An attempt to define a just detectable effect for airborne chemicals on the respiratory tract in mice

 We have attempted to define a just detectable effect (JDE) for three different types of reactions along the respiratory tract: (a) sensory irritation of the upper airways (S), (b) airflow limitation along the conducting airways (A), and (c) pulmonary irritation at the alveolar level (P1 or P). Each type of reaction, S, A, P1 or P, was recognized by analyzing the breathing pattern of unanesthetized mice held in body plethysmographs. A rule-based computer program analyzed each breath during a period of 3.75 h and classified each breath as normal (N) or falling in any of the above categories (i.e., S, A, P1 or P). Eight groups of four mice were used for sham exposures: exposed to water vapor. These data sets were used, as sham exposure data, to define the variation which can occur with time in order to define an expected range of normal variation. Once this range was established, we defined JDE values for each type of effect and used such values to evaluate the results obtained in exposed animals. Eight groups of four mice were exposed to a mixture of airborne chemicals, machining fluid G (MFG), at concentrations from 0.17 to 55 mg/m³. Data sets for individual animals and for each group of animals exposed to MFG were analyzed to determine if and when a particular effect occurred. It was possible to recognize the effects of low exposure concentrations on groups of exposed animals or individual animals within each group. This procedure will be valuable when investigating the effect of airborne chemicals and when it is impossible to generate high exposure concentrations to define concentration-response relationships. Received: 4 October 1995/Accepted: 20 December 1995     

Development of new reactions and their pharmaceutical application based on the molecular structure characteristics

The author and his group have been developing new reactions based on molecular structure characteristics; sigma-symmetric bifunctional molecules, dipole-dipole repulsion, active amide structures, latent active species, orbital-orbital interactions, nonbonded interactions, strained structures, allenic structures, etc. Various new reactions such as asymmetric aminolyses and Dieckmann-type cyclizations of prochiral sigma-symmetric dicarboxylic diamides, asymmetric aldol-type reactions onto gamma-hydroxybutenolides and asymmetric imine alkylations onto omega-acetoxy lactams using chiral Sn(II) enolates, asymmetric Pummerer-type reactions, cascade reactions and endo-mode cyclizations exploiting alpha,beta-unsaturated allenic esters and ketones, base- and palladium-promoted ring-expansion reactions, and syntheses of alpha-substituted serines and 1-azabicyclo [1.1.0] butane have been achieved. These new reactions were applied to the synthetic development of new seed and lead compounds (SH-enzyme inhibitors, tumor inhibitors, and antibiotics) with the aim of synthesizing new drugs. Asymmetric syntheses of (+)-Prelog-Djerassi lactonic acid methyl ester, (+)-carbacyclin, ISP-I (myriocin), (+)-conagenin, Geissman-Waiss lactone, biapenem (a new carbapenem antibiotic), thienamycin-like gamma-lactam, and bicyclic alkaloids were also achieved by utilizing these reactions. Evaluation and molecular design of the seed and model compounds for angiotensin II receptor antagonists, tumor inhibitors, and antibiotics have been investigated on the basis of QRSA and/or nonbonded S...X (X=O, N, S, halogens) interaction concepts.     

In vitro skin penetration of fragrances: Trapping the evaporated material can enhance the dermal absorption of volatile chemicals

This study compared the evaporation and skin absorption profiles of four fragrance chemicals in in vitro skin penetration studies performed in conditions of airflows of low velocity with and without trapping of the evaporated volatiles. The presence of a trapping chamber above the skin surface slowed down the evaporation of the chemicals, possibly due to formation of a gaseous stagnant layer of greater thickness than the one existing at the skin surface in the real-life conditions of multidirectional and/or turbulent flows. In addition, the use of a trapping chamber considerably influenced the distribution of the fragrance chemicals in the skin layers and resulted in 2- to 8-fold increase of the doses available for systemic absorption. Such unrealistic overestimation of the percutaneous absorption can significantly impact the risk assessment of topically applied volatile chemicals and can lead to defining unrealistic margins of safety.     

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.     

An attempt to study the effects of chemical structure on the affinity and efficacy of compounds related to acetylcholine

Two sets of series of compounds, RN⁺Me₃, RN⁺Me₂Et, RN⁺MeEt₂, RN⁺Et₃, and R'N⁺Me₃, R'N⁺Me₂Et, R'N⁺MeEt₂, R'N⁺Et₃, have been prepared, in which R is a 2-(diphenylacetoxy)ethyl, 2-(benziloyloxy)ethyl, 2-(2,2-diphenylethoxy)ethyl, 3-(diphenylmethoxy)propyl or 3,3-diphenylbutyrylmethyl group, and R' is a 2-acetoxyethyl, 2-ethoxyethyl, 3-methoxypropyl or butyrylmethyl group: compounds of the first set therefore differ from those of the second set in that they contain a diphenylmethyl group (or a benziloyl group) in place of a methyl group. The former compounds are antagonists of acetylcholine whereas most of the latter act like acetylcholine. The affinity constants of the former compounds for the acetylcholine receptors of the guinea-pig ileum have been determined and the equipotent molar ratios relative to acetylcholine have been measured for the latter compounds. The variation of the affinity constant with the constitution of the onium group in the antagonists (the diphenylmethyl compounds) was sufficiently consistent from one series to another for it to seem likely that corresponding changes in affinity with the constitution of the onium group would occur in the agonists. From the relative activity of the agonists and with this knowledge of relative affinity it was possible to assess the effects of their structure on efficacy. Substitution of one methyl in the onium group by an ethyl group in these compounds increased affinity but decreased efficacy. The replacement of a second methyl by a second ethyl group had little effect on affinity but decreased efficacy still further. The replacement of the ester link in acetylcholine by a 4-ether oxygen atom (as in the diphenylmethoxypropyl and methoxypropyl compounds) did not appreciably reduce affinity but markedly reduced efficacy, whereas the replacement of the ester link by a 3-ether oxygen atom (as in the diphenylethoxyethyl and ethoxyethyl compounds) markedly reduced affinity but did not reduce efficacy. The diphenylbutyrylmethyl compounds had low affinity and the butyrylmethyl compounds had low efficacy. We conclude that the action of acetylcholine at the postganglionic parasympathetic receptors in the guinea-pig ileum depends upon the presence of the 4-carbonyl group (and presumably the onium group) for affinity and on the 3-ether oxygen atom and the trimethylammonium group for efficacy.     

An attempt to structurally convert mu-selective morphine toward delta-receptor binding: dimerization based on enkephalin conformation

In order to elucidate the substrate specificities for mu- and delta-opioid receptors, dimerization of the mu-specific morphine molecule was attempted, based on the hypothesis of the possible relationship between the molecular conformation of endogenous enkephalin and its selectivity for each opioid receptor. The NOR2 ([normorphine]N-CH2-CH2-N[normorphine]) thus synthesized exhibited agonist activity showing a preference for binding with the delta-opioid receptor, compared with the parent morphine molecule. Antagonist activity was also observed for NOR3 ([normorphine]N-CH2-CH2-CH2-N[normorphine]).     

An attempt to modulate the microporous diffusion of a model polypeptide by altering its secondary structure

The purpose of the present study was to determine whether intentional alteration of the secondary structure of a model polypeptide, conantokin-G, influenced the rate and extent of aqueous pore diffusion across a synthetic microporous membrane. Use of a microporous synthetic membrane allowed for analysis of polypeptide transport without the confounding variables of protein binding, acid- and/or enzyme-mediated degradation, endocytotic uptake, and enzymatic inactivation associated with a biological membrane. Conantokin-G was intentionally changed from its native random coil structure to the alpha-helix structure using calcium, and both structures were verified using circular dichroism. The alpha-helix structure of conantokin-G was retained even after additional free calcium was removed by equilibrium dialysis. Over the concentration range of 1.25 to 20 mM, there was a linear relationship between the solution calcium concentration and the percent of the alpha-helix conformer present. The apparent permeability, the apparent aqueous diffusion coefficient with and without inclusion of the Renkin function, and the hydrodynamic radii estimated by diffusion and a computer-software program were calculated for the random coil and alpha-helix structures of conantokin-G. Calcium-mediated conversion of conantokin-G to its alpha-helix structure did not significantly (p >.05) change its apparent permeability across a microporous membrane. It is suggested that perhaps complete conversion to the alpha-helix structure of only a fraction of the conantokin-G molecules (only 0.45 or 45% of the molecules can be converted to the alpha-helix structure at Ca(2+) concentrations >or= 20 mM) may have limited the extent of transport of the alpha-helix conformer.     

Discrimination of neuroleptics by means of their interaction with amfonelic acid: an attempt to characterize the test

The non-amphetamine stimulant amfonelic acid (AFA) is known to enhance the effects of haloperidol, trifluoperazine and spiperone but not those of the atypical neuroleptics clozapine, thioridazine, and sulpiride, on the striatal levels of 3,4-dihydroxyphenylacetic acid. Consequently, the interaction between neuroleptics and AFA has been proposed as a test to discriminate typical and atypical neuroleptics. In this study, these findings were confirmed. Essentially the same results were obtained when the levels of homovanillic acid were measured. However, striatal dopamine levels were decreased similarly by combinations of AFA with typical and atypical neuroleptics. A comparison of the results with 17 neuroleptics with their reported clinical liability to cause extrapyramidal symptoms supported the idea that the test may discriminate drugs with a better ratio of therapeutic vs side effects. A series of antidepressants and alpha-noradrenergic antagonists possessing antidopaminergic properties was found to perform like atypical neuroleptics, i.e. their effects on dopamine metabolites were not enhanced by AFA.     

The effect of water solubility of solutes on their flux through human skin in vitro: an extended Flynn database fitted to the Roberts-Sloan equation

The edited Flynn database (n=62) for determining the effect of the physicochemical properties of solutes on their skin absorption has been extended (n=114) to give a database for which solubilities of the solutes in water, S(AQ), and their maximum fluxes from water through human skin in vitro, J(MAQ), are known or can be calculated. Besides the six major contributors to the original and edited Flynn database, nine more contributors have been included in the extended database to give 15 contributors. As in the edited Flynn database, data for solutes that were significantly ionized or for experiments using different thicknesses of skin were not excluded from the extended database so that the diversity of the original database was maintained. The extended database was fit to five equations where the independent variables were solubility in octanol (S(OCT)), in water (S(AQ)) or molecular weight (MW) and combinations of those three variables; and the dependent variable was J(MAQ). The best fit was obtained from the Roberts-Sloan (RS) equation: logJ(MAQ) = x + ylogS(OCT) + (1 - y)logS(AQ) - z MW, x = -2.574, y = 0.586, z = 0.00440, r(2) = 0.887, S.D. = 0.399, F = 139. This result is comparable to the best fit published using permeability coefficients, P, as the dependent variable, but gives greater insight into the factors affecting permeation. J(MAQ) is more important clinically because it described how much is permeating per unit area and time, while P is in the units of speed (cmh(-1)). Because of the dependence of J(MAQ) on S(AQ), the selection of new drugs with improved topical delivery should include considerations of their S(AQ) in their design.     

Cluster analysis of the dermal permeability and stratum corneum/solvent partitioning of ten chemicals in twenty-four chemical mixtures in porcine skin

Assumptions based on absorption from single solvent systems may be inappropriate for risk assessment when chemical mixtures are involved. We used K-means and hierarchical cluster analyses to identify clusters in stratum corneum partitioning and porcine skin permeability datasets that are distinct from each other based on mathematical indices of similarity and dissimilarity. Twenty-four solvent systems consisting of combinations of water, ethanol, propylene glycol, methyl nicotinate and sodium lauryl sulfate were used with 10 solutes, including phenol, p-nitrophenol, pentachlorophenol, methyl parathion, ethyl parathion, chlorpyrifos, fenthion, simazine, atrazine and propazine. Identifying the relationships between solvent systems that have similar effects on dermal absorption formed the bases for hypotheses generation. The determining influence of solvent polarity on the partitioning data structure supported the hypothesis that solvent polarity drives the partitioning of non-polar solutes. Solvent polarity could not be used to predict permeability because solvent effects on diffusivity masked the effects of partitioning on permeability. The consistent influence of the inclusion of propylene glycol in the solvent system supports the hypothesis that over-saturation due to solvent evaporation has a marked effect on permeability. These results demonstrated the potential of using cluster analysis of large datasets to identify consistent solvent and chemical mixture effects.     

An attempt to correlate kinetic data on the drug receptor interaction with the chemical structures of cholinomimetic agents

The rate of action of several cholinergic drugs has been determined using isolated, electrically driven guinea pig atria. The association and dissociation rate constants of the drug receptor complex were evaluated permitting estimation of the dissociation constant K.     

The use of pH measurements to predict the potential of chemicals to cause acute dermal and ocular toxicity.

Regulatory guidelines for the assessment of acute dermal and ocular toxicity refer to the need to take the pH values of chemicals into consideration, since the acidic and basic properties of chemicals are known to play a role in the generation of acute dermal and ocular lesions. However, not all test guidelines provide an objective interpreting pH measurements in terms of acute skin or eye toxicity. The aim of this study was to develop classification models based on pH data for predicting the potential of chemicals to cause skin corrosion, skin irritation and eye irritation. The possible application of these models in the context of tiered testing strategies is discussed.     

Modification of lipid peroxidation by some known substituted imidazole derivatives. An attempt to correlate their antioxidant activity with lipophilicity.

The antioxidant activity of two antiallergic agents, oxatomide, astemizole and two neuroleptic drugs, penfluridol, pimozide, structurally related, was tested, using heat-inactivated rat hepatic microsomal fraction and linoleic acid. Lipid peroxidation was induced by Fe2+/ascorbic acid and Fe2+, respectively, and evaluated spectrophotometrically as 2-thiobarbituric acid reactive material. It was found that these compounds inhibited significantly the peroxidation of microsomal membranes in a concentration-dependent manner, as well as the peroxidation of linoleic acid. The lipophilicity of the tested compounds, expressed as RM values derived from reversed phase thin layer chromatography (RPTLC), was determined and the relationship between antioxidant activity and lipophilicity was discussed.     

Relationships of the molecular structure of aldosterone derivatives with their binding affinity for mineralocorticoid receptor

The molecular structures of 19-nor-11-deoxycorticosterone (III) and 21-hydroxypregna-4,11-diene-3,20-dione (IV) were determined by X-ray crystallographic analysis and the factors affecting the binding affinities for the mineralocorticoid receptor were examined with six aldosterone derivatives (I-VI) containing these two compounds. The most important factor was found to be the steric one; affinity increased with increasing flatness of the structure. The electronic factor may be a minor influence although a good relationship was found between the affinity and the 13C-NMR chemical shift of the C(5) atom. The factor playing no role in the binding is the hydrophobic one.     

The effect of water solubility of solutes on their flux through human skin in vitro: a prodrug database integrated into the extended Flynn database

To develop more effective compounds as enhancers, O-acylmenthol derivatives which were expected to be enzymatically hydrolyzed into nontoxic metabolites by esterases in the living epidermis were synthesized from l-menthol and pharmaceutical excipient acids (lactic acid, cinnamic acid, salicylic acid and oleic acid) in this study. Their promoting activity on the percutaneous absorption of five model drugs, 5-fluorouracil (5-FU), isosorbide dinitrate (ISDN), lidocaine (LD), ketoprofen (KP), and indomethacin (IM), which were selected based on their lipophilicity represented by log K(O/W), were tested in vitro across full thickness rat skin with each of the evaluated drugs in saturated donor solution. 2-Isopropyl-5-methylcyclohexyl 2-hydroxypanoate (M-LA) provided the highest increase of accumulation of 5-FU (3.74-fold) and LD (4.19-fold) in the receptor phase while 2-isopropyl-5-methylcyclohexyl cinnamate (M-CA) was ineffective for most of the drugs; Both 2-isopropyl-5-methylcyclohexyl 2-hydroxybenzoate (M-SA) and (E)-2-isopropyl-5-methylcyclohexyl octadec-9-enoate (M-OA) had better promoting effects on the drugs with low water-solubility. The four O-acylmenthol enhancers produced parabolic relationship between the lipophilicity (log K(O/W)) of the model drugs (5-FU, ISDN, KP, IM) and their enhancement ratio of the permeation coefficient (ER(P)), indicating that the lipophilicity of the penetrants has significant effect on the permeation results, r = 0.989 (P=0.144) for M-LA, r = 0.965 (P = 0.216) for M-CA, r = 0.786 (P = 0.630) for M-SA, and r = 0.996 (P = 0.088) for M-OA.     

Protective effects of equol and their polyphenolic isomers against dermal aging: Microarray/protein evidence with clinical implications and unique delivery into human skin

Abstract

Context: Equol is a polyphenolic/isoflavonoid molecule that can be expressed as isomers. However, the characteristics of the equol isomers on dermal gene/protein expression and human skin percutaneous absorption remain unknown.

Objective: Perform a comprehensive investigation on equol as: R-equol, racemic equol or S-equol to determine their differential expression of skin-related genes, quantify collagen expression and determine percutaneous absorption in human skin.

Methods: Quantified: (i) gene expression/mRNA levels via gene array technology using human skin equivalents with equol exposure at 1.2% in qPCR experiments, (ii) in vitro collagen expression in human fibroblasts, and (iii) percutaneous absorption by Franz cell techniques.

Results: In the qPCR studies, only three genes displayed the greatest significant expression by S-equol, whereas 16 genes displayed the greatest significant levels (either stimulation or inhibition) by R-equol and/or racemic equol, such as extracellular matrix proteins (i.e., collagen and elastin), nerve growth factor, aging genes [FOS, 100 A8 and A9 calcium-binding proteins, 5α-reductase type 1, and matrix metalloproteinases (1, 3, and 9)], and inflammatory genes (e.g., interleukin-1 alpha, interleukin-6, and cyclooxygenase-1). Collagen type I expression in fibroblasts was greater with racemic versus S-equol treatment at 1 and 10 nM. Percutaneous absorption demonstrated high sequestering in keratinocytes with subsequent accumulation/release over time.

Discussion and conclusion: Overall, these results illustrate the significant differences in mirror-image molecules or isomers of equol where R-equol and/or racemic equol are better molecules for skin gene expression compared to S-equol and the percutaneous absorption of equol represents a unique epidermal reservoir delivery mechanism.