A sustained release formulation of novel quininib-hyaluronan microneedles inhibits angiogenesis and retinal vascular permeability in vivo.

Pathologic neovascularisation and ocular permeability are hallmarks of proliferative diabetic retinopathy and age-related macular degeneration. Current pharmacologic interventions targeting VEGF are effective in only 30-60% of patients and require multiple intraocular injections associated with iatrogenic infection. Thus, our goal is to develop novel small molecule drugs that are VEGF-independent are amenable to sustained ocular-release, and which reduce retinal angiogenesis and retinal vascular permeability. Here, the anti-angiogenic drug quininib was formulated into hyaluronan (HA) microneedles whose safety and efficacy was evaluated in vivo. Quininib-HA microneedles were formulated via desolvation from quininib-HA solution and subsequent cross-linking with 4-arm-PEG-amine prior to freeze-drying. Scanning electron microscopy revealed hollow needle-shaped particle ultrastructure, with a zeta potential of -35.5mV determined by electrophoretic light scattering. The incorporation efficiency and pharmacokinetic profile of quininib released in vitro from the microneedles was quantified by HPLC. Quininib incorporation into these microneedles was 90%. In vitro, 20% quininib was released over 4months; or in the presence of increasing concentrations of hyaluronidase, 60% incorporated quininib was released over 4months. Zebrafish hyaloid vasculature assays demonstrated quininib released from these microneedles significantly (p<0.0001) inhibited ocular developmental angiogenesis compared to control. Sustained amelioration of retinal vascular permeability (RVP) was demonstrated using a bespoke cysteinyl leukotriene induced rodent model. Quininib-HA microparticles significantly inhibited RVP in Brown Norway rats one month after administration compared to neat quininib control (p=0.0071). In summary, quininib-HA microneedles allow for sustained release of quininib; are safe in vivo and quininib released from these microneedles effectively inhibits angiogenesis and RVP in vivo.

Combination of the sodium-glucose cotransporter-2 inhibitor empagliflozin with orlistat or sibutramine further improves the body-weight reduction and glucose homeostasis of obese rats fed a cafeteria diet.

The present study assessed the potential of the sodium glucose-linked transporter (SGLT)-2 inhibitor empagliflozin to decrease body weight when administered alone or in combination with the clinically effective weight-loss agents orlistat and sibutramine in obese rats fed a cafeteria diet. Female Wistar rats were exposed to a cafeteria diet to induce obesity. Empagliflozin was dosed once daily (10, 30, and 60 mg/kg) for 28 days. Combination studies were subsequently performed using a submaximal empagliflozin dose (10 mg/kg) with either sibutramine or orlistat. Body weight, food, and water intake were recorded daily. The effect of drug treatment on glucose tolerance, relevant plasma parameters, and carcass composition was determined. Empagliflozin dose-dependently reduced body weight, plasma leptin, and body fat though increased urinary glucose excretion. The combination of empagliflozin and orlistat significantly reduced body weight compared to animals treated with either drug alone, and significantly improved glucose tolerance, plasma insulin, and leptin compared to vehicle-treated controls. The effect of sibutramine to improve glycemic control in an oral glucose-tolerance test was also significantly increased, with empagliflozin and combination treatment leading to a reduction in carcass fat greater than that observed with either drug alone. These data demonstrate that empagliflozin reduces body weight in cafeteria-fed obese rats. In combination studies, empagliflozin further improved the body-weight or body-fat loss of animals in comparison to orlistat or sibutramine alone. Such studies may indicate improved strategies for the treatment of obese patients with prediabetes or type 2 diabetes.

Effects of the DPP-4 inhibitor, linagliptin, in diet-induced obese rats: a comparison in naive and exenatide-treated animals.

BACKGROUND: To assess the chronic effect of the DPP-4 inhibitor, linagliptin, alone, in combination with exenatide, and during exenatide withdrawal, in diet-induced obese (DIO) rats. METHODS: Female Wistar rats were exposed to a cafeteria diet to induce obesity. Animals were then dosed with vehicle or linagliptin (3 mg/kg PO) orally once-daily for a 28 day period. In a subsequent study, rats received exenatide (either 3 or 30 microg/kg/day) or vehicle by osmotic mini-pump for 28 days. In addition, groups of animals were dosed orally with linagliptin either alone or in combination with a 3 microg/kg/day exenatide dose for the study duration. In a final study, rats were administered exenatide (30 microg/kg/day) or vehicle by osmotic mini-pump for eleven days. Subsequently, exenatide-treated animals were transferred to vehicle or continued exenatide infusion for a further ten days. Animals transferred from exenatide to vehicle were also dosed orally with either vehicle or linagliptin. In all studies, body weight, food and water intake were recorded daily and relevant plasma parameters and carcass composition were determined. RESULTS: In contrast to exenatide, linagliptin did not significantly reduce body weight or carcass fat in DIO rats versus controls. Linagliptin augmented the effect of exenatide to reduce body fat when given in combination but did not affect the body weight response. In rats withdrawn from exenatide, weight regain was observed such that body weight was not significantly different to controls. Linagliptin reduced weight regain after withdrawal of exenatide such that a significant difference from controls was evident. CONCLUSIONS: These data demonstrate that linagliptin does not significantly alter body weight in either untreated or exenatide-treated DIO rats, although it delays weight gain after exenatide withdrawal. This finding may suggest the utility of DPP-4 inhibitors in reducing body weight during periods of weight gain.

Some cannabinoid receptor ligands and their distomers are direct-acting openers of SUR1 K(ATP) channels.

Here, we examined the chronic effects of two cannabinoid receptor-1 (CB1) inverse agonists, rimonabant and ibipinabant, in hyperinsulinemic Zucker rats to determine their chronic effects on insulinemia. Rimonabant and ibipinabant (10 mg·kg⁻¹·day⁻¹) elicited body weight-independent improvements in insulinemia and glycemia during 10 wk of chronic treatment. To elucidate the mechanism of insulin lowering, acute in vivo and in vitro studies were then performed. Surprisingly, chronic treatment was not required for insulin lowering. In acute in vivo and in vitro studies, the CB1 inverse agonists exhibited acute K channel opener (KCO; e.g., diazoxide and NN414)-like effects on glucose tolerance and glucose-stimulated insulin secretion (GSIS) with approximately fivefold better potency than diazoxide. Followup studies implied that these effects were inconsistent with a CB1-mediated mechanism. Thus effects of several CB1 agonists, inverse agonists, and distomers during GTTs or GSIS studies using perifused rat islets were unpredictable from their known CB1 activities. In vivo rimonabant and ibipinabant caused glucose intolerance in CB1 but not SUR1-KO mice. Electrophysiological studies indicated that, compared with diazoxide, 3 µM rimonabant and ibipinabant are partial agonists for K channel opening. Partial agonism was consistent with data from radioligand binding assays designed to detect SUR1 K(ATP) KCOs where rimonabant and ibipinabant allosterically regulated ³H-glibenclamide-specific binding in the presence of MgATP, as did diazoxide and NN414. Our findings indicate that some CB1 ligands may directly bind and allosterically regulate Kir6.2/SUR1 K(ATP) channels like other KCOs. This mechanism appears to be compatible with and may contribute to their acute and chronic effects on GSIS and insulinemia.

Antibody-induced lysis of isolated rat epididymal adipocytes and complement activation in vivo.

OBJECTIVE: To identify human monoclonal antibodies selectively binding to human adipocytes and to evaluate their ability to induce lysis of isolated rat adipocytes in vitro and to reduce rat complement levels in vivo. RESEARCH METHODS AND PROCEDURES: Using phage display technology, human monoclonal antibodies binding to human adipocyte plasma membranes were identified. Three antibodies (Fat 13, Fat 37, and Fat 41) were selected based on their additional cross-reaction with rat adipocytes and reformatted as a rat chimeric IgG2bs. The ability of these antibodies, both singly and in combination, to induce lysis of rat epididymal adipocytes in vitro and the reduction of serum complement levels in vivo in the rat was evaluated. RESULTS: All antibodies caused similar time- and dose-dependent lysis of isolated rat adipocytes. Calculated mean EC(50) values (maximum percentage of lysis in parentheses) were 0.680 microg/mL (63.2%), 0.546 microg/mL (72.4%), and 0.391 microg/mL (73.7%) for Fat 13, Fat 37, and Fat 41, respectively. Combinations were no more effective than individual antibodies in inducing lysis. Anti-adipocyte antibodies (both singly and in combination) were also similarly effective in vivo. In rats, doses of monoclonal antibody up to 10 mg/kg intraperitoneal generally caused almost complete depletion of serum complement up to 24 hours after dosing recovering to baseline values by day 5. DISCUSSION: Individual and combinations of monoclonal anti-adipocyte antibodies produced a complement-dependent and concentration-dependent activity to lyse adipocytes in vitro and in vivo as measured by a dramatic depletion in serum complement.

Artifactual uncoupling by uncoupling protein 3 in yeast mitochondria at the concentrations found in mouse and rat skeletal-muscle mitochondria.

Western blots detected uncoupling protein 3 (UCP3) in skeletal-muscle mitochondria from wild-type but not UCP3 knock-out mice. Calibration with purified recombinant UCP3 showed that mouse and rat skeletal muscle contained 0.14 microg of UCP3/mg of mitochondrial protein. This very low UCP3 content is 200-700-fold less than the concentration of UCP1 in brown-adipose-tissue mitochondria from warm-adapted hamster (24-84 microg of UCP1/mg of mitochondrial protein). UCP3 was present in brown-adipose-tissue mitochondria from warm-adapted rats but was undetectable in rat heart mitochondria. We expressed human UCP3 in yeast mitochondria at levels similar to, double and 7-fold those found in rodent skeletal-muscle mitochondria. Yeast mitochondria containing UCP3 were more uncoupled than empty-vector controls, particularly at concentrations that were 7-fold physiological. However, uncoupling by UCP3 was not stimulated by the known activators palmitate and superoxide; neither were they inhibited by GDP, suggesting that the observed uncoupling was a property of non-native protein. As a control, UCP1 was expressed in yeast mitochondria at similar concentrations to that of UCP3 and at up to 50% of the physiological level of UCP1. Low levels of UCP1 gave palmitate-dependent and GDP-sensitive proton conductance but higher levels of UCP1 caused an additional GDP-insensitive uncoupling artifact. We conclude that the uncoupling of yeast mitochondria by high levels of UCP3 expression is entirely an artifact and provides no evidence for any native uncoupling activity of the protein.