Long-term stability of insulin glulisine loaded nanoparticles formulated using an amphiphilic cyclodextrin and designed for intestinal delivery.

Long-term stability is one of the main challenges for translation of therapeutic proteins into commercially viable biopharmaceutical products. During processing and storage, proteins are susceptible to denaturation. The aim of this work was to evaluate the stability of amphiphilic cyclodextrin-based nanoparticles (NPs) containing insulin glulisine. The stability of the NP dispersion was systematically evaluated following storage at three different temperatures (4 °C, room temperature (RT) and 40 °C). While the colloidal parameters of the NPs in terms of size and zeta potential were maintained (109 ± 9 nm, polydispersity index 0.272, negative zeta potential -25 ± 3 mV), insulin degraded over 60 days during storage. To enhance the shelf life of the product and to circumvent the need for cold-chain maintenance, a lyophilized formulation containing insulin glulisine NPs (1.75 mg/mL of NPs) and 25 mg/mL trehalose was produced. The freeze-dried powder extended the stability of the product for up to 30 days at ambient temperature and 90 days at 4 °C (with 95% and >80% insulin recovery, respectively). Following intra-intestinal administration of the freeze-dried formulation, while no lowering of blood glucose was seen, insulin glulisine was detected in both portal and systemic blood indicating that potential exists for further development of the formulation to simultaneously achieve prolonged stability and therapeutic efficacy.

Physicochemical, pharmacokinetic and pharmacodynamic analyses of amphiphilic cyclodextrin-based nanoparticles designed to enhance intestinal delivery of insulin.

Due to excellent efficacy, low toxicity, and well-defined selectivity, development of new injectable peptides is increasing. However, the translation of these drugs into products for effective oral delivery has been restricted due to poor oral bioavailability. Nanoparticle (NP) formulations have potential to overcome the barriers to oral peptide delivery through protecting the payload and increasing bioavailability. This study describes the rational design, optimization and evaluation of a cyclodextrin-based NP entrapping insulin glulisine for intestinal administration. A cationic amphiphilic cyclodextrin (click propyl-amine cyclodextrin (CD)) was selected as the primary complexing agent for NP development. Following NP synthesis, in vitro characterization was performed. The insulin glulisine NPs exhibited an average size of 109 ±â€¯9 nm, low polydispersity index (0.272) negative zeta potential (-25 ±â€¯3 mV), high association efficiency (71.4 ±â€¯3.37%) and an insulin loading of 10.2%. In addition, the NPs exhibited colloidal stability in intestinal-biorelevant media (SIF, supplemented-SIF 1% (w/v) and FaSSIF-V2) for up to 4 h. Proteolysis studies indicated that the NPs conferred protection to the entrapped insulin relative to free insulin. In vivo rat jejunal instillation studies demonstrated that the NPs mediated systemic insulin absorption, accompanied by a decrease in blood glucose levels. The relative bioavailability of the instilled insulin (50 IU/kg) from the NP was 5.5% compared to subcutaneous administration of insulin solution (1 IU/kg). The pharmacodynamic and pharmacokinetic data indicate that this cyclodextrin-based formulation may have potential for further research as an oral insulin dosage form.

SAR110894, a potent histamine H3-receptor antagonist, displays disease-modifying activity in a transgenic mouse model of tauopathy.

INTRODUCTION: Tau hyperphosphorylation and neurofibrillary tangles are histopathologic hallmarks of tauopathies. Histamine H3-receptor antagonists have been proposed to reduce tau hyperphosphorylation in preclinical models. METHODS: We evaluated the ability of SAR110894, a selective histamine H3-receptor antagonist, to inhibit tau pathology and prevent cognitive deficits in a tau transgenic mouse model (THY-Tau22). RESULTS: SAR110894 treatment for 6 months (but not 2 weeks) in THY-Tau22 mice decreased both tau hyperphosphorylation at pSer396-pSer404 (AD2 signal) in the hippocampus and the number of AT8 (pSer199/202-Thr205) positive cells in the cortex and decreased the formation of neurofibrillary tangles in the cortex, hippocampus, and amygdala. Macrophage inflammatory protein 1-alpha messenger RNA expression was decreased in the hippocampus. SAR110894 also prevented episodic memory deficits, and this effect was still detected after treatment washout. DISCUSSION: Long-term SAR110894 treatment could have potential disease modifying activity in neurodegenerative tauopathies.

Physiologically based pharmacokinetic modeling for sequential metabolism: effect of CYP2C19 genetic polymorphism on clopidogrel and clopidogrel active metabolite pharmacokinetics.

Clopidogrel is a prodrug that needs to be converted to its active metabolite (clopi-H4) in two sequential cytochrome P450 (P450)-dependent steps. In the present study, a dynamic physiologically based pharmacokinetic (PBPK) model was developed in Simcyp for clopidogrel and clopi-H4 using a specific sequential metabolite module in four populations with phenotypically different CYP2C19 activity (poor, intermediate, extensive, and ultrarapid metabolizers) receiving a loading dose of 300 mg followed by a maintenance dose of 75 mg. This model was validated using several approaches. First, a comparison of predicted-to-observed area under the curve (AUC)0-24 obtained from a randomized crossover study conducted in four balanced CYP2C19-phenotype metabolizer groups was performed using a visual predictive check method. Second, the interindividual and intertrial variability (on the basis of AUC0-24 comparisons) between the predicted trials and the observed trial of individuals, for each phenotypic group, were compared. Finally, a further validation, on the basis of drug-drug-interaction prediction, was performed by comparing observed values of clopidogrel and clopi-H4 with or without dronedarone (moderate CYP3A4 inhibitor) coadministration using a previously developed and validated physiologically based PBPK dronedarone model. The PBPK model was well validated for both clopidogrel and its active metabolite clopi-H4, in each CYP2C19-phenotypic group, whatever the treatment period (300-mg loading dose and 75-mg last maintenance dose). This is the first study proposing a full dynamic PBPK model able to accurately predict simultaneously the pharmacokinetics of the parent drug and of its primary and secondary metabolites in populations with genetically different activity for a metabolizing enzyme.

Clopidogrel pharmacodynamics and pharmacokinetics in the fed and fasted state: a randomized crossover study of healthy men.

Clopidogrel requires CYP450-mediated hepatic metabolism to form its active metabolite (clopi-H4). This randomized, placebo-controlled, crossover study was designed to characterize the effect of a high-fat or standard breakfast on adenosine diphosphate (ADP)-induced platelet aggregation and exposure to unchanged clopidogrel and clopi-H4 following clopidogrel (300-mg loading dose, 75 mg/d for 4 days) in 72 healthy men. At day 5 and as assessed by liquid chromatography-tandem mass spectrometry, unchanged clopidogrel area under the concentration- time curve from 0 to 24 hours (AUC(0-24)) increased 3.32-fold (90% confidence interval [CI], 2.88-3.84), and clopi-H4 AUC(0-24) decreased nonsignificantly by 12% (90% CI, 0.82-0.94) upon administration of clopidogrel with a standard breakfast. The estimated treatment difference in maximum platelet aggregation (MPA) induced by ADP 5 µM and assessed by light transmission aggregometry was 4.7%, with the 90% CI (0.9%-8.5%) contained within the prespecified equipotency range of ±15%. The mean ± standard deviation of day 5 inhibition of platelet aggregation was 49.7% ± 17.2% and 54.0% ± 13.3% in the fed and fasted states, respectively. Despite increased unchanged clopidogrel and slightly decreased clopi-H4 exposure following clopidogrel administration, the numerical increase in MPA in the fed versus fasted state was small and within the prespecified limit of equipotency. These findings confirm that clopidogrel can be taken with or without food.

An improved method for specific and quantitative determination of the clopidogrel active metabolite isomers in human plasma.

Pharmacokinetic analyses of clopidogrel are hampered by the existence of multiple active metabolite isomers (H1 to H4) and their instability in blood. We sought to retest the pharmacodynamic activities of the four individual active metabolite isomers in vitro, with the ultimate aim of determining the isomers responsible for clopidogrel activity in vivo. In vitro activity was evaluated by measuring binding of [³³P]-2-methylthio-ADP on P2Y12-expressing Chinese hamster ovary (CHO) cells and human platelets in platelet-rich plasma (PRP). A stereoselective method that used reverse-phase ultra high-performance liquid chromatography (UHPLC) and tandem mass spectrometry (MS) was developed to measure individual concentrations of the stable 3'-methoxyacetophenone (MP) derivatives of H1-H4. The new method was used to analyze plasma samples from clopidogrel-treated subjects enrolled in a phase I clinical trial. In vitro binding assays confirmed the previously observed biological activity of H4 (IC50: CHO-P2Y12: 0.12 µM; PRP: 0.97 µM) and inactivity of H3, and demonstrated that H1 was also inactive. Furthermore, H2 demonstrated approximately half of the biological activity in vitro compared with H4. Optimisation of UHPLC conditions and MS collision parameters allowed the resolution and detection of the four derivatised active metabolite isomers (MP-H1 to MP-H4). The stereoselective assay was extensively validated, and was accurate and precise over the concentration range 0.5-250 ng/ml. Only MP-H3 and MP-H4 were quantifiable in incurred clinical samples. Based on in vitro pharmacodynamic data and found concentrations, the active metabolite isomer H4 is the only diastereoisomer of clinical relevance for documenting the pharmacokinetic profile of the active metabolite of clopidogrel.

Coherent structures in dissipative particle dynamics simulations of the transition to turbulence in compressible shear flows.

We present simulations of coherent structures in compressible flows near the transition to turbulence using the dissipative particle dynamics method. The structures we find are remarkably consistent with experimental observations and direct numerical simulations (DNS) simulations of incompressible flows, despite a difference in Mach number of several orders of magnitude. The bifurcation from the laminar flow is bistable and shifts to higher Reynolds numbers when the fluid becomes more compressible. This work underlines the robustness of coherent structures in the transition to turbulence and illustrates the ability of particle-based methods to reproduce complex nonlinear instabilities.

Statistics of crumpled paper.

A statistical study of crumpled paper is allowed by a minimal 1D model: a self-avoiding line bent at sharp angles--in which the elastic energy resides--put in a confining potential. Many independent equilibrium configurations are generated numerically and their properties are investigated. At small confinement, the distribution of segment lengths is log-normal in agreement with previous predictions and experiments. At high confinement, the system approaches a jammed state with a critical behavior, whereas the length distribution follows a gamma law in which the parameter is predicted as a function of the number of layers in the system.

Lack of effect of food on the bioavailability of a new ketolide antibacterial, telithromycin.

Telithromycin is an innovative antibacterial designed for the treatment of community-acquired respiratory tract infections. This study assessed the effect of food on the bioavailability of a single oral dose of telithromycin 800 mg in healthy male subjects. Male volunteers aged 18-45 y were recruited for an open-label, single-dose, 2-period, cross-over study. In each trial period, subjects received a single oral dose of telithromycin 800 mg after an overnight fast, or after a standard high-fat breakfast. A washout period of 6-8 d separated the 2 study periods. All 18 subjects recruited (mean age 30.7 y) completed the study. Telithromycin was rapidly absorbed, reaching maximum plasma concentrations within a median of 2.50 and 2.25 h in the fasting and non-fasting states, respectively. There was no statistical difference between the non-fasting and fasting states for any of the pharmacokinetic parameters measured. The mean plasma telithromycin concentration versus time profiles for the non-fasting and fasting phases were almost superimposable. For the maximum plasma concentration and area under the curve from time 0 to infinity, the 90% confidence intervals for the mean non-fasting:fasting ratios were 83-116 and 101-123 mg x h/l, respectively; these are within 80-125% of the bioequivalence range. Telithromycin was well tolerated. The bioavailability, rate and extent of absorption of the new ketolide antibacterial telithromycin were unaffected by food.