Prediction of skin permeation of highly lipophilic compounds; in vitro model with a modified receptor phase

A skin permeation in vitro model for highly lipophilic compounds was developed, containing bovine serum albumin (BSA) in the receptor phase. Skin permeation rates of three homologous series of compounds varying within a wide range of hydrophobicity were measured. For the more water-insoluble compounds (log P_IM 3), it was demonstrated that the modified in vitro model proposed ensures a more efficient collection of the permeant in the receptor phase.     

Differences in adipose tissue distribution of basic lipophilic drugs between intraperitoneal and other routes of administration

1. Distribution of 14C-methadone in male rats was studied after administration of single i.p. doses. Highest concentrations were attained after 30 min in the decreasing order of abdominal adipose tissues, liver, lung, other organs. Concentrations in abdominal adipose tissues were 20 times higher than in subcutaneous adipose tissue. This is in contrast with what occurs with other routes of administrations, where only low concentrations are attained in both subcutaneous and abdominal adipose tissues. 2. The situation in the peritoneal cavity after i.p. injection was simulated in vitro by incubation of whole, excised abdominal adipose tissues of rats with methadone and other basic drugs (dibenzepine, alprenolol, opipramol, propranolol, chlorpheniramine, desipramine, imipramine and chlorpromazine) for 6 h at pH 7.4. These drugs were readily taken up (25 to 74% at equilibrium). 3. There was a positive correlation between uptake and lipophilicity of the drugs as measured by log P (octanol/water). Less lipophilic drugs such as amphetamine, morphine and chlorphentermine were not appreciably taken up from the incubation medium. The threshold log P-value for appreciable adipose tissue uptake is around 2. 4. It is concluded from these data and related studies that basic lipophilic drugs are not stored in adipose tissues in vivo, except when given via the i.p. route. In the latter case the storage appears to result from non-systemic, diffusional uptake from the peritoneal cavity.     

Organochlorine compounds in adipose tissue of Greenlanders and southern Danes.

Abdominal fat tissue samples from the general population of Greenland and from southern Denmark were analyzed for o,p'-DDE and p,p'-DDE [1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene], p,p,-DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl)-ethane], o,p'-DDT and p,p'-DDD [1,1-dichloro-2,2-bis(p-chlorophenyl)ethane], lindane (1,2,3,4,5,6-hexachlorocyclohexane), aldrin (1,2,3,4,10,10-hexachloro-1,4,4a,5,8,8a-hexahydro-endo-exo-1,4:5,8-dimethanonaphthalene), dieldrin (1,2,3,4,10,10-hexachloro-6,7-epoxy-1,4,4a,5,6,7,8,8a-octahydro-endo-exo-1,4:5,8-dimethanonaphthalene), heptachlor (1,4,5,6,7,8,8-heptachloro-3a,4,7,7a-tetrahydro-4,7-methanoindene), heptachlor epoxide, and polychlorinated biphenyls (PCBs). Fat tissue from Greenlanders contained significantly higher amounts of p,p'-DDE (p less than or equal to 0.05), p,p'-DDT (p less than or equal to 0.01), and total DDT (SIGMA DDT, the sum of DDT and its metabolites) (p less than or equal to 0.01) than southern Danes. Lindane, aldrin-like residue, dieldrin heptachlor-like residue, heptachlor epoxide, and PCBs were present in adipose tissue of both groups and there were no significant differences between the groups. The p,p'-DDE level in Greenland was lower than that in the United States, eastern Europe, and India. Among Greenlanders of different ages the highest sigma DDT was found in the age group of 22-45 yr; the content of PCBs increased with age. Among southern Danes the highest sigma DDT was found in a higher age group than in Greenlanders. Among Greenlanders the content of aldrin-like residue decreased with age and that of dieldrin increased with age. A significant correlation was found (on the basis of wet weight) between p,p'-DDE and PCB content in southern Danes (p less than or equal to 0.02). The correlation between sigma DDT and PCB content was also significant in this population (p less than or equal to 0.01). These two relationships were not significantly correlated for the Greenlanders. The DDE/PCB and sigma DDT/PCB ratios were higher in Greenlanders than in southern Danes. These ratios are age-dependent and are highest in the age group 22-45 yr among the Greenlanders. A low DDE/PCB ratio and a low sigma DDT/PCB ratio have been proposed as markers for industrialization. In Greenlanders p,p'-DDE represented about 70% of sigma DDT. For southern Danes this level was about 90%. The data obtained are presented and discussed on the basis of both lipid and wet weight levels.     

Different lipids remodeling signature demonstrated heterogeneity of white adipose tissue browning

OBJECTIVE White adipose tissue(WAT)browning confers beneficial effects on metabolic diseases.However, visceral adipose tissue(VAT) is not as susceptible to browning as subcutaneousadipose tissue(SAT).Therefore, interpreting the heterogeneity of VAT and SAT in brown remodeling is extremely important. METHODS We first investigated the effects of beta-3 adrenergic stimulation by CL316, 243 on systemic metabolism. Then, highcoverage targeted lipidomics approach with multiple reaction monitoring(MRM) was utilized to provide extensive detection of lipid metabolites in VAT and SAT using an Exion ultra performance liquid chromatography(UPLC)coupled with Sciex QTRAP 6500 Plus. RESULTS Beta-3 adrenergic stimulation notably ameliorated the systemic metabolism. Moreover, comprehensive lipidomics analysis elucidated different lipids adaptation between VAT and SAT in adrenergic-induced browning. CL316, 243 induced browning heterogeneity of VAT and SAT with more dramatic alteration of total lipid classes and individual molecular lipid species in VAT rather than SAT, though VAT is resistant to browning. Specifically, CL316, 243 stimulation differentially affected glycerides content in VAT and SAT and boosted the abundance of more glycerophospholipids species in VAT than in SAT. Besides, CL316,243 increased sphingolipids in VAT without changes in SAT, meanwhile, elevated cardiolipin species more prominently in VAT than in SAT. Further study uncovered that the lipids remodeling heterogeneity of VAT and SAT may attribute to a more significant alteration of lipids metabolism genes in VAT rather than SAT in response to CL316,243 treatment. CONCLUSION Our datasets provide acomprehensive analysis of lipids metabolic heterogeneity between VAT and SAT browning and may provide promising lipid targets to motivate WAT browning.     

Adrenergic lipolysis in human adipose tissue in vitro.

In human adipose tissue in vitro, dose-response curves of lipolytic agents in releasing free fatty acids and glycerol into an albumine-containing medium were followed. Norepinephrine and adrenaline produced about 20% of the maximal effect of isoproterenol, isopropylnorsynephrine about 40%, theophylline more than 100%. Found pD2 values were approximately 7.4 for isoproterenol and norepinephrine, approximately 5.9 for isopropylnorsynephrine. Phentolamine (1 times 10-5 M) elevated the maximal norepinephrine effects up to the isoproterenol maxima. Phenoxybenzamine (1 times 10-5 M) had significantly lower potentiating effects. Phenylephrine depressed lipolytic actions of isoproterenol (1 times 10-6 M) showing the same pI2 value 3.0 as well in human as in rat adipose tissue and exerted per se weak lipolytic effects also. It is concluded neither potentiating actions of alpha-blockers nor depressing effects of phenylephrine in human adipose tissue seem related to affecting adrenergic alpha-receptors. The existence of antilipolytic actions of adrenergic alpha-receptors is questioned.     

Organochlorine compounds in human adipose tissue from north Texas.

This paper reports a preliminary study that was conducted to determine the concentrations of organochlorine compounds in the adipose tissue of residents of North Texas. Thirty-five human adipose tissue samples were obtained during autopsy between 1987 and 1988 from persons who had no known occupational exposure to organochlorine pesticides. These samples were analyzed by electron-capture gas chromatographic methods for the presence of beta-benzene hexachloride, o,p'-DDE, p,p'-DDE, o,p'-DDT, p,p'-DDT, dieldrin, oxychlordane, and heptachlor epoxide. The findings indicate greater than 97% occurrence for each compound with the exception of o,p'-DDE and o,p'-DDT, each of which occurred in 54% of the population sampled. Statistical analyses of the data showed strong positive correlations between adipose tissue concentrations and age for oxychlordane, heptachlor epoxide, p,p'-DDT, p,p'-DDE, and total DDT (the normalized sum of DDT and its analogues) (p less than or equal to .05). Statistically significant differences (p less than or equal to .05) in geometric mean concentrations between all age groups were found for total DDT and p,p'-DDE. However, the group means comparison test was only significantly different between the 41-60 yr and 61 and over age groups for p,p'-DDT, oxychlordane, and heptachlor epoxide. No statistically significant difference was found between sexes in this study. The geometric means of pesticide concentrations in adipose tissue were compared with those reported in previous U.S. Environmental Protection Agency surveys for the general population. This comparison clearly indicates a declining temporal trend in environmental exposure for banned DDT analogues; however, a consistent temporal trend exists for oxychlordane and heptachlor epoxide--pesticides whose uses are currently restricted but not proscribed.     

Decreased incorporation of glucose into lipids and increased lactate production by adipose tissue after long-term treatment of rats with D-fenfluramine

Male rats were treated with ten daily doses of 10 mg of D-fenfluramine/kg. Body weight decreased after days 1 and 2, but thereafter the weight gain paralleled that of the control rats. After the tenth injection there were decreases in the weights of the epididymal fat pads, their fat content, and the average size of the adipocytes after collaginase digestion. The rate of glucose uptake by incubated pieces of adipose tissue was maintained after D-fenfluramine treatment, and the production of lactate increased. The incorporation of glucose into fatty acids by adipose tissue pieces decreased by 65-74% after treatment with D-fenfluramine. This effect was not reversed by adding insulin or phenylisopropyladenosine to the incubations. D-Fenfluramine also decreased the incorporation of glucose into glyceride-glycerol, but this effect was less pronounced than that for fatty acid synthesis. Direct addition of D-fenfluramine to the incubation inhibited lipid synthesis from [14C]glucose but only at drug concentrations above 1 mM. It is concluded that the treatment of rats with D-fenfluramine modifies the metabolic balance of adipose tissue so as to direct glucose metabolism away from lipid synthesis and towards lactate production. This could be a significant mechanism in the overall loss of adipose tissue mass caused by the administration of D-fenfluramine.     

The toxicological significance of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds in human adipose tissue

Reports of polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzo-furans (PCDFs) in human tissues were reviewed to assess their toxicological significance. The predominance of 2,3,7,8-chlorinated congeners in human tissues and in the food chain, but not in other environmental matrices, suggests that the food chain is the major source of human residues. Exposures to unique distributions of congeners can result in recognizable patterns of excess 2,3,7,8-chlorinated PCDDs/PCDFs in humans. Current levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) in the general population can be accounted for by an average level of 133 or 27 ppq (parts per quadrillion) in food based on an estimated half-life in humans of 1 or 5 yr, respectively. 2,3,7,8-TCDD is more persistent in humans than in rodents or lagomorphs, resulting in higher body burdens in humans at comparable levels in the diet. Taken alone, this toxicokinetic difference would increase risks estimated for humans from toxicity in laboratory animals. However, humans appear to be less susceptible due to the following: less food is ingested per body mass, more 2,3,7,8-TCDD is sequestered in adipose tissue and away from target organs, and tissue susceptibility appears to be lower than in the most sensitive rodents and lagomorphs. The body burden of 2,3,7,8-TCDD in a Seveso woman receiving an apparently nontoxic dose was approximately 180 times the average body burden of 2,3,7,8-TCDD equivalents in the general population of industralized societies. The body burden of prisoners who were exposed dermally to a suspension of 2,3,7,8-TCDD and who developed severe chloracne was estimated to be as much as 38 times that of the Seveso woman. These comparisons suggest a considerable margin of safety for the general population.     

An in vitro model for assessing drug availability from lipophilic vehicles

An apparatus for studying the rate of release of a solute from a water-immiscible solvent into an acidic, aqueous liquid, followed by permeation through a simulated lipid membrane, is described. The object was to imitate the absorption of a drug after oral administration in a soft gelatin capsule. Rate constants of transfer were determined for a series of substituted benzoic acids, using octanol and isopropyl myristate as solvents. The quantity of solute in the acidic phase did not correlate with the solvent-water distribution coefficient, but was linearly related to the rate constant for transfer from solvent to water. A dynamic system was therefore postulated, rather than one in which the two phases are in equilibrium. In vivo studies on two of the solutes confirmed the in vitro observations. No simple relationship could be derived between blood concentrations and any in vitro parameter, but the rank order of magnitude of the blood concentrations fitted the postulated dynamic mechanism.     

An in vitro model for assessing drug availability from lipophilic vehicles.

An apparatus for studying the rate of release of a solute from a water-immiscible solvent into an acidic, aqueous liquid, followed by permeation through a simulated lipid membrane, is described. The object was to imitate the absorption of a drug after oral administration in a soft gelatin capsule. Rate constants of transfer were determined for a series of substituted benzoic acids, using octanol and isopropyl myristate as solvents. The quantity of solute in the acidic phase did not correlate with the solvent-water distribution coefficient, but was linearly related to the rate constant for transfer from solvent to water. A dynamic system was therefore postulated, rather than one in which the two phases are in equilibrium. In vivo studies on two of the solutes confirmed the in vitro observations. No simple relationship could be derived between blood concentrations and any in vitro parameter, but the rank order of magnitude of the blood concentrations fitted the postulated dynamic mechanism.     

Inhibition by glucocorticoids of prostaglandin release from adipose tissue in vitro.

1 When rabbit chopped adipose tissue was incubated with a lipolytic agent (adrenocorticotrophic hormone, ACTH1-24, 0.1 microng/ml) in Krebs solution, prostaglandin E2 was formed in the tissue and about the same amount was found in the medium. 2 In the presence of indomethacin (1 microng/ml) the appearance of prostaglandin E2 was almost abolished both in the tissue and in the medium. 3 When the incubation was carried out in the presence of hydrocortisone or betamethasone (1-10 microng)ml) the concentration of prostaglandin E2 leaking or carried into the medium was significantly reduced, whereas that remaining in the tissue was significantly increased. This action of the steroids was not reversed by increasing substrate (arachidonic acid) concentration in the medium. 4 The steroids did not affect lipolysis, nor did they influence prostaglandin metabolism since such activity was not detectable in the adipose tissue. 5 Anti-inflammatory steroids therefore did not reduce prostaglandin formation but increased the tissue/medium ratio, which supports the view that they inhibit the release of prostaglandins after these have been synthesized.     

Uptake of quaternary ammonium compounds into rat liver plasma membrane vesicles

In order to elucidate the mechanism by which quaternary ammonium compounds are transported across the rat hepatocyte plasma membrane, the transport of five quaternary ammonium compounds through rat hepatocyte plasma membrane vesicles was investigated. Transport is only observed when the organic cations possess a high lipophilicity. Uptake appeared to be a passive process and was not stimulated by a transmembrane electrical potential difference nor by the presence of an excess of anions like I^?. Taurocholate decreased the uptake. However in the presence of a catalytic amount of tetraphenylborate a transmembrane electrical potential difference (inside negative) stimulated the uptake.     

Permeability of lipophilic compounds in drug discovery using in-vitro human absorption model, Caco-2

Highly lipophilic compounds are often encountered in the early stages of drug discovery. The apparent permeability (Papp) of these compounds in Caco-2 cell could be underestimated because of considerable retention by the Caco-2 monolayer and non-specific binding to transwell surface. We have utilized a general approach for the determination of permeability of these compounds, which includes the addition of 1-5% DMSO in the apical (AP) and 4% bovine serum albumin (BSA) in the basolateral (BA) side. Two highly lipophilic and highly protein bound Schering compounds, SCH-A and SCH-B, exhibited poor recovery and low Papp in the conventional Caco-2 system that included 1% DMSO in the AP and BA sides. In contrast, both compounds were well absorbed in cynomolgus monkeys. Inclusion of BSA (up to 4%) in the BA side provided necessary absorptive driving force similar to in vivo sink conditions improving both recovery and Papp of these compounds as well as progesterone, a model highly lipophilic and highly protein bound compound. Whereas, the recovery and Papp of mannitol (high recovery, low permeability) and propranolol (high recovery, high permeability) remained unaffected. The presence of 4% BSA increased Papp of SCH-A, SCH-B, and progesterone by five-, four-, and three-fold, respectively. We also compared this approach with a second, based on the disappearance of the compound from the AP side, which resulted in a reasonable estimate of the permeability (23.3x10(-6) cm/s) for SCH-A. The results demonstrated that the reliable estimates of permeability of highly lipophilic compounds that are subjected to considerable retention by the cell monolayer and exhibit non-specific binding are obtained by the addition of BSA to the BA side.     

Uptake into mouse brain of four compounds present in the psychoactive beverage kava

A technique using gas chromatography-mass spectrometry and deuterated internal standards is described for the quantitation in brain tissue of four constituents of the intoxicating beverage kava. Dihydrokawain, kawain, desmethoxyyangonin, and yangonin were administered ip to mice at a dosage of 100 mg/kg. At specific time intervals (5, 15, 30, and 45 min), the mice were sacrificed and the brain concentrations of these four compounds determined. After 5 min, dihydrokawain and kawain attained maximum concentrations of 64.7 +/- 13.1 and 29.3 +/- 0.8 ng/mg wet brain tissue, respectively, and were rapidly eliminated. In contrast, desmethoxyyangonin and yangonin had poorly defined maxima corresponding to concentrations of 10.4 +/- 1.5 and 1.2 +/- 0.3 ng/mg wet brain tissue, respectively, and these compounds were more slowly eliminated from brain tissue. When crude kava resin was administered ip at a dosage of 120 mg/kg, the concentration in brain of kawain and yangonin markedly increased (2 and 20 times, respectively) relative to the values measured from their individual injection. In contrast, dihydrokawain and desmethoxyyangonin, after the administration of crude resin, remained at the percentage incorporation into brain tissue established for their individual ip injection.     

Advancing prediction of tissue distribution and volume of distribution of highly lipophilic compounds from a simplified tissue-composition-based model as a mechanistic animal alternative method

It has been reported that values of tissue-plasma ratios (K(p)) and resulting volume of distribution at steady state (V(ss)) are substantially overpredicted for several highly lipophilic drugs. This effect was observed particularly with the published version of the tissue-composition-based model, which used experimentally determined unbound fraction in plasma (fu(p)) as input for drugs. The reasons for the unreasonably high V(ss) predictions were investigated in this study for 14 highly lipophilic compounds with a log n-octanol-water partition coefficient (log P(ow)) of at least 5.8. Here, we argue that the experimentally determined fu(p) is inaccurate for these compounds, which affected the prediction of K(p) and V(ss). Alternatively, the tissue-plasma ratio of neutral lipids (nl) equivalent was used as the main factor governing K(p), and hence V(ss), in addition to log P(ow). The average fold error of deviation between the predicted and observed human V(ss) is 124 for the published model, whereas it significantly decreased to 1.5 for the proposed model. The sensitivity analysis confirmed the importance of nl content and drug lipophilicity. Overall, this study proposes a generic and simplified tissue-composition-based model for highly lipophilic drugs and chemicals, which is a step forward toward improving prediction of V(ss) into physiologically based pharmacokinetic (PBPK) models.     

大鼠脂肪干细胞的体外分离培养及对脂肪颗粒移植的影响

目的分析体外大鼠脂肪干细胞(ADSCs)混合脂肪移植后的存活情况。方法无菌条件下切取大鼠腹股沟脂肪组织,消化、分离培养得到第3代ADSCs,行成骨诱导(茜素红染色)和成脂诱导(油红O染色)。用CM-Dil荧光标记第3代ADSCs,24只大鼠每只背部皮下3处分别植入1.5ml脂肪颗粒、1.5ml荧光标记的ADSCs(密度为5×107个细胞/ml)和0.9ml荧光标记的ADSCs+0.6ml脂肪颗粒。术后2周、1个月和3个月每次取出8只大鼠的移植物,石蜡切片HE染色观察病理变化,冰冻切片荧光显微镜下观察ADSCs定位。结果 ADSCs成骨诱导2周后茜素红染色阳性,成脂诱导3周后油红O染色阳性。ADSCs与脂肪颗粒混合移植能明显改善脂肪组织的病理学变化。结论体外分离培养的大鼠ADSCs具有成骨、成脂分化的潜能,能改善脂肪颗粒移植时的脂肪组织的液化吸收现象。     

Retention of lipophilic compounds on laboratory tubing

¹⁴C-Labeled pesticidal carbamates were retained in polyvinyl chloride and latex components of intestinal perfusion apparatus approximately proportionate to their rate of disappearance from the perfusate during absorption experiments. Simulated control perfusions, equilibration of the perfusion system with the perfusate and analysis of the recovered substrate established that such a problem had been encountered. Precautions should be taken when-ever compounds with a high lipid partition coefficient are exposed to polymeric materials in an aqueous medium at very low concentrations.