficacy. Pharmacokinetics (PK) studies of celgosivir in mice showed that it rapidly metabolized to castanospermine. Simulation of the PK data with the survival data for the various doses of celgosivir tested suggests that the steady-state minimum concentration is a critical parameter to note in choosing dose and schedule. These results influenced the selection of the dose regimen for a proof-of-concept clinical trial of celgosivir as a treatment against dengue fever.

Design and synthesis of orally available MEK inhibitors with potent in vivo antitumor efficacy.

The structure-based design, synthesis, and biological evaluation of two novel series of potent and selective MEK kinase inhibitors are described herein. The elaboration of a lead pyrrole derivative to a bicyclic dihydroindolone core provided compounds with high potency and good drug-like pharmaceutical properties. Further scaffold modification afforded a series of dihydroindolizinone inhibitors, including an orally available advanced preclinical MEK inhibitor with potent in vivo antitumor efficacy.

Novel, potent and orally bioavailable indolizidinone-derived inhibitors of the hepatitis C virus NS3 protease.

A novel, potent, and orally bioavailable class of product-like inhibitors of the HCV NS3 protease was discovered by constraining the P2-P3 amide bond and the P3 hydrocarbon substituent to the protease-bound conformation. This preorganization was accomplished by incorporation of the P2-P3 amide into a six-membered ring attached to the P2-proline 5-position. Isothermal calorimetric characterization of the role of hydrocarbon substitution of this six-membered ring, upon binding the HCV NS3 protease, was found to be exclusively entropic in nature. The synthesis, preliminary SAR and pharmacokinetic profiling of this compact, indolizidinone-derived scaffold are described.

Celgosivir treatment misfolds dengue virus NS1 protein, induces cellular pro-survival genes and protects against lethal challenge mouse model.

Dengue virus (DENV) infections continue to spread aggressively around the world. Here we demonstrate that celgosivir (6-O-butanoyl castanospermine), strongly inhibits all four DENV serotypes. We show by fluorescence microscopy that the antiviral mechanism of celgosivir, is in part, due to misfolding and accumulation of DENV non-structural protein 1 (NS1) in the endoplasmic reticulum. Moreover, celgosivir modulates the host's unfolded protein response (UPR) for its antiviral action. Significantly, celgosivir is effective in lethal challenge mouse models that recapitulate primary or secondary antibody-dependent enhanced DENV infection. Celgosivir treated mice showed enhanced survival, reduced viremia and robust immune response, as reflected by serum cytokine analysis. Importantly, survival increased even after treatment was delayed till 2 days post-infection. Together the present study suggests that celgosivir, which has been clinically determined to be safe in humans, may be a valuable candidate for clinical testing in dengue patients.

Comparative disposition and metabolism of paraherquamide in sheep, gerbils, and dogs.

The disposition and metabolism of paraherquamide (PHQ), a potent and broad-spectrum anthelminthic, were examined in sheep, dogs, and gerbils. The metabolism of PHQ in these species was extensive and marked by significant species differences both in vitro and in vivo. In sheep and gerbils, PHQ metabolism occurs mainly at the pyrrolidine moiety, generating several metabolites that, for the most part, retained nematodicidal activity in vitro. In dogs, the dioxepene group was also extensively metabolized, ultimately resulting in formation of a catechol and loss of pharmacological activity. After oral administration of [3H]PHQ to intact sheep, gerbils, and dogs, the majority of the administered radioactivity was recovered in feces. Intact PHQ accounted for 0% (dogs) to approximately 30% (sheep and gerbils) of drug-related material in feces. A detailed investigation of the composition of the intestinal content of sheep indicated that a significant amount of the dose was still present in the rumen 24 h after dose and that PHQ underwent significant dehydration in the cecum. The oral pharmacokinetic parameters of PHQ in sheep and dogs suggest that its absorption is rapid in both species but that its apparent elimination rate is significantly higher in the dog (t(1/2) approximately 1.5 h) than it is in sheep (t(1/2) approximately 8.5 h). The short elimination half-life and the absence of PHQ or other active components in the dog gastrointestinal tract provide a potential explanation of the lack of efficacy of PHQ in this species.

Secretory PLA2 inhibitor indoxam suppresses LDL modification and associated inflammatory responses in TNFalpha-stimulated human endothelial cells.

BACKGROUND AND PURPOSE: Secretory phospholipase A2 (sPLA2) is implicated in atherosclerosis, although the effects of specific sPLA2 inhibitors have not been studied. We investigated the effects of the indole analogue indoxam on low-density lipoprotein (LDL) modification by sPLA2 enzymes of different types and on the associated inflammatory responses in human umbilical vein endothelial cells (HUVEC). EXPERIMENTAL APPROACH: LDL modification was assessed by measuring the contents of two major molecular species of lysophosphatidylcholine (LPC) using electrospray ionization-liquid chromatography/mass spectrometry. The proinflammatory activity of the modified LDL was evaluated by determining monocyte chemoattractant protein-1 (MCP-1) mRNA expression and transcriptional factor nuclear factor-kappaB (NF-kappaB) activity in HUVEC. KEY RESULTS: Indoxam dose-dependently inhibited palmitoyl- and stearoyl-LPC production in LDL incubated with snake venom sPLA2 (IC50 1.2 microM for palmitoyl-LPC, 0.8 microM for stearoyl-LPC). MCP-1 mRNA expression and NF-kappaB activity were enhanced by venom sPLA2-treated LDL, which was completely suppressed by indoxam but not by thioetheramide-PC, a competitive sPLA2 inhibitor. Indoxam also suppressed LPC production in LDL treated with human synovial type IIA sPLA2. Tumour necrosis factor alpha (TNFalpha) increased type V sPLA2 expression in HUVEC. Indoxam dose-dependently suppressed LPC production in native and glycoxidized LDL treated with TNFalpha-stimulated HUVEC. Indoxam suppressed MCP-1 mRNA expression and NF-kappaB activity in TNFalpha-stimulated HUVEC incubated with native or glycoxidized LDL. CONCLUSIONS AND IMPLICATIONS: Indoxam prevented sPLA2-induced LPC production in native and glycoxidized LDL as well as LDL-induced inflammatory activity in HUVEC. Our results suggest that indoxam may be a potentially useful anti-atherogenic agent.

Assessment of basic indirect pharmacodynamic response models with physiological limits.

Many physiological factors are regulated by homeostatic mechanisms to maintain normal body function. Empirical lower Rl (Model I and IV) or upper Rh limits (Model II and III) were included in current basic indirect response (IDR) models to account for the additional role of physiological limits (IDRPL). Various characteristics of these models were evaluated with simulations and explicit equations. The simulations reveal that the expanded models exhibit most properties of basic indirect response models, such as slow response initiation, lag time between the kinetic and dynamic peaks, a large dose plateau, and shift in Tmax with dose. The proposed models always produce lesser net responses than predicted by basic IDR models. Simulations demonstrate that addition of a parameter limit which is close to the baseline has a great influence on the overall and maximum responses and fitted model parameters. Only stimulatory IDRPL Models III and IV allow resolution of all model parameters in the absence of clear indications or predetermined values of the lower or upper limits. However, all four models are able to resolve model parameters when subgroups with different baselines are simultaneously fitted. These models create new interpretations of the determinants of baseline conditions which can be important in assessing inter-subject variability in responses. The applicability of IDRPL models is demonstrated using several examples from the published literature. Indirect response models with physiological limits will be useful in characterizing drug responses for turnover systems which are maintained within a certain range.