Enthalpies and constants of dissociation of several neutral and cationic acids in aqueous and methanol/water solutions at various temperatures

The acidic dissociation enthalpies and constants of anilinium, protonated tris(hydroxymethyl)aminomethane (HTris⁺), benzoic and acetic acids, have been determined at several temperatures in pure water and in methanol/water mixtures by potentiometry and by isothermal titration microcalorimetry (ITC). The pK_a values determined by both techniques are in accordance when the dissociation process involves large amounts of heat. However, for the neutral acids the ITC technique gave slightly lower pK_a values than those from potentiometry at the highest temperatures studied due to the small amounts of heat involved in the acidic dissociation. The dissociation enthalpies have been determined directly by calorimetry and the obtained values slightly decrease with the increase of temperature. Therefore, only a rough estimation of the dissociation enthalpies can be obtained from potentiometric pK_a by means of the Van’t Hoff approach.     

马钱子碱脂质体的大鼠离体透皮性能和兔皮肤刺激性研究

采用薄膜-超声分散法制备马钱子碱脂质体,并考察了其对大鼠离体皮肤的透过性能及对家兔皮肤的刺激性。结果表明,制品平均粒径(150±25)nm、包封率61%、载药量6%。体外透皮试验表明,与乳膏相比,制品透皮速率较慢,皮肤层中药物贮存量较高。兔皮肤刺激性试验表明,1日给予2次时对皮肤基本无刺激,但连续用药2周,则会产生水肿、溃疡等局部不良反应。     

崖柏精油的急性经口毒性、皮肤刺激性和致敏性实验

摘 要 目的：评价崖柏精油的急性经口毒性、皮肤刺激性和致敏性,为其临床安全用药提供依据。方法: 急性经口毒性采用霍恩（Horn’s）法测定。选用昆明种小鼠40只随机分为4组,给予不同剂量崖柏精油（21.50,10.00,4.64,2.15 g· kg^-1）,观察14 d。记录死亡数,查表求得LD₅₀,并记录死亡时间及中毒表现等。皮肤刺激性采用健康成年的白色家兔9只随机分为3组,分别给予崖柏精油100%浓度、50%浓度、25%浓度。采用食用植物油作为阴性对照。观察药物贴敷后1,24,48,72 h受试部位皮肤红斑、水肿的反应并评分。采用迟发型致敏反应最大剂量法进行崖柏精油致敏性测定。取健康白色豚鼠30 只,随机平均分为3 组：给药组,空白对照组（给予植物油基质）、阳性对照组（1 %的2,4-二硝基氯苯）。经皮内诱导、局部诱导阶段后,观察崖柏精油激发24 h及48 h 的皮肤变态反应情况并进行评分。 结果: 崖柏精油21.50 g· kg^-1可使小鼠全部死亡,10.00,4.64 g· kg^-1组可使动物部分死亡,2.15 g· kg^-1组动物无死亡,查表求得崖柏精油对雌雄性小鼠的LD₅₀分别为7.94,9.26 g· kg^-1。皮肤刺激性结果显示,崖柏精油（100%浓度）贴敷后1 h有强刺激性,而稀释后的药物刺激性逐渐减弱,25%浓度崖柏精油无明显刺激性。致敏性结果显示,阳性对照组可使豚鼠皮肤出现中度或重度融合性红斑及水肿,具有明显致敏性。而崖柏精油组未见明显红斑和水肿,显示无致敏性。结论:崖柏精油具有明显皮肤刺激性,急性经口毒性属实际无毒,未见致敏性。     

EpiSkin™和Epikutis®模型在体外皮肤刺激性和腐蚀性检测应用中的比较

目的 对Episkin™和Epikutis®模型在体外皮肤刺激性和腐蚀性检测各方面进行对比。方法 参考体外皮肤刺激实验（OECD指导原则439）和体外皮肤腐蚀实验（OECD指导原则431）,对2种模型在质量控制、检测方法、结果判断、运输等方面进行探讨。结果 2种模型在质量控制、检测方法、结果判断等方面存在差异,在实验操作性和成本上也各有优缺点,但2种模型均能满足体外皮肤刺激性和腐蚀性实验要求。结论 2种模型均能很好地用于体外皮肤刺激性和腐蚀性检测。     

The Relationship between pKa and Skin Irritation for a Series of Basic Penetrants in Man

The Relationship between pK_a and Skin Irritation for a Seriesof Basic Penetrants in Man. BERNER, B., WILSON, D. R., STEFFENS,R. J., MAZZENGA, G. C., HINZ, R., GUY, R. H., AND MAIBACH, H.I. (1990). Fundam. Appl. Toxicol 15, 760–766. For a seriesof bases, which penetrate through human skin in vitro at similarrates (0.056–0.49 µM/cm²/hr), penetrant pK_ais shown to correlate with erythema, edema, and color meterreadings. As estimates of irritation, erythema, edema, and rednessmeasurements are highly linearly correlated. For the selectedseries, irritation becomes significant for bases with a pK_a> 8. The irritation potential of acids with pK_a 4 has beenpreviously reported; pK_a appears highly predictive of acuteskin irritation for acids and bases in man.     

Phosphatidylserine as a Determinant for the Tissue Distribution of Weakly Basic Drugs in Rats

Interorgan variation in tissue distribution of weakly basic drugs such as quinidine, propranolol, and imipramine was investigated as a function of binding to phosphatidylserine (PhS) in tissues. Tissue distributions of these drugs were determined using 10 different tissues at a steady-state plasma concentration and were expressed as tissue-to-plasma partition coefficients (K _p values). The concentration of PhS in the tissue was determined by two-dimensional thin-layer chromatography. Plotting of K _p values, except for brain, against the tissue PhS concentrations showed a linear relationship, indicating that PhS is a determinant in the interorgan variation of these tissue distributions. Further, differences in tissue distribution among the drugs was considered to be due to the difference in binding potency to PhS. Drug binding parameters to individual standard phospholipid were determined using a hexane-pH 4.0 buffer partition system. Binding was highest to PhS, and a linear relationship was found between the log nK [product of the number of binding sites (n) and the association constant (K) for PhS binding] obtained in vitro and K _p values of drugs in tissues in vivo. The empirically derived equation, K _p = 14.3 × (log nK) × (PhS cone.) – 8.09, was found to predict K _p values in vivo of weakly basic drugs. Thus, a determinant of interorgan variation in the tissue distribution of the weakly basic drugs studied was the tissue concentration of PhS and the drug binding affinity to PhS.     

Systemic delivery of timolol after dermal application: Transdermal flux and skin irritation potential in the rat and dog

Timolol, a beta-adrenergic antagonist, was evaluated for transdermal flux with rat skin in vitro and with the dog in vivo. Skin irritation after dermal application of timolol was assessed in the rat in vivo. Drug flux across rat skin in vitro ranged between 2 and 110 µg cm^–2 hr^–1, dependent on the formulation. The transdermal flux of timolol in the dog was greater than 10 µg cm^–2 hr^–1. This estimate was based on the degree of antagonism of isoproterenol challenge following transdermal administration of timolol relative to that obtained following intravenous administration of timolol. Irritation was observed in the rat after occluded dermal application of timolol free base but was not observed when the concentration of the drug in the formulation was decreased.     

Kinetic Analysis on the Skin Disposition of Cytotoxicity as an Index of Skin Irritation Produced by Cetylpyridinium Chloride: Comparison of In Vitro Data using a Three-Dimensional Cultured Human Skin Model with In Vivo Results in Hairless Mice

PURPOSE: The aim of this study was to kinetically and dynamically analyze in vitro cytotoxicity as an index of skin irritation by use of a three-dimensional cultured human skin model and to compare the in vitro assay data with data from living animals. METHODS: A cationic surfactant, cetylpyridinium chloride (CPC), was selected as a model irritant. Living skin equivalent-high (LSE-high) and hairless mice were used for the in vitro and in vivo tests, respectively. Skin irritation dermatodynamics was evaluated by calorimetric thiazoyl blue (MTT) conversion assay both for in vitro and in vivo tests, whereas dermatokinetics of CPC in LSE-high and mouse skin were evaluated using HPLC. RESULTS: The time course of cell viability in the skin after application of CPC to intact skin was distinctly different from that of stratum-corneum-stripped skin in both LSE-high and hairless mice. Biphasic behavior characterized by two first-order rates with an inflection time point was observed in intact skin, whereas cell viability monoexponentially decreased immediately after CPC application in stripped skin. The time courses of cell viability in the skin and dermatodynamics were closely related to that of dermatokinetics of CPC. CONCLUSION: The present study demonstrates that the in vitro cytotoxic profile was similar to the in vivo cytotoxicity test and that dermatodynamics was related to dermatokinetics of CPC.     

In Vitro Skin Permeation and Bioassay of Chlorhexidine Phosphanilate,a New Antimicrobial Agent

An in vitro technique was developed to study the permeation and antimicrobial activity of graded concentrations of a new antibacterial agent, chlorhexidine phosphanilate (CHP), in cream formulations using Franz diffusion cells. Formulations containing from 0.2 to 2% CHP were quantitatively applied to intact excised skin and to skin from which the stratum corneum and partial epidermis had been enzymatically removed. Receptor fluids from diffusion cells were sampled over time and assayed by HPLC methods for chlorhexidine and phosphanilic acid; 24- and 48-hr samples of the diffusate from studies with damaged skin were also bioassayed using clinical isolates of appropriate microbial species. Through intact skin almost no permeation of CHP was observed over 48 hr. The failure of CHP to penetrate intact human skin suggests that normal stratum corneum is the rate-limiting barrier to penetration by this antimicrobial agent. In damaged skin lacking stratum corneum barrier, the release of CHP from the formulation becomes the rate-determining step. Coincident with penetrating damaged skin, CHP dissociates, and the molar ratio of the chlorhexidine and phosphanilate moieties in the diffusate changes to favor phosphanilic acid. The extent of changes in the permeation rates of both moieties of CHP was directly related to the CHP concentration in cream. Both CHP moieties were found to reach equilibrium in the dermis within 24 hr after application. It was also observed that CHP creams down to 0.2% concentration yielded diffusates with activity exceeding the minimum inhibitory concentration of all test microorganisms within 24 hr.     

Ring Structure and Aromatic Substituent Effects on the pKa of the Benzoxaborole Pharmacophore

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Chemical Stability of Pentostatin (NSC-218321), a Cytotoxic and Immunosuppressant Agent

Pentostatin, an unusual nucleoside of natural origin, has been used for the treatment of hairy cell leukemia, as an immunosuppressant agent, and as an inhibitor of adenosine deaminase. The studies of the physicochemical properties and solution stability of pentostatin (1) are important to the development of a parenteral formulation for extensive preclinical and clinical testing. Pentostatin displayed apparent pK _a values at 25 ± 0.1°C and ionic strength of 0.15 M of 2.03 ± 0.03 and 5.57 ± 0.14 (spectrophotometric) and 5.50 ± 0.02 (potentiometric) for N₁ and the amidine nitrogen in the seven-membered ring, respectively, which are the most likely protonation sites. The rates of degradation of pentostatin were determined as a function of pH, buffer concentration, and temperature. In the pH range 1.0–4.0, pentostatin undergoes acid-catalyzed glycosidic cleavage leading to the formation of the base compound, 2, and 2-deoxyribose. A carbonium ion mechanism in which C–N bond cleavage was the rate-determining step was consistent with the data. In the pH range 6.5–10.5, the imine bond at C₅ position in pentostatin is hydrolyzed to form the corresponding formamide. Pentostatin hydrolysis in this pH range was independent of pH. At pH >11, pentostatin decomposes to nonchromophoric products probably through multiple-step base-catalyzed hydrolytic mechanisms. Pentostatin appears to be quite stable after reconstitution of a lyophilized experimental dosage form. Care must be taken if pentostatin is extensively diluted with 5% dextrose in water, as pentostatin stability is compromised at pH values less than 5.     

The aqueous stability of bupropion

In this study the aqueous stability of bupropion was determined and the pH-degradation profile was obtained. The effects of hydrogen ion, solvent and hydroxide ion concentration are discussed with particular emphasis on the kinetics of degradation of bupropion. Kinetics and degradation of bupropion were determined by HPLC-UV and LC–MS analysis both utilising high pH chromatographic methods. Degradation of bupropion in aqueous solutions follows first-order reaction kinetics. The pH-degradation profile was determined using non-linear regression analysis. The micro- and macro-reaction constants for degradation are presented. Bupropion was most stable in aqueous solutions below pH 5. Degradation was catalyzed by water but mainly by hydroxide ion on the unionised form of bupropion. The energy of activation for decomposition in aqueous solution pH 10.7 I = 0.055 was determined to be 53 kJ mol⁻¹ with a frequency factor of 6.43 × 10¹⁰ s⁻¹. The degradants of bupropion were positively identified and a mechanism of degradation is proposed. The inherent instability of bupropion above pH 5 has implications for its therapeutic use, formulation, pharmacokinetics and use during analysis and storage.     

Dermal penetration of propylene glycols: Measured absorption across human abdominal skin in vitro and comparison with a QSAR model

The dermal penetration of undiluted monopropylene glycol (MPG) and dipropylene glycol (DPG) has been measured in vitro using human abdominal skin under conditions of infinite dose application, and the results compared with predictions from the SKINPERM QSAR model (ten Berge, 2009). The measured steady-state penetration rates (J_ss) for MPG and DPG were 97.6 and 39.3 μg/cm²/h, respectively, and the permeability coefficients (K_p) were 9.48 × 10⁻⁵ cm/h for MPG and 3.85 × 10⁻⁵ cm/h for DPG. In comparison, the SKINPERM model slightly over-predicted J_ss and K_p for MPG and DPG by between 2.6- and 5.1-fold, respectively. The model predictions of 254 μg/cm²/h and 24.6 × 10⁻⁵ cm/h for MPG, and 202 μg/cm²/h and 19.8 × 10⁻⁵ cm/h for DPG were in fairly good agreement with the measured values. Further, the model predicted a J_ss of 101 μg/cm²/h and a K_p of 9.9 × 10⁻⁵ cm/h for the homologue tripropylene glycol. Assuming that the measured J_ss was the same under conditions of finite dose application (taken to be 10 μL/cm²) and was maintained over a 24-h period (both conservative assumptions), the relative dermal absorption of the applied dose was estimated to be 23% (0.96%/h) for MPG and 9% (0.39%/h) for DPG. However, the extrapolation for MPG may be further overestimated due to possible residence in the stratum corneum under infinite conditions of exposure that would not be applicable to a finite loading dose.     

FDA的“评估简化新药申请的仿制透皮和局部给药系统可能的刺激性和致敏性的供企业用的指导原则草案”介绍

美国食品药品监督管理局（FDA）于2023年4月发布了“评估简化新药申请仿制透皮和局部给药系统可能的刺激性和致敏性的供企业用的指导原则草案”,全面而又具体地阐明FDA对仿制透皮和局部给药系统（TDS）可能的刺激性和致敏性人体内研究的设计和实施的建议。其中包括一般原则（一般考虑）、研究设计和实施、统计分析（刺激性分析和致敏性分析）、辅料TDS和阳性对照TDS以及部分（切割）TDS等。而中国目前还没有类似的指导原则,详细介绍FDA该指导原则主要内容,期望对中国仿制TDS刺激性和致敏性人体内研究与监管有所裨益。     

Potentiometric determination of ionisation constants for diphacinone and chlorophacinone in a dioxane–water cosolvent system

The purpose of this study was to determine the ionisation constants of two poorly soluble compounds, namely diphacinone and chlorophacinone, potentiometrically in 1,4-dioxane–water mixtures with ibuprofen used as a standard. In this study, Gran's method was employed for the calibration of glass electrode in cosolvent systems with pH measurements based on the concentration scale (p_cH). Aqueous pK_a values for the tested compounds were obtained by extrapolation on a Yasuda–Shedlovsky plot. It was demonstrated that the pK_a for ibuprofen determined using this method was consistent with those reported in literature. The technique was applied successfully to the two indandione derivatives, diphacinone and chlorophacinone. The present study demonstrated that the use of an organic cosolvent is effective in improving the solubility of compounds allowing potentiometric determination of ionisation constants that are otherwise difficult in aqueous solutions.     

pH-partition profiles of 4-(3-oxo-1,2-benzisothiazolin-2-yl)phenyl and phenoxyalkanoic acids

The 1,2-benzisothiazolin-3-one nucleus is well known in the medicinal chemistry literature for the variety of biological effects exerted by its derivatives. In the present paper, the dependence of the n-octanol/buffer distribution coefficient (D) on pH of four 4-(3-oxo-1,2-benzisothiazolin-2-yl)phenyl and phenoxyalkanoic acids was investigated, employing the reference shake-flask method. From the analysis of the pH-partition profiles in the chosen partition system, the logP(AH), the logP(A(-)) and the pK(a) values for each compound were determined. The physico-chemical data obtained were compared to the pK(a) and logP values of the corresponding phenyl and phenoxyalkanoic acids, and an estimation of the lipophilic and electronic contribution of the 1,2-benzisothiazolin-3-one substituent in the para-position is proposed. The 1,2-benzisothiazolin-3-one nucleus behaves as a lipophilic, moderately electron-withdrawing group.     

A pharmacological method to estimate the pK I of competitive inhibitors of agonist uptake processes in isolated tissues

Summary An equation is derived from a mathematical model (proposed by Furchgott) which, under certain circumstances, estimates the pK _I (-log dissociation constant) of a competitive inhibitor of agonist uptake by utilizing the sensitization of isolated tissues, to the substrate-agonist, by uptake inhibition. The method is theoretically more sound and appears to be improved by the use of potency-ratios of the substrate-agonist and an agonist which is not a substrate for the uptake process since this allows for the detection and correction of receptor and toxic effects of uptake inhibitors. The pK _I values of cocaine, desmethylimipramine and imipramine for the neuronal uptake of norepinephrine were estimated by this method in guinea-pig tracheae and left atria. Also, the pK _I values for 17 -oestradiol, corticosterone, clonidine and metanephrine for the extraneuronal uptake of isoproterenol were estimated in guinea-pig tracheae (and cat left atria for 17 -oestradiol). All estimates were consistent with literature pK _I values obtained biochemically with radiolabelled substrates.     

Kinetic evidence for a common binding site for substrates and inhibitors of the neuronal noradrenaline carrier

Summary The neuronal noradrenaline uptake mechanism (uptake₁) has been further characterized. For a number of substrates of uptake, the half-saturating concentration (K _m) and the maximal initial transport rate (V _max) were determined. Furthermore, the dissociation constants (K _D) for binding of these substrates to the desipramine binding site of the neuronal noradrenaline carrier were measured. The uptake experiments were done on rat phaeochromocytoma cells (PC12 cells), the binding experiments on purified plasma membranes of PC12 cells. The substrates differed markedly in respect of V _max, K _m, and K _D. Neither K _m and V _max nor K _D and V _max were found to be correlated. However, the discrepancy between K _m and K _D expressed as the ratio, K_m/K_D, was negatively correlated with V _max (r = – 0.9315, n = 7, p < 0.01).For the interpretation of these results a model on the basis of the steady-state assumption has been proposed for uptake₁. From the mathematics of that model the following conclusions can be drawn. (1) The half-saturating substrate concentration (K _m) is not identical with the dissociation constant for the binding of a substrate to the substrate recognition site (K _D). (2) The discrepancy between K _m and K _D is expected to be negatively correlated with the maximal initial transport rate of the substrate (V _max).The experimental results are in good agreement with the proposed model for uptake,. Especially the negative correlation between K _m/K _D and V_max supports the hypothesis that desipramine inhibits uptake, via binding to the substrate recognition site of the neuronal noradrenaline carrier.This study was supported by the Deutsche Forschungsgemeinschaft (SFB 176) Send offprint requests to E. Schömig at the above address     

鹅不食草油鼻用微乳温敏凝胶释药系统鼻黏膜刺激性及主要脏器影响研究

目的 考察鹅不食草油鼻用微乳温敏凝胶剂对大鼠鼻腔黏膜的刺激作用及主要脏器的形态学变化。方法 大鼠随机分成空白对照组、空白微乳温敏凝胶组及鹅不食草油鼻用微乳温敏凝胶组,考察大鼠鼻腔给药后鼻黏膜及主要脏器形态学结构变化,评价鹅不食草油鼻用微乳温敏凝胶的初步安全性。结果 大鼠鼻黏膜组织切片显示各组的鼻中隔软骨上均有完整纤毛和正常的黏膜细胞存在,未见明显的血管充血现象,也均未见明显的黏膜、组织细胞坏死、脱落及出血现象;主要脏器组织切片图结构显示大鼠鼻腔给药后与空白对照组及空白微乳温敏凝胶组一样均未见组织细胞结构病变、水肿、坏死等变化。结论 鹅不食草油鼻用微乳温敏凝胶释药系统对大鼠鼻黏膜无明显刺激性,对主要脏器未见明显损伤,提示其应用具有用药的安全性。     

The Electrical Characteristics of Human Skin in Vivo

Purpose. The objectives of this study were to investigate the impedance properties of human skin in vivo and to examine the effect of iontophoresis upon them. Methods. Having established the intra- and inter-individual variation in basal values of skin impedance, the effect of varying iontophoretic current density, ionic strength and counter-ion on the rate of recovery of skin impedance after iontophoresis was investigated. Results. Passage of an iontophoretic current caused a significant reduction in the magnitude of the skin impedance. Increasing the current density caused an even greater reduction in the value of the skin impedance and slowed the rate of recovery. Reduction of the ionic strength resulted in an increase in the rate of recovery following iontophoresis. A significant increase in the rate of recovery was observed when CaCl₂ replaced NaCl as the electrolyte. Although visual inspection revealed the presence of greater erythema when CaCl₂ was used, there was an absence of the mild sensation experienced by volunteers when using NaCl. The last part of the study established a correlation between transepidermal water loss and impedance analysis as complementary methods for probing skin barrier function in vivo. The data were fitted to an equivalent circuit consisting of a resistor in parallel with a constant-phase element and a mechanistic model proposed to explain the electrical properties of the skin. Conclusions. The first comprehensive investigation of the effect of iontophoresis on the electrical properties of human skin in vivo has been described. It would appear from the results, and from their interpretation, that impedance spectroscopy may be an effective method to quantify the impact of iontophoresis on the skin, and to determine the extent to which proposed drug delivery regimens will perturb skin barrier function.