An Investigation into the Influence of Counterion on the RS‐Propranolol and S‐Propranolol Skin Permeability

The effects of two contra‐ions, namely benzoate (Bz) and oleate (Ol), on the in vitro human skin permeability of propranolol racemate (RS‐PR) or S‐enantiomer (S‐PR) were studied. Saline solution (SS) or mineral oil (MO) were selected as vehicles. The MO increased the permeability coefficient (K_p) of PR‐Bz (pK_p ≈ 4) of about four times with respect to SS (pK_p ≈ 8) probably due to the ion pair formation. The steady‐state flux of S‐enantiomers resulted about twofold higher than that of racemates according to their lower melting temperatures with the exception of (S)‐PR‐Ol and (RS)‐PR‐Ol vehicled in SS which not resulted statistically different. This anomalous result could be explained considering the behavior of (RS)‐PR‐Ol or (S)‐PR‐Ol in aqueous solutions: these salts formed ion pairs which associated to form aggregates up to a concentration of 20 µg/mL as verified by light scattering. Therefore, their effective concentrations in SS resulted similar and justified the overlapped skin permeation profiles. All three considered variables, namely counterion, vehicle, and chirality, resulted mutually interfering on and deeply influenced the passive diffusion process of PR. © 2009 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 99: 1217–1224, 2010     

Solid Lipid Particles for Oral Delivery of Peptide and Protein Drugs III — the Effect of Fed State Conditions on the In Vitro Release and Degradation of Desmopressin

The effect of food intake on the release and degradation of peptide drugs from solid lipid particles is unknown and was therefore investigated in vitro using different fed state media in a lipolysis model. Desmopressin was used as a model peptide and incorporated into solid lipid particles consisting of trimyristin (TG14), tripalmitin (TG16), and tristearin (TG18), respectively. Fasted state and fed state media with varying phospholipid and bile salt concentrations, as well as fed state media with milk and oleic acid glycerides, respectively, were used as the release media. The presence of oleic acid glycerides accelerated the release of desmopressin significantly from all solid lipid particles both in the presence and absence of lipase. The presence of oleic acid glycerides also reduced the degradation rate of desmopressin, probably due to the interactions between the lipids and the protease or desmopressin. Addition of a medium chain triglyceride, trilaurin, in combination with drug-loaded lipid particles diminished the food effect on the TG18 particles, and trilaurin is therefore proposed to be a suitable excipient for reduction of the food effect. Overall, the present study shows that strategies to reduce food effect, such as adding trilaurin, for lipid particle formulations should be considered as drug release from such formulations might be influenced by the presence of food in the gastrointestinal tract.     

Possibilities and Limiting Factors for the Use of Dissolution as a Quality Control Tool to Detect Presence of Crystallinity for Amorphous Solid Dispersions: An Experimental and Modeling Investigation

In this article, experiments on tablets containing a model compound, grazoprevir, were conducted to explore how media selection for a quality control dissolution method can influence the sensitivity for the dissolution method toward drug crystallinity detection in an amorphous solid dispersion formulation. The experiment shows that under ideal nonsink conditions with respect to crystalline solubility, dissolution can indeed be predictive of crystallinity in the formulation. However, the limit of detection for crystallinity with quality control dissolution can change based on inherent variabilities in the drug product. In addition, it is demonstrated that the method's sensitivity and accuracy might be reduced if the crystalline particles are sufficiently small with respect to the solid dispersion particles. To further demonstrate the limits of the dissolution method, a dissolution model was also explored to simulate and predict the sensitivity of the dissolution response toward crystallinity based on solubility in the media and particle size of the crystals.     

Solid-State Stability of Human Insulin I. Mechanism and the Effect of Water on the Kinetics of Degradation in Lyophiles from pH 2–5 Solutions

Purpose. Previous studies have established that in aqueous solution at low pH human insulin decomposition proceeds through a cyclic anhydride intermediate leading to the formation of both deamidated and covalent dimer products. This study examines the mechanism and kinetics of insulin degradation in the amorphous solid state (lyophilized powders) as a function of water content over a similar pH range. Methods. Solutions of 1.0 mg/mL insulin were adjusted to pH 2–5 using HC1, freeze-dried, then exposed to various relative humidities at 35°C. The water content within the powders was determined by Karl Fischer titration, and the concentrations of insulin and its degradation products were determined by HPLC. Degradation kinetics were determined by both the initial rates of product formation and insulin disappearance. Results. Semi-logarithmic plots of insulin remaining in lyophilized powders versus time were non-linear, asymptotically approaching non-zero apparent plateau values, mathematically describable by a reversible, first-order kinetic model. The rate of degradation of insulin in the solid state was observed to increase with decreasing apparent pH (pH) yielding, at any given water content, solid-state pH-rate profiles parallel to the solution pH-rate profile. This pH dependence could be accounted for in terms of the fraction of the insulin A21 carboxyl in its neutral form, with an apparent pKa of 4, independent of water content. Aniline trapping studies established that the mechanism of degradation of human insulin in lyophilized powders between pH 3–5 and at 35°C involves rate-limiting intramolecular nucleophilic attack of the Asn_A21 C-terminal carboxylic acid onto the side-chain amide carbonyl to form a reactive cyclic anhydride intermediate, which further reacts with either water or an N-terminal primary amino group (e.g., Phe_B1, and Gly_Al) of another insulin molecule to generate either deamidated insulin (Asp_A21) or an amide-linked covalent dimer (e.g., [Asp_A21-Phe_B1] or [Asp_A21-Gly_A1]), respectively. The rate of insulin degradation in lyophilized powders at 35°C increases with water content at levels of hydration well below the suspected glass transition and approaches the rate in solution at or near the water content (20–50%) required to induce a glass transition. Conclusions. The decomposition of human insulin in lyophilized powders between pH 3–5 is a water induced solid-state reaction accelerated by the plasticization effect of sorbed water. The formation of the cyclic anhydride intermediate at A21 occurs readily even in the glassy state, presumably due to the conformational flexibility of the A21 segment even under conditions in which the insulin molecules as a whole are largely immobile.     

Effect of silicone oil on the microstructure,gelation and rheological properties of sorbitan monostearate–sesame oil oleogels

Oleogels contain oil or a non-polar liquid which is gelled with an agent called an organogelator. The aim of this study was to evaluate the effects of the addition of silicone oil (cyclopentasiloxane) to the gelation process and to the properties of sorbitan monostearate (SMS)–sesame oil oleogel and compared with that of SMS–sesame oil oleogel and SMS–cyclopentasiloxane oleogel. Three different oil phases; sesame oil phase, cyclopentasiloxane phase and a mixture of cyclopentasiloxane and sesame oil, were used to prepare oleogels with SMS gelator. The critical gelling concentrations (CGC) for oleogels were determined using different concentration of SMS in a range of 5%–22% (w/w). The characterization of the developed oleogels was done using Fourier transform infrared spectroscopy (FTIR), polarized light microscope, rheometer, X-ray diffraction (XRD) and differential scanning calorimetry (DSC). The addition of cyclopentasiloxane reduced the CGC of SMS–sesame oil oleogel from 20% to 10% (w/w). In microscopic characterization, the oleogels with a mixture of oil phases showed the longer and thicker three-dimensional gel network than that of oleogels with sesame oil and cyclopentasiloxane. FTIR studies demonstrated that this network formation was mainly due to hydrogen bonding. Rheological measurements revealed that the combination of cyclopentasiloxane and sesame oil produced strong gel with higher complex modulus values and longer linear viscoelastic region than oleogels prepared with sesame oil and cyclopentasiloxane. In addition, oleogels with the combination of the two oils had higher enthalpy (ΔH_m) and entropy (ΔS_m) thus could increase thermodynamic stability of the oleogels. Therefore, the addition of cyclopentasiloxane can improve the physical, thermal properties and stability of SMS–sesame oil oleogel, provide greater sensory profile and better product aesthetics. The developed oleogel can be a novel carrier for topical drug delivery.     

Effect of K+ and Rb+ on the action of verapamil on a voltage-gated K+ channel,hKv1.3: implications for a second open state?

Background and purpose:

Verapamil blocks current through the voltage-gated K⁺ channel K_v1.3 in the open and inactivated state of the channel but not the closed state. The binding site for verapamil was proposed to be close to the selectivity filter and the occupancy of the selectivity filter might therefore influence verapamil affinity.

Experimental“ approach:

We investigated the influence of intra- and extracellular K⁺ and Rb⁺ on the effect of verapamil by patch-clamp studies, in COS-7 cells transfected with hK_v1.3 channels.

Key results:

Verapamil affinity was highest in high intracellular K⁺ concentrations ([K⁺]_i) and lowest in low [Rb⁺]_i, indicating an influence of intracellular cations on verapamil affinity. Experiments with a mutant channel (H399T), exhibiting a strongly reduced C-type inactivated state, demonstrated that part of this changed verapamil affinity in wild-type channels could be caused by altered C-type inactivation. External K⁺ and Rb⁺ could influence verapamil affinity by a voltage-dependent entry into the channel thereby modifying the verapamil off-rate and in addition causing a voltage-dependent verapamil off-rate.

Conclusions and implications:

Recovery from verapamil block was mainly due to the voltage-dependent closing of channels (state-dependent block), implying a second open state of the channel. This hypothesis was confirmed by the dependency of the tail current time course on duration of the prepulse. We conclude that the wild-type hK_v1.3 channel undergoes at least two different conformational changes before finally closing with a low verapamil affinity in one open state and a high verapamil affinity in the other open state.     

Transport of active flavonoids,based on cytotoxicity and lipophilicity: An evaluation using the blood–brain barrier cell and Caco-2 cell models

This in vitro study aims to evaluate and compare transmembrane transport of eight cardio-cerebrovascular protection flavonoids including puerarin, rutin, hesperidin, quercetin, genistein, kaempferol, apigenin and isoliquiritigenin via the rat blood–brain barrier cell and Caco-2 cell monolayer models, based on the data of cytotoxicity and lipophilicity. The cytotoxicity of the flavonoids to rat brain microvessel endothelial cell was determined by the MTT assay. The apparent permeability coefficients (P_app) of the flavonoids were calculated from the unilateral transport assays in Transwell system with simultaneous determination using a high performance liquid chromatography. The results showed that the cytotoxicity and oil–water partition coefficient of the flavonoids modified by the number and position of the glycoside and hydroxyl group were the key determinant for the transmembrane transport. The P_app values of the flavonoids reduced adversely when the numbers of glycoside and hydroxyl groups of the flavonoids increased accordingly. The tested flavonoids exhibited time-dependent P_app values in these models. The efflux mechanism related with P-glycoprotein also existed with the polar flavonoids; verapamil could enhance the permeation of rutin and quercetin via inhibition of P-glycoprotein. We propose that genistein and isoliquiritigenin with the permeation priority in vitro Caco-2 and BBB cell model could be better as the drug candidates for cardio-cerebral vascular protection. These findings provided important information for establishing the transport relationship for the flavonoid compounds and evaluating the potential oral bioavailability and brain distribution of the flavonoids.     

Efflux transporter breast cancer resistance protein dominantly expresses on the membrane of red blood cells,hinders partitioning of its substrates into the cells,and alters drug–drug interaction profiles

1. Red blood cell (RBC) partitioning is important in determining pharmacokinetic and pharmacodynamic properties of a compound; however, active transport across RBC membranes is not well understood, particularly without transporter-related cell membrane proteomics data.

2. In this study, we quantified breast cancer resistance protein (BCRP/Bcrp) and MDR1/P-glycoprotein (P-gp) protein expression in RBCs from humans, monkeys, dogs, rats and mice using nanoLC/MS/MS, and evaluated their effect on RBC partitioning and plasma exposure of their substrates. BCRP-specific substrate Cpd-1 and MDR1-specific substrate Cpd-2 were characterized using Caco-2 Transwell® system and then administered to Bcrp or P-gp knockout mice.

3. The quantification revealed BCRP/Bcrp but not MDR1/P-gp to be highly expressed on RBC membranes. The knockout mouse study indicated BCRP/Bcrp pumps the substrate out of RBCs, lowering its partitioning and thus preventing binding to intracellular targets. This result was supported by a Cpd-1 and Bcrp inhibitor ML753286 drug–drug interaction (DDI) study in mice. Because of enhanced partitioning of Cpd-1 into RBCs after BCRP/Bcrp inhibition, Cpd-1 plasma concentration changed much less extent with genetic or chemical knockout of Bcrp albeit marked blood concentration increase, suggesting less DDI effect.

4. This finding is fundamentally meaningful to RBC partitioning, pharmacokinetics and DDI studies of BCRP-specific substrates.     

Investigation of the Hemostatic Effect of a Transdermal Patch Containing 0.55?mg Ethinyl Estradiol and 2.1?mg Gestodene Compared with a Monophasic Oral Contraceptive Containing 0.03?mg Ethinyl Estradiol and 0.15?mg Levonorgestrel: An Open-Label,Randomized, Crossover Study

Background

Transdermal delivery of contraceptives offers several advantages over combined oral contraceptives (COCs), including effective absorption and the provision of relatively constant serum concentrations. Ethinyl estradiol (EE) and the progestin gestodene are well-absorbed through the skin and, therefore, well-suited for use in a transdermal contraceptive patch.

Objective

The objective of this study was to investigate the impact of a once-weekly transparent, transdermal patch delivering low doses of EE and gestodene equivalent to a COC containing 0.02 mg EE and 0.06 mg gestodene on hemostasis parameters compared with a monophasic COC containing 0.03 mg EE and 0.15 mg levonorgestrel.

Methods

In this single-center, open-label, randomized, crossover study, 30 women (aged 18–35 years) received three cycles of each treatment, separated by a two-cycle washout period. The primary outcome measure was the absolute change from baseline in prothrombin fragments 1 + 2 and d-dimer.

Results

For both treatments, prothrombin fragments 1 + 2 remained stable during the first treatment period, and increased only slightly in the second period (mean absolute change 0.025 and 0.028 nmol/L in the novel Bayer patch and COC groups, respectively). Increases in d-dimer were observed in both periods (mean absolute change 107.0 ± 147.2 ng/L for the novel Bayer patch and 113.7 ± 159.0 ng/L for the COC). There were no statistically significant treatment differences in prothrombin 1 + 2 or d-dimer (p = 0.667 and p = 0.884, respectively) and no statistically significant treatment sequence or period effects.

Conclusion

A COC containing 0.03 mg EE and 0.15 mg levonorgestrel and the novel Bayer patch have comparable influence on hemostatic endpoints. Both treatments were well-tolerated by subjects.