Covalent inhibition of recombinant human carboxylesterase 1 and 2 and monoacylglycerol lipase by the carbamates JZL184 and URB597

Carboxylesterase type 1 (CES1) and CES2 are serine hydrolases located in the liver and small intestine. CES1 and CES2 actively participate in the metabolism of several pharmaceuticals. Recently, carbamate compounds were developed to inhibit members of the serine hydrolase family via covalent modification of the active site serine. URB597 and JZL184 inhibit fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively; however, carboxylesterases in liver have been identified as a major off-target. We report the kinetic rate constants for inhibition of human recombinant CES1 and CES2 by URB597 and JZL184. Bimolecular rate constants (k_inact/K_i) for inhibition of CES1 by JZL184 and URB597 were similar [3.9 (±0.2) × 10³ M^?1 s^?1 and 4.5 (±1.3) × 10³ M^?1 s^?1, respectively]. However, k_inact/K_i for inhibition of CES2 by JZL184 and URB597 were significantly different [2.3 (±1.3) × 10² M^?1 s^?1 and 3.9 (±1.0) × 10³ M^?1 s^?1, respectively]. Rates of inhibition of CES1 and CES2 by URB597 were similar; however, CES1 and MAGL were more potently inhibited by JZL184 than CES2. We also determined kinetic constants for spontaneous reactivation of CES1 carbamoylated by either JZL184 or URB597 and CES1 diethylphosphorylated by paraoxon. The reactivation rate was significantly slower (4.5×) for CES1 inhibited by JZL184 than CES1 inhibited by URB597. Half-life of reactivation for CES1 carbamoylated by JZL184 was 49 ± 15 h, which is faster than carboxylesterase turnover in HepG2 cells. Together, the results define the kinetics of inhibition for a class of drugs that target hydrolytic enzymes involved in drug and lipid metabolism.     

Studies on percutaneous penetration of chemicals – Impact of storage conditions for excised human skin

According to international guidelines skin penetration experiments can be carried out using freshly excised or frozen stored skin. However, this recommendation refers to data obtained in experiments with human cadaver skin. In our study, the percutaneous penetration of the occupationally relevant chemicals anisole, cyclohexanone and 1,4-dioxane was investigated for freshly excised as well as for 4 and 30 days at ?20 °C stored human skin using the diffusion cell technique. As indicator for the impairment of skin barrier by freezing cholesterol dissolution was determined in the solvents in exposure chambers of diffusion cells. Considering the percutaneously penetrated amounts, the following ranking was determined: 1,4-dioxane > anisole > cyclohexanone (decline to a factor of 5.9). The differences of fluxes between freshly excised and frozen stored skin (4 and 30 days) were not significant (p > 0.05). Cholesterol dissolved from the skin indicates no significant differences between freshly excised and frozen stored skin. This study shows that freezing of human skin for up to 30 days does not alter the skin barrier function and the permeability of chemicals.     

The pharmacokinetics of orally administered S-carboxymethyl-l-cysteine in the dog,calf and sheep

The aim of this study was to investigate the feasibility of employing S-carboxymethyl-l-cysteine as a treatment of chronic obstructive pulmonary disease in dogs. To this end the pharmacokinetic parameters of orally administered S-carboxymethyl-l-cysteine were determined in the dog, cow and sheep. Six healthy beagle dogs, six endogenous Greek sheep and four Holstein Fresian calves were orally dosed with 10 mg/kg body weight of S-carboxymethyl-l-cysteine. No significant differences in T_max and T_1/2 were reported between the species. However, significantly higher AUC_(0–last), 21.56 ± 6.67 μg h ml^?1 and AUC_(0–∞), 21.63 ± 6.68 μg h ml^?1 were seen in the dogs compared to the sheep and calves. The calculated V_D was significantly higher in the sheep (10.4 ± 2.7 L kg^?1) and the calves (3.8 ± 0.7 L kg^?1) compared to the dogs (1.0 ± 0.6 L kg^?1). The rank order of increasing C_L was sheep (3.4 ± 2.7 L h^?1 kg^?1) > calves (2.7 ± 0.4 L h^?1 kg^?1) > dogs (0.5 ± 0.2 L h^?1 kg^?1). The result for the dogs was significantly lower that the calculated C_L for the sheep and calves.All these results indicate that the oral administration of S-carboxymethyl-l-cysteine may be useful during the therapeutic management of chronic obstructive pulmonary disease in dogs.     

Human skin penetration of cobalt nanoparticles through intact and damaged skin

Cobalt nanoparticles (CoNPs) are produced for several industrial and biomedical applications but there is a lack of data on human cutaneous absorption. Cobalt is also a skin sensitizer that can cause allergic contact dermatitis. Co applied as NPs, due to their small size and high surface, can penetrate into the skin in higher amount that bulk material. The aim of this study was to evaluate the absorption of Co applied as NPs in both intact and damaged skin. Experiments were performed using Franz cells and 1.0 mg cm^?2 of CoNPs was applied as donor phase for 24 h. Mean Co content of 8.5 ± 1.2 ng cm^?2 and 1.87 ± 0.86 μg cm^?2 were found in the receiving solutions of Franz cells when the CoNPs suspension was applied on intact skin and on damaged skin, respectively. Twenty-four hours Co flux permeation was 76 ± 49 ng cm^?2 h^?1 in damaged skin with a lag time of 2.8 ± 2.1 h. This study suggests that Co applied as NPs is able to penetrate the human skin in an in vitro diffusion cell system.     

Binding of new aminopropan-2-ol compounds to bovine serum albumin, α1 acid glycoprotein and rat serum using equilibrium dialysis and LC/MS/MS

BackgroundThe binding of three new aminopropan-2-ol compounds briefly called 2F109, ANBL and TWo8 with potential cardiovascular activity to bovine serum albumin (BSA), α₁-acid glycoprotein (AGP) and to rat serum was studied. The chemical structures of these compounds are related to carvedilol. They possess an antiarrhythmic and hypotensive activity, and β- and α-adrenolytic mechanism of action. All analogues are weak bases with pKa values 8.65,8.85 and 8.26 for 2F109, ANBL and TWo8, respectively, and they possess lipophilic character (log P > 1.9584).MethodsThe extent of protein binding was determined using equilibrium dialysis in the range 2.5 – 900 μM, and 2.5 – 300 μM for binding of investigated compounds to BSA and AGP, respectively, and the quantitative measurement was done by LC/ESI-MS/MS assay.ResultsThe studied compounds bound to a single class of binding sites on BSA which was characterized by low affinity (K_d for 2F109 = 8.49 × 10^–5 M, for ANBL = 1.92 × 10^–5 M, and for TWo8 = 1.71 × 10^–5 M) and low capacity(n = 0.53 for 2F109,0.132 for ANBL and 0.13 for TWo8). The binding of 2F109, ANBL and TWo8 to AGP revealed one class of binding sites, with moderate affinity (K_d for 2F109 = 4.67 × 10^–6 M, for ANBL = 3.48 × 10^–5 M, and for TWo8 = 1.13 × 10^–5 M) and higher capacity (n = 2.21 for 2F109, 2.76 for ANBL and 2.28 for TWo8).ConclusionThe obtained data indicate that 2F109, ANBL and TWo8 moderately bind to BSA (34.2 – 71.2%) with low capacity (K_a = 6.21 × 10³–7.61 × 10³ M^–1)and strongly bind to AGP(71.5–85.5%)with moderate affinity (K_a = 7.94 × 104⁴.73 ×10⁵ M^–1).     

Determination of lamivudine and zidovudine permeability using a different ex vivo method in Franz cells

IntroductionThe major processes that control the absorption of orally administered drugs are dissolution and gastrointestinal permeation. These processes depend on two main properties: solubility and permeability. Based on these characteristics, the Biopharmaceutical Classification System (BCS) was proposed as a tool to assist in biowaiver and bioavailability prediction of drugs.MethodsThe purpose of the present study was to evaluate the permeability of lamivudine (3TC) and zidovudine (AZT) using a different ex vivo method in Franz cells. A segment of jejunum was inserted in a Franz cells apparatus, in order to assess drug permeability in the apical–basolateral (A–B) and basolateral–apical (B–A) directions. Each drug was added to the donor chamber, collected from the acceptor chamber and analyzed by HPLC. Fluorescein (FLU) and metoprolol (METO) were used as low and high permeability markers, respectively.ResultsThe apparent permeability (P_app) results for the A–B direction were: P_{app FLU A–B} = 0.54 × 10^? 4 cm·s^? 1, P_{app METO A–B} = 7.99 × 10^? 4 cm·s^? 1, P_{app 3TC A–B} = 4.58 × 10^? 4 cm·s^? 1 and P_{app AZT A–B} = 5.34 × 10^? 4 cm·s^? 1. For the B–A direction, the P_app results were: P_{app FLU B–A} = 0.56 × 10^? 4 cm·s^? 1, P_{app METO B–A} = 0.25 × 10^? 4 cm·s^? 1, P_{app 3TC B–A} = 0.24 × 10^? 4 cm·s^? 1 and P_{app AZT B–A} = 0.19 × 10^? 4 cm·s^? 1.DiscussionFor the A–B direction, the P_app results of fluorescein and metoprolol show low and high permeability, respectively, indicating that the membranes were appropriate for permeability studies. For the A–B direction, the P_app results of 3TC and AZT suggest that these antiretroviral drugs have permeability values close to metoprolol. Nevertheless, for the B–A direction the P_app results do not suggest efflux mechanism for any of the drugs. Thereby, the different ex vivo methods using Franz cells can be successfully applied in drug permeability studies, in particular for drug biopharmaceutical classification.     

Comparative evaluation of two dye probes in the rat everted gut sac model for unambiguous classification of P-gp substrate and inhibitor

IntroductionP-glycoprotein (P-gp) plays a crucial role in beta-amyloid efflux from the blood–brain barrier thus becoming a promising pharmacological target in the treatment of Alzheimer's disease (AD). The increase of P-glycoprotein expression and activity by a P-gp inducer could be an effective pharmacological strategy in slowing or halting the progression of AD. Commonly used in vitro methods to classify a P-gp interacting molecule as substrate, inhibitor, modulator or inducer are not always confirmed by in vivo experiments. Here we validate the new dye-probe beta-amyloid (1–40) HiLyte Fluor? TR-labeled (Ab-HiLyte) (Anaspec) P-gp mediated transport in the ex vivo rat everted gut sac assay by using MC18 or MC266, a fully characterized P-gp inhibitor and substrate, respectively, and compare it with the commonly used dye rhodamine.MethodsMale Wistar rats' everted intestines were divided into sacs, each sac was filled with 10 μM Ab-HiLyte with or without 50 μM of MC18 or MC266. Ab-HiLyte concentrations in mucosal fluid were measured spectrophotometrically at 594 nm at each appropriate time.ResultsThe Ab-HiLyte P-gp mediated efflux had a K = 1.00 × 10^? 2 min^? 1 and t_1/2 = 68.74 min, while in the presence of MC18, the Ab-HiLyte efflux turned out to be reduced by an order of magnitude (K = 1.65 × 10^? 3 min^? 1) and the half life is extremely increased (t_1/2 = 419 min). A P-gp substrate, like MC266, determines no change in the efflux of Ab: the kinetic constant and the half life turned out to be unmodified (K = 1.81 × 10^? 2 min^? 1 and t_1/2 = 38.28 min).DiscussionThe results demonstrate that the new dye probe, Ab-HiLyte, could be a probe of choice to unequivocally distinguish between a P-gp substrate and an inhibitor. This is particularly important as different groups obtain a controversial classification of the same compound.     

Using transdermal iontophoresis to increase granisetron delivery across skin in vitro and in vivo: Effect of experimental conditions and a comparison with other enhancement strategies

The objectives of the study were (i) to investigate the effect of experimental parameters on the iontophoretic transport of granisetron, (ii) to identify the relative contributions of electromigration (EM) and electroosmosis (EO), (iii) to determine the feasibility of delivering therapeutic amounts of drug for the treatment of chemotherapy-induced nausea and vomiting and (iv) to test the in vitro results in a simple animal model in vivo. Preliminary in vitro studies using aqueous granisetron formulations investigating the effect of drug concentration (5, 10, 20 and 40 mM) and current density (0.1, 0.2, 0.3 mA cm^?2) were performed using porcine ear skin. As expected, cumulative delivery in vitro at the 20 and 40 mM concentrations was significantly greater than that at 5 and 10 mM, which were not statistically different (p < 0.05). Increasing the applied current density from 0.1 to 0.3 mA cm^?2 resulted in a ～4.2-fold increase in iontophoretic flux. Furthermore, in the absence of Na⁺ in the formulation, no dependence of iontophoretic flux on drug concentration was reported (at a granisetron concentration of 40 mM, the transport rate was 2.93 ± 0.62 μg cm^?2 min^?1). Co-iontophoresis of acetaminophen was used to show that EM was the predominant transport mechanism accounting for 71–86% of total granisetron delivery. In vivo studies in Wistar rats (40 mM granisetron; application of 0.3 mA cm^?2 for 5 h with Ag/AgCl electrodes and salt bridges) showed an average iontophoretic input rate (k_input) of 0.83 ± 0.26 μg min^?1 and a maximum plasma concentration (C_max) of 0.092 ± 0.004 μg ml^?1. Based on these results and given the known pharmacokinetics, transdermal iontophoresis could achieve therapeutic drug levels for the management of chemotherapy-induced emesis using a reasonably sized (4–6 cm²) patch.     

Human Skin is Permselective for the Small,Monovalent Cations Sodium and Potassium but not for Nickel and Chromium

The molar conductance of excised human skin (Λ_skin) immersed in electrolyte solutions comprising four cationic (Na⁺, K⁺, Ni^2 +, and Cr^3 +) and five anionic (Cl^?, NO₃^?, SO₄^2 ?, CrO₄^2 ?, and Cr₂O₇^2 ?) species was determined as a function of concentration in Franz diffusion cells. Cation transport numbers for four of these electrolytes were measured in Franz cells by the electromotive force method. Parallel experiments were conducted in solutions alone to establish the validity of the technique. Molar conductance decreased with increasing concentration, following the Kohlrausch law, over a 4–12-fold concentration range. Molar conductance and cation transport values at infinite dilution were extrapolated from these data and used to estimate ionic conductances at infinite dilution. These values were subsequently used to calculate limiting ion mobilities and diffusivities in solution and skin. Results for skin showed the expected increase in cation permselectivity for monovalent cations and a 40–110-fold reduction in effective diffusivities with respect to those in solution. However, Ni^2 + and Cr^3 + were relatively less mobile in skin than in solution. Salt diffusivities calculated from ionic mobilities in skin provided a partial explanation for the difference in allergenic potency of NiCl₂compared with NiSO₄ and Cr^3 + versus Cr^6 + salts.     

Effect of melamine on potassium currents in rat hippocampal CA1 neurons

As an industrially synthesized chemical, melamine has been applied in a wide range of areas. However, many questions on the adverse effect and toxicity of melamine have been emerged, recently. In this investigation, the cytotoxicity of melamine on PC12 cells was evaluated. Furthermore, the effect of melamine on the transient outward potassium current (I_A) and the delayed rectifier potassium current (I_K) in hippocampal CA1 pyramidal neurons of rat was studied using whole-cell patch-clamp technique. The results showed that melamine-induced cell death in a concentration and time-dependent manner, and produced a concentration-dependent inhibition in amplitudes of I_A and I_K at any concentrations (5 × 10⁻⁴, 5 × 10⁻⁵, and 5 × 10⁻⁶ g/ml). Moreover, at higher concentration (5 × 10⁻⁴ g/ml), melamine had observable effects of the steady-state inactivation of I_A, that is melamine shifted inactivation curve of I_A towards hyperpolarization. The spontaneous firing frequency was increased as well. These results suggest that the regulation of I_A and I_K induced by melamine would make neurons display aberrant firing properties and abnormal neuronal discharge, which could be a possible underlying mechanism for the melamine-induced neurotoxicity.     

In vivo sustained dermal delivery and pharmacokinetics of interferon alpha in biphasic vesicles after topical application

Biphasic vesicles, a novel nanostructured lipid-based delivery system show potential for topical application of interferon alpha (IFN α) for the treatment of human papillomavirus (HPV) infections (anogenital warts). Dermal delivery of IFN α encapsulated in biphasic vesicles (BPV-IFN α), applied topically to the skin, was characterized in a guinea pig model.BPV-IFN α (1 g, 2 MIU/g) was topically applied either as a single or multiple treatments on the skin of guinea pigs. As a comparison with currently used regimens, IFN α solution was administered intravenously or intradermally. Skin and serum samples were collected over 96 h, IFN α levels were determined by an antiviral assay, and half-life (t_1/2) and elimination (k) rates were calculated.Topical BPV-IFN α treatment resulted in maximum skin levels (about 100,000 U/100 cm²) of IFN α within 6 h and maintained for 72–96 h. Clearance from the skin after intradermal injections was initially fast (t_1/2 0.62 h, k 1.1179 h⁻¹), followed by a slower steady decrease after 6 h. After intravenous and intradermal administration, IFN α was rapidly cleared from the serum, t_1/2 0.75 h, k 0.9271 h⁻¹ and t_1/2 1.28 h, k 0.5421 h⁻¹, respectively, whereas after topical application, IFN α levels remained below 100 U/mL. Topical application of BPV- IFN α resulted in sustained delivery of biologically active IFN α locally into skin with minimal systemic exposure.     

Intracellular disposition of arabinogalactan and asialofetuin in HepG2 cells

The binding and uptake of arabinogalactan and asialofetuin in HepG2 cells was kinetically characterized using ^I25I-labeled ligands. The number of binding sites (n) and the association constant (K) of arabinogalactan was 1.9 × 10⁵ ± 1.2 × 10⁵ sites/cell and 5.0 × 10⁶ ± 3.9 × 10⁶ M^? 1, respectively, whereas the n and K_a of asialofetuin was 2.7 × 10⁵ ± 1.1 × 10⁵ sites/cell and 1.1 × 10⁷ ± 0.7 × 10⁷ M^? 1, respectively. These results suggest that the binding capacity of HepG2 cells for arabinogalactan is lower than that for asialofetuin. Moreover, the amount of arabinogalactan uptake by HepG2 cells was lower than that of asialofetuin. Thus, asialofetuin was preferentially bound and internalized by hepatoma cells compared to arabinogalactan.     

Upgrading bio-oil produced from the catalytic pyrolysis of sugarcane (Saccharum officinarum L) straw using calcined dolomite

In this study, the catalytic pyrolysis of sugarcane straw (SCS) into bio-oil and chemicals using calcined dolomite was applied for upgraded bio-oil production. Experiments were performed in a custom-built SS316 tube reactor, and the effects of the pyrolysis parameters, including the different dolomite calcination conditions, temperature (400-600 °C), biomass feed rate (0.3–1.2 kg h^?1), sweeping gas flow rate (80–200 cm³ min^?1) and average size distribution (250–1000 µm), were systematically investigated. The results showed that the SCS catalytic pyrolysis process obtained liquid yields of 36.15 wt%, gas yields of 52.09 wt% and solid yields of 11.76 wt% when using a pyrolysis temperature, biomass feed rate, nitrogen sweep gas, and average biomass size of 450 °C, 0.6 kg h^?1, 80 cm³ min^?1 and 500 µm, respectively, with 10 wt% calcined dolomite. The calcined dolomite influenced the bio-oil components from the carbonylation and the cracking of volatile vapor and resulted in an upgraded bio-oil with a lower oxygen content, higher gross calorific value and decreased acid corrosion.     

Extractive spectrophotometric assay of cyclizine in a pharmaceutical formulation and biological fluids

Two highly sensitive and simple spectrophotometric methods were developed to quantitate the drug cyclizine (CYC) in its pure form and in a pharmaceutical formulation. The two methods involved ion-associate formation reactions (method A) with mono-acid azo dyes, i.e., sudan (I) and sudan (II), as well as ion-pair reactions (method B) with bi-azo dyes, i.e., sudan (III), sudan (IV) and sudan red 7B (V). The reactions were extracted with chloroform, and the extraction products were quantitatively measured at 480, 550, 500, 530 and 570 nm using reagents I–V, respectively. The reaction conditions were monitored and optimised. The Beer plots for reagents I–V showed linear relationships for the concentrations of 4.2–52.0, 5.4–96.0, 3.5–43.0, 4.4–80.0 and 0.6–18.0 μg mL^?1, respectively, with molar absorptivities of 2.2 × 10⁴, 4.1 × 10⁴, 3.6 × 10⁴, 2.5 × 10⁴ and 1.3 × 10⁴ L mol^?1 cm^?1, respectively. Sandell sensitivities and detection limits were calculated and analysed. The implementation of the two methods to the analysis of a commercial tablet (Valoid) succeeded, and the recovery study suggested that there was no interference from common excipients in the tablet. Regarding the accuracy and precision of the methods, a statistical comparison of the results was performed using Student’s t-test and the F-test at the 95% confidence level. The accuracy and precision of the proposed methods were not significantly different.     

Na+–H+ exchange inhibition attenuates ischemic injury in rat random pattern skin flap: The role of mitochondrial ATP-sensitive potassium channels

Necrosis of distal portion of skin flaps due to ischemia still remains a problem in plastic surgery. Following ischemia, a cascade of deleterious events including over-activity of Na⁺–H⁺ Exchanger (NHE) takes place. In present study we evaluated the effect of the potent NHE inhibitor, 5-(N-ethyl-N-isopropyl) amiloride (EIPA) on ischemic tissue injury in a skin flap model, and investigated the role of mitochondrial ATP-sensitive K⁺ channels (K_ATP) in this phenomenon. Seventy-eight rats were randomly divided into thirteen treatment groups (6 rats each). Four groups received different doses of EIPA in the flap. EIPA/GLY group received an effective dose of a K_ATP channel blocker, glibenclamide (GLY, 0.3 mg/kg) intraperitoneally (i.p.) 30 min before raising the flap, and a local effective dose of EIPA (0.1 mM) immediately after raising the flap. EIPA/diazoxide group (EIPA/DIA) received a sub-effective dose of diazoxide (7.5 mg/kg i.p.) 30 min before raising the flap and a local sub-effective dose of EIPA (0.075 mM). EIPA 0.1 and 0.2 mM significantly increased flap survival area compared to control group (56.01±6.1%, P<0.001). The protective effect of EIPA (0.1 mM) was abolished by administration of glibenclamide (0.3 mg/kg i.p.). Co-administration of a sub-effective dose of EIPA (0.075 mM), with a sub-effective dose of diazoxide (7.5 mg/kg i.p.) significantly improved flap survival (P<0.05). We demonstrated that the NHE inhibitor, EIPA can increase random pattern skin flap survival. Administration of diazoxide potentiates this effect, while glibenclamide abolishes that, implicating that the protective effect of EIPA is mediated through mitochondrial-K_ATP channels.     

Berberine possesses muscarinic agonist-like properties in cultured rodent cardiomyocytes

Berberine, a natural product alkaloid, has been shown to display a wide array of pharmacological effects. Generally, the mechanism of action of each of these effects has not been well described. The aim of the present study is to test the hypothesis that some of berberine's cardiovascular effects are mediated through activation of cardiac M2 muscarinic cholinergic receptors. In our studies, we tested the ability of berberine to alter the contraction rate of cultured neonatal rodent cardiomyocytes. In these spontaneously contracting primary cultured cells, berberine reduced the contraction rate in a manner independent of β-adrenergic receptor blockade but sensitive to pertussis toxin, a Gi/o G protein inhibitor. Muscarinic antagonists completely blocked the effect of berberine on contraction rate of cardiomyocytes, whereas the effect of berberine was not opposed by antagonists to opioid, adenosine or α-adrenergic receptors. Further, berberine bound to muscarinic receptors of adult mouse heart membranes with relatively high affinity (K_i = 5.4 × 10^?6 M) comparable to that of the classic muscarinic agonist, carbachol, and to muscarinic M2 receptors exogenously expressed in HEK 293 cells (K_i = 4.9 × 10^?6 M). Therefore, the findings of the present study suggest that berberine is a muscarinic agonist at M2 receptors, potentially explaining some of its reported cardiovascular effects.     

Percutaneous absorption of herbicides derived from 2,4-dichlorophenoxyacid: Structure–activity relationship

Ethyl to octyl esters of 2,4-dichlorophenoxy-acetic acids (2,4DAA), 2,4-dichlorophenoxy-propionic acids (2,4DPA) or 2,4-dichlorophenoxy-butyric acids (2,4DBA) are present in the most commonly used herbicides. Their use involves a significant risk of skin exposure, but little is known about the percutaneous flux of these substances. Studies have shown that percutaneous transition of esters may be dependent on their hydrolysis by esterases present in the skin. In this study, we describe ex vivo percutaneous absorption of seven pure esters (methyl to decyl) with a 2,4DA structure for rats (n = 6) and humans (n = 7). Esters were applied at 50 μL cm⁻² to dermatomed skin (approximately 0.5 mm thick) for 24 h. The enzymatic constants for hydrolysis of each ester by skin esterases were determined in vitro using skin homogenates from both species. Structure–activity relationships linking the evolution of the ex vivo percutaneous flux of esters and the 2,4D structure with enzymatic (V_max; K_m) and/or physical parameters (molecular weight, molecular volume, size of the ester, log(k_ow)) were examined to develop a good flux estimation model. Although the percutaneous penetration of all of the esters of the 2,4D family are “esterase-dependent”, the decreasing linear relationship between percutaneous penetration and hyrophobicity defined by the logarithm for the octanol–water partition coefficient (log(k_ow)) is the most pertinent model for estimating the percutaneous absorption of esters for both species. The mean flux of the free acid production by the esterases of the skin is not the limiting factor for percutaneous penetration. The rate of hydrolysis of the esters in the skin decreases linearly with log(k_ow), which would suggest that either the solubility of the esters in the zones of the skin that are rich in esterases or the accessibility to the active sites of the enzyme is the key factor. The structure–activity relationship resulting from this study makes it possible, in humans and in rats, to make a good estimate of the ex vivo percutaneous fluxes for all pure esters of this family of herbicides.     

Plasmonic gold nanoparticles possess the ability to open potassium channels in rat thoracic aorta smooth muscles in a remote control manner

Colloidal gold nanoparticles (AuNPs) of ~ 5 nm core size and Zeta-potential of − 35 mV, having absorption maximum and plasmon resonance in the range of 510–570 nm, were studied as a potential K⁺-channel opener in vascular smooth muscle (SM) cells. Experimental design of the study comprised SM contractile recordings.When externally applied to the organ bath, AuNPs (10^− 6 − 3 × 10^− 4 M) led to decrease in amplitude of norepinephrine-induced contractions in a concentration-dependent and endothelium-independent manner in SM thoracic aorta, with mean value of pD₂ (− log EC₅₀) 4.2 ± 0.03, E_max = 55 ± 4%. Being added to the bath solution in concentration of 10^− 4 M, AuNPs significantly increased whole cell peak outward current at + 70 mV from 32 ± 2 pA/pF to 59 ± 5 pA/pF (n = 14, P < 0.05). External irradiation using a 5 mW/532 nm green laser, to facilitate plasmon resonance, led to an increment in the AuNPs-induced macroscopic outward potassium current (I_K) from 59 ± 5 pA/pF to 74 ± 1 pA/pF (n = 10, P < 0.05). Paxilline (500 nM), when added to the external bathing solution, significantly decreased AuNPs-induced increment of I_K in SM cells. Single channel recordings provided a direct confirmation of BK_Ca activation by AuNPs at the single-channel level. Application of AuNPs to the bath potentiated BK_Ca activity with a delay of 1–2 min, as was seen initially by more frequent channel openings followed by the progressive appearance of additional open levels corresponding to multiple openings of channels with identical single-channel amplitudes. Eventually, after 10–15 min in the presence of AuNPs and especially when combined with the green laser illumination, there was a massive increase in channel activity with > 10 channels evident. When irradiated by laser, AuNPs significantly increased the amplitude of maximal AuNPs-induced relaxation from 55 ± 5% to 85 ± 5% (n = 10, P < 0.05) while the sensitivity of SM to AuNPs was without changes.In summary, plasmonic AuNPs possess the ability to activate BK_Ca channel opening in vascular SM. Laser irradiation facilitates this effect due to local plasmon resonance that, in turn, further increases BK_Ca channel activity causing SM relaxation.     

Formulation,optimization and evaluation of transferosomal gel for transdermal insulin delivery

The present study deals with the development of transferosomal gel containing insulin by reverse phase evaporation method for painless insulin delivery for use in the treatment of insulin dependent diabetes mellitus. The effect of independent process variables like ratio of lipids (soya lecithin:cholesterol), ratio of lipids and surfactants, and ratio of surfactants (Tween 80:sodium deoxycholate) on the in vitro permeation flux (μg/cm²/h) of formulated transferosomal gels containing insulin through porcine ear skin was optimized using 2³ factorial design. The optimal permeation flux was achieved as 13.50 ± 0.22 μg/cm²/h with drug entrapment efficiency of 56.55 ± 0.37% and average vesicle diameter range, 625–815 nm. The in vitro insulin permeation through porcine ear skin from these transferosomal gel followed zero-order kinetics (R² = 0.9232–0.9989) over a period of 24 h with case-II transport mechanism. The in vitro skin permeation of insulin from optimized transferosomal gel by iontophoretic influence (with 0.5 mA/cm² current supply) also provided further enhancement of permeation flux to 17.60 ± 0.03 μg/cm²/h. The in vivo study of optimized transferosomal gel in alloxan-induced diabetic rat has demonstrated prolonged hypoglycemic effect in diabetic rats over 24 h after transdermal administration.     

Microneedle-assisted delivery of verapamil hydrochloride and amlodipine besylate

The aim of this project was to study the effect of stainless steel solid microneedles and microneedle rollers on percutaneous penetration of verapamil hydrochloride and amlodipine besylate.Verapamil, 2-(3,4-dimethooxyphenyl)-5-[2-(3,4 dimethoxyphenyl)ethyl-methyl-amino]-2-propan-2-yl-pentanenitrile is a calcium channel blocker agent that regulates high blood pressure by decreasing myocardial contractilty, heart rate and impulse conduction. Amlodipine, (R, S)-2-[(2-aminoethoxy) methyl]-4-(2-chlorophenyl)-3-ethoxycarbonyl-5-methoxycarbonyl-6-methyl-1, 4-dihydropyridine, is a calcium channel blocker that is used for the management of hypertension and ischemic heart disease. Passive penetration of verapamil and amlodipine across the skin is low. In vitro studies were performed with microneedle-treated porcine ear skin using vertical static Franz diffusion cells (PermeGear, Hellertown, PA, USA). The receiver chamber contained 5 ml of PBS (pH7.4) and was constantly maintained at 37 °C temperature with a water circulation jacket. The diffusion area of the skin was 1.77 cm². The donor compartment was loaded with 1 ml of the solution containing 2.5 mg/ml of amlodipine besylate. The donor chamber was covered with parafilm to avoid evaporation. Passive diffusion across untreated porcine skin served as control. Aliquots were taken every 2 h for 12 h and analyzed by liquid chromatography–mass spectrometry. Transcutaneous flux of verapamil increased significantly from 8.75 μg/cm²/h to 49.96 μg/cm²/h across microneedle-roller treated porcine skin. Percutaneous flux of amlodipine besylate following the use of stainless steel microneedles was 22.39 μg/cm²/h. Passive flux for the drug was 1.57 μg/cm²/h. This enhancement of amlodipine flux was statistically significant. Transdermal flux of amlodipine with microneedle roller was 1.05 μg/cm²/h in comparison with passive diffusion flux of 0.19 μg/cm²/h. The difference in flux values was also statistically significant. Stainless steel solid microneedles and microneedle rollers increased percutaneous penetration of verapamil hydrochloride and amlodipine besylate. It may be feasible to develop transdermal microneedle patches for these drugs.