Discovery of transdermal penetration enhancers by high-throughput screening

Although transdermal drug delivery is more attractive than injection, it has not been applied to macromolecules because of low skin permeability. Here we describe particular mixtures of penetration enhancers that increase skin permeability to macromolecules (approximately 1-10 kDa) by up to approximately 100-fold without inducing skin irritation. The discovery of these mixtures was enabled by an experimental tool, in vitro skin impedance guided high-throughput (INSIGHT) screening, which is >100-fold more efficient than current tools. In vitro experiments demonstrated that the mixtures delivered macromolecular drugs, including heparin, leutinizing hormone releasing hormone (LHRH) and oligonucleotides, across the skin. In vivo experiments on hairless rats with leuprolide acetate confirmed the potency and safety of one such mixture, sodium laureth sulfate (SLA) and phenyl piperazine (PP). These studies show the feasibility of using penetration enhancers for systemic delivery of macromolecules from a transdermal patch.     

Investigation of substituted 6-aminohexanoates as skin penetration enhancers

Skin penetration enhancers are compounds used to facilitate the transdermal delivery of drugs that are otherwise not sufficiently permeable. Through a synthetic route implementing two series of esters, we generated transdermal penetration enhancers by a multi-step reaction with substituted 6-aminohexanoic acid. We present the synthesis of all newly prepared compounds here with structural confirmation accomplished by (1)H NMR, (13)C NMR, IR and mass spectroscopy (MS). The lipophilicity (logk) of all compounds was determined via RP-HPLC and their hydrophobicity (logP/ClogP) was also calculated using two commercially available programs. Ab initio calculations of geometry and molecular dynamic simulations were employed to investigate the 3-dimensional structures of selected compounds. The transdermal penetration-enhancing activity of all the synthesized esters were examined in vitro and demonstrated higher enhancement ratios than oleic acid. Compounds 2e (C(10) ester chain) and 2f (C(11) ester chain) exhibited the highest enhancement ratios. It can be concluded that the series non-substituted at the C((2)) position by a ω-lactam ring showed significantly higher activity than those with azepan-2-one. None of the prepared compounds penetrated through the skin. All of the investigated agents demonstrated minimal anti-proliferative activity using the SK-N-MC neuroepithelioma cell line (IC(50)>6.25μM), suggesting these analogs would have a low cytotoxic profile when administered in vivo as chemical penetration enhancers. The correlation between the chemical structure of the studied compounds and their lipophilicity is discussed in regards to transdermal penetration-enhancing activity.     

Cardiovascular effects and pharmacokinetics of the carboxylic ionophore monensin in dogs and rabbits

Monensin, a carboxylic ionophore, produces strong pressor, positive chronotropic effects and elevates the blood glucose level when injected intravenously (100 μg/kg) into pentobarbital anesthetized dogs or administered orally (2 mg/kg) to conscious dogs. Intravenously administered monensin disappeared from the blood rapidly with a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of ca. 2.5 min and, in the conscious dogs, ingested monensin showed a peak plasma level 90 min after feeding; this coincided with the time of maximum increase in arterial blood pressure and blood glucose. In conscious rabbits, although higher doses of monensin were administered, 200 μg/kg intravenously and 10 mg/kg orally, its cardiovascular effects were less than observed in the dog and were slower in onset. This correlated with slower clearing of injected monensin from the blood ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 8}\text{min}$) and slower entry of ingested monensin from the gut into the blood. Rabbit plasma and tissue levels were higher 17 hr after oral ingestion of monensin than six hr after ingestion.     

Cardiovascular and hemorheological effects of three modified human hemoglobin solutions in hemodiluted rabbits

The cardiovasculareffects of human albumin (Alb) and three human hemoglobin(Hb) solutions, dextran-benzene-tetracarboxylate Hb,-crosslinked Hb, ando-raffinose-polymerized Hb werecompared in anesthetized rabbits undergoing acute isovolemichemodilution with Hct reduction from 41.4 ± 2.7 to 28.8 ± 1.6%. The impact of the vasoconstricting properties of Hb was examinedby measuring heart rate (HR), mean arterial pressure (MAP), abdominalaortic, and femoral arterial blood flow, vascular resistance (VR), and aortic distension during the first 3 h after hemodilution. The impactof the hemorheological parameters was assessed by measurements ofhemodiluted blood viscosity. In contrast to Alb, the Hb solutions elicited an immediate increase in MAP (20-38%). The effects of Alb and Hb solutions on HR, as well as on aortic and femoral arterial blood flow, were similar. VR decreased with Alb (20-28%) andincreased with all three Hb solutions (30-90%), but the MAP andVR rising trends were different with each Hb solution. Aorticdistension decreased in Hb groups compared with the Alb group for thefirst 60 min. The viscosity of hemodiluted blood was similar for all groups at high shear rates but was dependent on the viscosity of thesolutions at low shear rates. We conclude that the vasoconstriction elicited by the Hb solutions overrides the vasodilation associated withviscosity changes due to hemodilution and would be the major factorresponsible to the cardiovascular changes.     

Strain-dependent effects of cognitive enhancers in the mouse

A series of cognitive enhancers (CEs) have been reported to increase spatial memory in rodents, information on behavioral effects, however, is limited. The aim of the study was therefore to examine the behavioral effects of three CEs in two well-documented inbred mouse strains. C57BL/6J and DBA/2 mice were administered intraperitonial. D-cycloserine (DCS; NMDA receptor agonist), 1-(4-Amino-5-chloro-2-methoxyphenyl)-3-[1-butyl-4-piperidinyl]-1-propanone hydrochloride (RS67333; 5HT4-receptor agonist), and (R)-4-{[2-(1-methyl-2-pyrrolidinyl)ethyl]thio}phenol hydrochloride (SIB-1553A; beta-4-nicotinic receptor agonist) and tested in the open field (OF), elevated plus maze (EPM), neurological observational battery and rota-rod. Cognitive performance was tested in the Morris water maze. All compounds modified behavioral performance in the OF, DCS showed an anxiolytic effect in the EPM, and differences in the observational battery were observed i.e. vestibular drop was decreased by SIB-1553A and RS67333 treatment in C57BL/6J and increased with DCS treatment in DBA/2 mice. In the rota rod SIB-1553A improved motor performance. DCS effects on learning and memory was comparable to controls whereas the other compounds impaired performance in the Morris water maze. In conclusion, behavioral testing of CEs in the mouse revealed significant changes that may have to be taken into account for evaluation of CEs, interpretation of cognitive studies and warrant further neurotoxicological studies. Moreover, strain-dependent differences were observed that in turn may confound results obtained from behavioral and cognitive testing.     

Physical and chemical permeation enhancers in transdermal delivery of terbutaline sulphate

Conclusions  The efficacy of a magnetic field to act as a permeation enhancer was demonstrated. Because in vitro and in vivo performance of F1 and F2 were comparable, the substitution of chemical enhancers by magnetic field in transdermal delivery systems appears to be possible.     

Identification of siRNA delivery enhancers by a chemical library screen

Most delivery systems for small interfering RNA therapeutics depend on endocytosis and release from endo-lysosomal compartments. One approach to improve delivery is to identify small molecules enhancing these steps. It is unclear to what extent such enhancers can be universally applied to different delivery systems and cell types. Here, we performed a compound library screen on two well-established siRNA delivery systems, lipid nanoparticles and cholesterol conjugated-siRNAs. We identified fifty-one enhancers improving gene silencing 2–5 fold. Strikingly, most enhancers displayed specificity for one delivery system only. By a combination of quantitative fluorescence and electron microscopy we found that the enhancers substantially differed in their mechanism of action, increasing either endocytic uptake or release of siRNAs from endosomes. Furthermore, they acted either on the delivery system itself or the cell, by modulating the endocytic system via distinct mechanisms. Interestingly, several compounds displayed activity on different cell types. As proof of principle, we showed that one compound enhanced siRNA delivery in primary endothelial cells in vitro and in the endocardium in the mouse heart. This study suggests that a pharmacological approach can improve the delivery of siRNAs in a system-specific fashion, by exploiting distinct mechanisms and acting upon multiple cell types.     

Cardiovascular, metabolic and endocrine effects of chemical sympathectomy and of adrenal demedullation in fetal sheep

A procedure in fetal sheep for causing peripheral sympathectomy by regular intravascular guanethidine sulphate administration and for causing adrenal demedullation by intragland injection of acid formalin is reported. Demedullation substantially removed adrenaline from the fetal circulation, but has a small effect only on noradrenaline. Plasma noradrenaline levels were depressed by 50% when demedullated fetuses were also subject to peripheral sympathectomy by guanethidine sulphate treatment. This provides some evidence that the paraganglia in the sheep fetus contributes to resting plasma catecholamines. Furthermore the ability of adrenal demedullation to increase markedly this pool of extra-adrenal chromaffin tissue indicates that in the fetus adrenal activity regulates the growth of these para-aortic bodies. In response to sympathectomy plasma vasopressin concentrations rose substantially, whilst adrenal demedullation caused a small rise. Demedullation and sympathectomy depressed fetal plasma glucose and elevated plasma cortisol. In both sympathectomised and adrenal demedullated fetuses resting heart rate and blood pressure was not depressed. However in those with a depleted peripheral nervous system periods of cardiovascular instability were apparent after 2-3 days of treatment with guanethidine sulphate. Hence there were regular episodes where fetal blood pressure and heart rate fell sharply followed 60-90s later by very large increases in blood pressure sustained for up to 10 min and associated with substantial production of plasma vasopressin and catecholamines. These results show that fine cardiovascular control in the fetus requires an intact sympathetic system as the endocrine system is too slow responding to effectively maintain reflex vascular control.     

Polycationic lipophilic-core dendrons as penetration enhancers for the oral administration of low molecular weight heparin

Two polycationic lipophilic-core carbohydrate-based dendrons 2a-b and five polycationic lipophilic-core peptide dendrons 3-6, containing four arginine or lysine terminal residues, were synthesized and then tested in rats as penetration enhancers for the oral delivery of low molecular weight heparin. Better results were obtained with dendrons containing terminal lysine residues than terminal arginine. A significant anti-factor Xa activity was obtained when low molecular weight heparin was coadministered with dendron 5.     

Cardiovascular, respiratory and glottic effects of sodium salicylate administered into different parts of the circulation in rabbits.

The authors studied the effect of sodium salicylate administered into different parts of the circulatory system on various cardiovascular, respiratory and glottic parameters in Pentobarbital-anaesthetized rabbits. The results show that apnoea, bradycardia and hypotension, followed by hypertension, can also be caused by the extrathoracic action of salicylate. Cardiovascular responses induced by injecting salicylate into the carotid circulation are qualitatively the same, even after vagotomy, as in injection into the femoral vein. Salicylate injected into the common carotid artery, the internal carotid artery or the femoral vein causes inspiratory apnoea in rabbits, with powerful electrical activity of the diaphragm and an intrapleural pressure shift to marked inspiratory values. Laryngoconstriction occurs simultaneously, despite inspiratory apnoea. The injection of salicylate into the common carotid artery after bilateral vagotomy induces expiratory (not inspiratory) apnoea, indicating that the vagi play an important role in the origination of inspiratory apnoea in rabbits.     

渗透剂对高效氯氰菊酯毒力和三种鳞翅目害虫上表皮超微结构的影响

以甜菜夜蛾Spodoptera exigua (Hübner)、小菜蛾Plutella xylostella (L.)和棉铃虫Helicoverpa armigera (Hübner)为试虫,通过室内生物测定技术,研究了氮酮、噻酮和N-甲基-2-吡咯烷酮等3种渗透剂对高效氯氰菊酯毒力的影响;利用扫描电镜观察了甜菜夜蛾和小菜蛾4龄幼虫对照组和渗透剂处理组间上表皮超微结构的变化。结果表明：含3.0% N-甲基-2-吡咯烷酮的高效氯氰菊酯对小菜蛾幼虫LD₅₀为0.0074 μg/头、毒力系数为2.0,对高效氯氰菊酯增效作用明显,而对其他两种试虫没有增效作用;氮酮和噻酮对高效氯氰菊酯对3种鳞翅目幼虫毒力均无增效作用。甜菜夜蛾对照组上表皮蜡质层和护蜡层清晰可见,渗透剂处理组上表皮护蜡层有不同程度的消失;而小菜蛾对照组和N-甲基-2-吡咯烷酮处理组上表皮护蜡层没有明显变化,只有氮酮处理组上表皮的护蜡层消失。以上结果提示,渗透剂对上表皮护蜡层的影响不是其对高效氯氰菊酯增效作用的机制。     

Dynamics of proteins and lipids in the stratum corneum: effects of percutaneous permeation enhancers

We have spin labeled the stratum corneum (SC) with a lysine specific reagent, succinimidyl-2,2,5,5-tetramethyl-3-pirroline-1-oxyl-carboxylate spin label (SSL), to assess the dynamics and hydration degree of SC proteins by electron paramagnetic resonance (EPR) spectroscopy taking measurements directly from the intact tissue. Treating the SC with two percutaneous penetration enhancers, 8 M urea or 20% (v/v) 1-methyl-2-pyrrolidone (1 MP), destabilizes the proteins thus promoting more mobile and solvent-exposed protein conformations. Upon SC lipid depletion the nitroxide side chain becomes more solvent exposed, suggesting that the removal of hygroscopic substances in the extraction process favors more hydrated protein conformations. On the other hand, the treatments with 8 M urea or 40% (v/v) 1 MP did not alter significantly the fluidity in the SC lipid domain as assessed by the probe 5-doxyl stearic acid; these permeation enhancers, specially 1 MP, seem to increase the probe solubility in the solvent leading to a considerable fraction of spin label to be removed from the lipid domain.