Biopharmaceutics: Topical Delivery of Keloid Therapeutic Drug,Tranilast, by Combined Use of Oleic Acid and Propylene Glycol as a Penetration Enhancer: Evaluation by Skin Microdialysis in Rats

Topical delivery of tranilast (N-(3,4-dimethoxycinnamoyl)anthranic acid), an inhibitor of collagen synthesis and a therapeutic drug for keloid and hypertrophic scar, was examined, in rats, with oleic acid alone or a combination of oleic acid and propylene glycol as penetration enhancer. Evaluation was by measurement of the concentration of tranilast in plasma and in the dialysate from skin microdialysis. When tranilast at a dose of 1.5 mg was applied topically as an ethanol solution containing 5% polyvinylpyrrolidone on a dorsal skin surface (2.25 cm²), the maximum concentration of tranilast in skin dialysate was approximately 2 μM. When 10 or 20% oleic acid was added to the same ethanol solution the maximum concentration of tranilast in the dialysate increased to 10–20 μM, and this value was further increased to 60 μM by the addition of a combination of oleic acid (10 or 20%) and propylene glycol (10%) to the solution. With the combination of oleic acid and propylene glycol the area under the plot of the concentration of tranilast in skin dialysate against time between 0 and 4 h (AUC_0–4) was more than 400-fold that after intravenous administration. The transdermal bioavailability of tranilast as assessed by the AUC_0–4 of tranilast in plasma, was 0.2% of the dose applied in the ethanol solution, 3–5% of that applied in the ethanol solution containing oleic acid, and 14–16% of that applied in the ethanol solution containing both oleic acid and propylene glycol. These results suggest that the topical delivery of tranilast with an absorption enhancer such as a mixture of oleic acid and propylene glycol might be a more effective medication than oral administration of tranilast for the treatment of keloid and hypertrophic scar.     

Evaluation of Benzylpenicillin as an Internal Standard for Measurement of Piperacillin Bone Concentrations Via Microdialysis

Microdialysis is a pharmacokinetic tool that can be advantageous when obtaining tissues’ pharmacokinetic information. Since absolute extracellular tissue concentrations are needed in pharmacokinetic studies, calibrating the microdialysis system is necessary. The internal standard method is superior when compared to other calibration methods. However, thorough evaluation of the internal standard is required before it can be used. In vitro experiments and an in vivo study on pigs (n = 8) were conducted to assess the relative recoveries by gain and by loss for piperacillin, both with and without a benzylpenicillin concentration of 5 µg/mL. Furthermore, the in vivo setup allowed for an evaluation of piperacillin cancellous bone and subcutaneous tissue concentrations in a single 8 h dosing interval. Ultra-high performance liquid chromatography (UHPLC) was used to determine piperacillin and benzylpenicillin concentrations. Relative recovery by loss for benzylpenicillin and relative recovery by gain for piperacillin were similar in in vitro and in vivo. Presence of benzylpenicillin did not affect the relative recovery for piperacillin. Relative recovery, pharmacokinetic parameters and fT>MIC were similar when comparing the retrodialysis by drug and the internal standard calibration methods (p > 0.31). Mean fT>MIC (16 µg/mL) for plasma, cancellous bone and subcutaneous tissue were 232 min, 255 min and 295 min, respectively.Our findings suggest that benzylpenicillin is suitable as an internal standard for piperacillin in microdialysis studies. Mean fT>MIC (16 µg/mL) for plasma, cancellous bone, and subcutaneous tissue reached a target of 50% fT>MIC under the investigated conditions (mean range: 52%–66%); however, the target was not obtained in all pigs in all compartments. Moreover, 100% fT>MIC was not obtained in any case, suggesting that different strategies must be taken into consideration if higher targets are employed.     

In-vivo Microdialysis Study of the Distribution of Cisplatin into Brain Tumour Tissue after Intracarotid Infusion in Rats With 9L Malignant Glioma

Simultaneous brain microdialysis in tumour and non-tumour tissues has been used for kinetic determination of the local distribution of an anticancer agent, cisplatin, in rats. Rat brain was implanted with 9L malignant glioma and cisplatin (3.5 mg kg^?) was administered as a selective intracarotid infusion for 30 min to rats prepared for brain microdialysis. The amount of platinum in the dialysate collected from tumour and non-tumour brain tissues was determined by atomic absorption spectrophotometry, as representative of cisplatin. Total and free platinum concentrations in plasma were also measured. Free platinum is accumulated preferentially in the tumour tissue and the brain tumour distribution coefficient (the ratio of brain tumour platinum AUC to plasma free platinum AUC, where AUC is the area under the platinum concentration-time curve) was 0.69, although there was little distribution into normal brain tissue. Drug binding to plasma proteins was 65%. It is concluded that simultaneous microdialysis is an easy and available method for assessing in-vivo local pharmacokinetics and distribution of cisplatin in tumour and non-tumour tissues of the brain.     

Biopharmaceutics: Effect of Ointment Bases on Topical and Transdermal Delivery of Salicylic Acid in Rats: Evaluation by Skin Microdialysis

Microdialysis has been used to determine the concentration of salicylic acid in skin tissue and plasma periodically for 4 h to evaluate the effect of ointment bases on topical and transdermal delivery of salicylic acid. The ointment bases examined were solbase (water-soluble), poloid and white petrolatum (oleaginous), hydrophilic poloid (water in oil (w/o) type emulsion lacking water) and absorptive ointment (w/o-type emulsion containing water). The ointments (0.1 g) containing 25 μmol salicylic acid were applied for 2 h to the surface of rat skin (1 cm²) with (intact) or without the stratum corneum. For intact skin, the extent of topical delivery from different ointments, evaluated by the area under the concentration-time curve (AUC) of salicylic acid in the skin tissue (AUCskin), increased in the order solbase. white petrolatum, poloid, hydrophilic poloid. absorptive ointment. The ratio of AUC_skin (topical delivery) to the AUC of salicylic acid in plasma (AUC_plasma, transdermal delivery) varied remarkably among the different bases, the greatest ratio being observed for absorptive ointment. When the ointments were applied to skin surface without stratum corneum, AUC_skin for solbase was much higher (about 45 times that for intact skin), whereas only a small (two-fold) increase was observed for poloid and hydrophilic poloid and the increase was negligible for white petrolatum and absorptive ointment. For skin without the stratum corneum, the ratio AUC_skin/AUC_plasma for the different ointments was comparable, although the magnitudes of AUC_skin and AUC_plasma still varied substantially. The variance of AUC values arises as a result of the different rates of release of salicylic acid from the bases. These results indicate that: the topical and transdermal delivery of salicylic acid in intact skin varies substantially among different ointment bases, and the greatest topical delivery is observed for absorptive ointment; use of absorptive ointment increases the retention of salicylic acid in the stratum corneum; and the stratum corneum functions strongly as a penetration barrier for solbase, moderately for poloid and hydrophilic poloid, and less for absorptive ointment and white petrolatum.     

Study on Brain Interstitial Fluid Distribution and Blood-Brain Barrier Transport of Baclofen in Rats by Microdialysis

Purpose. This study was performed to examine the distribution in the brain interstitial fluid (ISF) and the blood-brain barrier (BBB) transport of baclofen in rats by a microdialysis technique. Methods. Following an i.v. bolus administration and/or the constant i.v. infusion of baclofen to the microdialysis cannula-bearing anesthetized rats, the concentrations of baclofen in the hippocampal ISF, whole brain tissue, cerebrospinal fluid (CSF), and plasma were determined by high-performance liquid chromatography (HPLC). Data were kinetically analyzed to estimate the transport parameters, i.e., the influx clearance (CL_in) from plasma to brain and the efflux rate constant (k_eff) from brain to plasma, and the steady-state volume of distribution in the brain (V_d). Results. The concentrations of baclofen in ISF, whole brain tissue, and CSF at the pseudo-steady state were almost 30-fold lower than the plasma unbound concentration, suggesting the restricted distribution of baclofen in the brain. The estimated values of CL_in and k_eff were 0.00157 ± 0.00076 ml/min/g of brain and 0.0872 ± 0.0252 min^–1, respectively. The efflux clearance (CL_out) calculated by multiplying k_eff by V_d (0.816 ± 0.559 ml/g of brain) was 0.0712 ± 0.0529 ml/min/g of brain, and it was significantly 40-fold greater than the CL_in value and fully greater than the convective flow in ISF. Furthermore, no significant concentration gradient was observed between ISF and CSF. These results suggest that the CL_out value mainly reflects the efflux clearance through the BBB. Additionally, the hippocampal ISF/plasma concentration ratio of baclofen was markedly increased by both systemic administration of probenecid and its direct instillation into ISF. Conclusions. The restricted distribution of baclofen in the brain ISF may be ascribed to the efficient efflux from the brain through the BBB which is regulated possibly by a probenecid-sensitive organic anion transport system.     

In-vivo Microdialysis Measurement of 5-Hydroxytryptamine and its Metabolites, 5-Hydroxyindoleacetic Acid and N-Acetyl 5-Hydroxytryptamine,in Rat Blood: Effects of Histamine-receptor Antagonists

The blood concentrations of 5-hydroxytryptamine (5-HT) and its metabolites, 5-hydroxyindoleacetic acid (5-HIAA) and N-acetyl 5-HT were assayed by in-vivo microdialysis and a highly sensitive HPLC procedure that was originally developed to analyse CNS mediators. We investigated the effects of histamine-receptor antagonists on 5-HT metabolism and its release into the blood of rats. The mean basal levels of 5-HT, 5-HIAA and N-acetyl 5-HT in the blood measured by in-vivo microdialysis were 77.2 ± 9.4, 20.3 ± 1.5 and 1.89 ± 0.15 pmol mL^?1, respectively. These levels were not significantly affected by an intraperitoneal injection of saline, and remained at constant levels for at least 8 h after administration of saline. After an intraperitoneal injection of 5-HT hydrochloride (0.5 mg kg^?1), 5-HT was soon detected in the blood of the jugular vein. 5-HIAA also quickly appeared in the blood and declined monoexponentially from 60 min after injection. In contrast, N-acetyl 5-HT slowly appeared in the blood and it reached a maximal level at 270 min. The 5-HT and N-aceryl 5-HT levels in dialysates from rat jugular vein were significantly increased by intraperitoneal pyrilamine (2.0 mg kg^?1), (+)-chlorpheniramine (2.0 mg kg^?1) and cimetidine (20.0 mg kg^?1). However, there was no increase in the 5-HIAA concentration after an intraperitoneal injection of these histamine-receptor antagonists, demonstrating that the 5-HT released from various cells containing 5-HT was predominantly metabolized to N-acetyl 5-HT by N-acetyltransferase. Moreover, thioperamide did not affect the basal levels of 5-HT, 5-HIAA or N-acetyl 5-HT. Because the recovered 5-HT, 5-HIAA and N-acetyl 5-HT in the dialysate is directly proportional to the free fraction in the blood, in-vivo microdialysis is a reliable method of examining 5-HT metabolism and its release into the blood.     

Determination of Isatin,an Endogenous Monoamine Oxidase Inhibitor,in Urine and Tissues of Rats by HPLC

• 1.We have previously identified isatin as one of the endogenous monoamine oxidase (MAO) inhibitors in the urine and the brain of stroke-prone spontaneously hypertensive rats (SHRSP), using gas chromatography-mass spectrometry (GC-MS).
• 2.In this study, we attempted to develop a convenient assay to determine isatin using high performance liquid chromatography with an ultraviolet detector (HPLC-UV). The standard curve for authentic isatin was linear at a range from 2 to 20 nmol per ml. The coefficient of variance was within 3% for both intra-assay and inter-assay. The sensitivity was 20 pmol per 10 μl of urine sample.
• 3.Isatin concentration correlated significantly and positively with endogenous MAO activity (tribulin-like activity) in both urine (r=0.924, P<0.001) and kidney extracts (r=0.862, P<0.01). There was a significant difference in urinary isatin between Wistar Kyoto rats (WKY) and SHRSP. Oral administration of isatin increased urinary isatin concentration and systolic blood pressure in WKY.
• 4.Determination of isatin using HPLC-UV may be useful for elucidating role of isatin in various conditions of stress and disease.

     

Use of Shed Snake Skin as a Model Membrane for in Vitro Percutaneous Penetration Studies: Comparison with Human Skin

The potential usefulness of shed snake skin as a model membrane for transdermal research was examined. There are similarities between shed snake skin and human stratum corneum in terms of structure, composition, lipid content, water permeability, etc. The permeability of various compounds and the contribution of several functional groups to the permeability were also found to be similar between shed snake skin and human skin. Moreover, the permeability of compounds through shed snake skin was increased by Azone, one of the most extensively studied transdermal penetration enhancers. Considering the similarities between shed snake skin and human skin, ease of storage and handling, and low cost, shed snake skin may offer a good model membrane for transdermal research.     

不同品种大黄油糊外用抗炎作用及对大鼠皮肤溃疡、痔疮模型的影响

目的：通过对大黄的外用实验研究,总结其外用功能。方法：观察三品种大黄油糊外用对大鼠蛋清性足跖肿胀、二甲苯致小鼠耳廓肿胀、大鼠创伤性皮肤溃疡、99．0％醋酸溶液致大鼠痔疮模型的影响。结果：大、小剂量掌叶大黄、唐古特大黄、药用大黄油糊均可显著抑制大鼠蛋清性足跖肿胀和二甲苯致小鼠耳廓肿胀,明显减少大鼠创伤性皮肤溃疡的面积、促进溃疡皮肤角质层、鳞状细胞层和局部病理组织的恢复,可显著加快大鼠肛周皮肤溃疡的愈合。结论：大黄可促进病理组织的修复,加快溃疡面积的愈合,外用有收敛生肌的功能。     

Minor Effect of Tolcapone,a Catechol- O-Methyltransferase Inhibitor,on Extracellular Dopamine Levels Modified by Amphetamine or Pargyline: A Microdialysis Study in Anaesthetized Rats

Abstract: In vivo microdialysis was used to examine the effects of tolcapone (30 mg/kg) on dopamine metabolism in amphetamine (5 mg/kg) and pargyline (75 mg/kg) treated rats. Amphetamine- or pargyline-induced decreases in the extracellular dihydroxyphenyl acetic acid (DOPAC) levels were counteracted by additional tolcapone. Tolcapone also decreased homovanillic acid effluxes below those caused by amphetamine or pargyline. However, dopamine effluxes were not further enhanced by additional tolcapone. These results show that central metabolism of dopamine can be modulated by catechol-O-methyltransferase (COMT) inhibition also without exogenous L-DOPA. However, extracellular dopamine levels are not easily increased.     

Antagonism by SB 204070 of 5-HT-evoked Contractions in the Dog Stomach: an In-vivo Model of 5-HT4 Receptor Function

The ability of 5-hydroxytryptamine (5-HT) to evoke contractile activity in the gastric Heidenhain pouch was measured in conscious dogs using a method in which 5-HT₄ receptor-antagonist activity can be measured in-vivo. At doses of 5-HT which evoked short-lived measurable responses (5 or 10 μg kg^?1, i.v.), it was found that this activity was greatly reduced by atropine (100 μg kg^?1, i.v.), but was unaffected by methysergide, methiothepin, ketanserin (each at 100 μg kg^?1, i.v.) or granisetron (10 or 100 μg kg^?1, i.v.). At best SDZ 205–557 2-diethylaminoethyl-[2-methoxy-4-amino-5-chloro] benzoate; 100 μg kg^?1, i.v.) reduced the action of 5-HT in 4/5 animals and increased it in the other but its effects were variable in magnitude and not consistently maintained. However, the more potent and selective 5-HT₄-receptor antagonist SB 204070 (1-butyl-4-piperidinylmethyl 8-amino-7-chloro-1,4-benzodioxan-5-carboxylate hydrochloride) dose-dependently antagonized the 5-HT-evoked contractions in all dogs tested. This action was reversible, but long-lasting with an effective half-life of 18·0 h when administered at 1 μg kg^?1. The estimated ID50 value was 0·55 μg kg^?1.     

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.     

Active Secretion of Drugs from the Small Intestinal Epithelium in Rats by P-Glycoprotein Functioning as an Absorption Barrier

Because the significance of P-glycoprotein in the in-vivo secretion of β-blockers in intestinal epithelial cells is unclear, the secretory mechanism for β-blockers and other drugs has been evaluated. Uptake of the β-blockers acebutolol, celiprolol, nadolol and timolol, and the antiarrhythmic agent, quinidine by the multidrug-resistant leukaemic cell line variant K562/ADM was significantly lower than that by drug-sensitive K562 cells, suggesting that these β-blockers are transported by P-glycoprotein out of cells. The reduced uptake of acebutolol by the drug-resistant K562/ADM cells was reversed by treating the cells with anti-P-glycoprotein monoclonal antibody, MRK16, whereas no such alteration in uptake was observed for drug-sensitive K562 cells. Acebutolol uptake by K562/ADM cells was, moreover, markedly enhanced, in a concentration-dependent manner, in the presence of the specific P-glycoprotein inhibitors, MS-209 and cyclosporin. Caco-2 cells were used for evaluation of the role of P-glycoprotein in intestinal permeability to drugs in-vitro. Basolateral-to-apical transport of acebutolol was twice that in the reverse direction. A similar polarized flux was also observed in the transport of vinblastine, but not in that of acetamide or mannitol. When in-vivo intestinal absorption was evaluated by the rat jejunal loop method, with simultaneous intravenous administration of a P-glycoprotein inhibitor, cyclosporin, intestinal absorption of both acebutolol and vinblastine increased 2.6- and 2.2-fold, respectively, but no such enhancement was observed in the absorption of acetamide. The effect of cyclosporin on the intestinal absorption of several drugs was further examined, and the extent of the contribution of P-glycoprotein as an absorption barrier to those drugs was evaluated. ATP depletion by occlusion of the superior mesenteric artery resulted in a clear increase in epithelial permeability to vinblastine, but not to 3-O-methylglucose or acetamide, indicating that vinblastine is secreted by ATP-dependent P-glycoprotein into the lumen. These findings demonstrate that P-glycoprotein plays a role as an absorption barrier by transporting several drugs from intestinal cells into the lumen.     

Evaluation of Guanfacine as a Potential Medication for Alcohol Use Disorder in Long-Term Drinking Rats: Behavioral and Electrophysiological Findings

One of the main treatment challenges in alcohol use disorder (AUD) is the high rate of craving in combination with decreased cognitive functioning including impaired decision making and impulse control that often lead to relapse. Recent studies show that guanfacine, an α-2-adrenoceptor agonist and FDA-approved ADHD medication, attenuates stress-induced relapse of several drugs of abuse including alcohol. Here we evaluated guanfacine''s effects on voluntary alcohol intake, the alcohol deprivation effect (ADE), alcohol seeking behavior, and cue/priming-induced reinstatement in Wistar rats that had voluntarily consumed alcohol for at least 2 months before treatment. In addition, guanfacine''s ability to regulate glutamatergic neurotransmission was evaluated through electrophysiological recordings in medial prefrontal cortex (mPFC) slices prepared from long-term drinking rats (and alcohol-naive controls) that had received three daily guanfacine (0.6 mg/kg/day) or vehicle injections in vivo. Guanfacine decreased alcohol intake in high, but not low, alcohol-consuming rats and the effects were generally more long lasting than that of the AUD medication naltrexone. Repeated guanfacine treatment induced a long-lasting decrease in alcohol intake, persistent up to five drinking sessions after the last injection. In addition, guanfacine attenuated the ADE as well as alcohol seeking and cue/priming-induced reinstatement of alcohol seeking. Finally, subchronic guanfacine treatment normalized an alcohol-induced dysregulated glutamatergic neurotransmission in the mPFC. These results support previous studies showing that guanfacine has the ability to improve prefrontal connectivity through modulation of the glutamatergic system. Together with the fact that guanfacine appears to be clinically safe, these results merit evaluation of guanfacine''s clinical efficacy in AUD individuals.     

Sold as Heroin: Perceptions and Use of an Evolving Drug in Baltimore,MD

Since 2001, heroin-related overdose deaths in the United States have risen six-fold, a rise unaccounted for by the expanding user population. Has heroin become a more dangerous drug? Reports of fentanyl and its analogs, often concealed in or sold as heroin, have also increased sharply. This article investigates heroin injectors’ perceptions and experiences of changes in the heroin supply in the East Coast city of Baltimore, Maryland, currently facing an epidemic in heroin- and fentanyl-related overdose deaths. Unusually, Baltimore’s heroin market is divided between two types: “Raw,” believed to be Colombian in origin and relatively pure, and the more adulterated “Scramble” (raw heroin traditionally blended with quinine and lactose). Users reported that Scramble heroin, while gaining market share, has become a highly unstable product, varying dramatically in appearance, intensity of onset, duration of action, and effect. Some considered that Scramble was no longer “heroin,” but was heavily adulterated or even replaced, mentioning fentanyl, benzodiazepines, and crushed opioid pills as additives. There was intense awareness of overdose as a present danger in users’ lives, which they linked to the recent adulteration of the heroin supply. Responses to this perceived adulteration varied, including information gathering, attraction, avoidance, taking precautions, and acceptance.     

Use of traditional herbal medicine as an alternative in dental treatment in Mexican dentistry: a review

Context: Herbal therapies are used worldwide to treat health conditions. In Mexico, generations have used them to treat gingivitis, periodontitis, mouth infections, and discoloured teeth. However, few studies have collected scientific evidence on their effects.

Objective: This study aimed at searching and compiling scientific evidence of alternative oral and dental treatments using medicinal herbs from Mexico.

Methods: We collected various Mexican medicinal plants used in the dental treatment from the database of the Institute of Biology at the National Autonomous University of Mexico. To correlate with existing scientific evidence, we used the PubMed database with the key term ‘(scientific name) and (oral or dental)’.

Results: Mexico has various medical herbs with antibacterial and antimicrobial properties, according to ancestral medicinal books and healers. Despite a paucity of experimental research demonstrating the antibacterial, antimicrobial, and antiplaque effects of these Mexican plants, they could still be useful as an alternative treatment of several periodontal diseases or as anticariogenic agents. However, the number of studies supporting their uses and effects remains insufficient.

Discussion and conclusion: It is important for the health of consumers to scientifically demonstrate the real effects of natural medicine, as well as clarify and establish their possible therapeutic applications. Through this bibliographical revision, we found papers that testify or refute their ancestral uses, and conclude that the use of plants to treat oral conditions or to add to the dental pharmacological arsenal should be based on experimental studies verifying their suitability for dental treatments.     

Drug Metabolism: Activation of 11 β-Hydroxysteroid Dehydrogenase by Dehydroepiandrosterone Sulphate as an Anti-hypertensive Agent in Spontaneously Hypertensive Rats

The anti-hypertensive properties of dehydroepiandrosterone sulphate (DHEAS) have been investigated by studying its effects on blood pressure, on serum concentrations of corticosterone and dehydrocorticosterone, and on 11 β-hydroxysteroid dehydrogenase (11 β-HSD) activity in spontaneously hypertensive rats (SHR). SHR were given intraperitoneal injections of DHEAS (10 mg day^?1 for 70 days) from six to 16 weeks of age. The blood pressure–time curve was significantly (P<0.05) suppressed immediately after administration of DHEAS. There was no difference between the heart rates of control and DHEAS groups. Serum concentrations of corticosterone and dehydrocorticosterone in the DHEAS group were significantly (P < 0.05) lower than those of the control group. The dehydrocorticosterone/corticosterone concentration ratio was, however, significantly (P < 0.05) higher in the DHEAS group, suggesting that treatment with DHEAS enhanced the overall interconversion of corticosterone to dehydrocorticosterone. The activity of 11 β-HSD in specific organs of the DHEAS group was affected, characteristic changes being increases in the kidney (14–58%), decreases in the liver (11–27%) and no change in the testis. Direct addition of DHEAS to 11 β-HSD preparations from the kidneys of control SHR had the same effect as that observed in the in-vivo experiments. The fall in serum corticosterone in the DHEAS group is considered to be related, at least partly, to increased activity of kidney 11 β-HSD. The inverse correlation of kidney 11 β-HSD activity with serum corticosterone and blood pressure (—r = 0.628, P < 0.01, and —r = 0.478, P < 0.05, respectively) suggest that DHEAS delayed the development of hypertension in SHR by selective promotion of kidney 11 β-HSD activity which in turn resulted in lower serum concentrations of corticosterone and its minimal aldosterone-like activity.     

Investigation of Protein-Surfactant Interactions by Analytical Ultracentrifugation and Electron Paramagnetic Resonance: The Use of Recombinant Human Tissue Factor as an Example

Purpose. The purpose of this work is to utilize electron paramagnetic resonance (EPR) spectroscopy in conjunction with analytical ultracentrifugation (AUC) to investigate the binding of surfactants to proteins with a transmembrance domain. As an example these methods have been used to study the interaction of a nonionic surfactant, C12E8, to recombinant human tissue factor (rhTF) in liquid formulations. The complementary nature of the two techniques aids in data interpretation when there is ambiguity using a single technique. In addition to binding stoichiometries, the possibility of identifying the interacting domains by using two forms of rhTF is explored. Methods. Two recombinant, truncated forms of human tissue factor were formulated in the absence of phospholipids. Neither of the recombinant proteins, produced in E. coli, contains the cytoplasmic domain. Recombinant human tissue factor 243 (rhTF 243) consists of 243 amino acids and includes the transmembrane sequences. Recombinant human tissue factor 220 (rhTF 220), however, contains only the first 221 amino acids of the human tissue factor, lacking those of the transmembrane region. EPR and AUC were used to investigate the interactions between these two forms of rhTF and polyoxyethylene 8 lauryl ether, C12E8. Results. Binding of C12E8 to rhTF 243 is detected by both EPR spectroscopy and AUC. Although a unique binding stoichiometry was not determined, EPR spectroscopy greatly narrowed the range of possible solutions suggested by the AUC data. Neither technique revealed an interaction between rhTF 220 and C12E8. Conclusions. The complementary nature of EPR spectroscopy and AUC make the combination of the two techniques useful in data interpretation when studying the interactions between rhTF and C12E8. By utilizing these techniques in this study, the binding stoichiometry of rhTF 243 to C12E8 ranges from 1.2:1 to 1.3:0.6 based on an aggregation number of 120. This binding is consistent with previously reported activity data that showed an increase in clotting rate when rhTF 243 is in the presence of C12E8 micelles. From the rhTF 220 data, it can further be concluded that the transmembrane domain of rhTF is necessary for interactions with C12E8.     

Use of fluorometry in assessing the efficacy of a cation-sensitive gel as an ophthalmic vehicle: comparison with scintigraphy.

Gelrite, a heteropolysaccharide that forms a gel in the presence of cations, was tested in humans for its efficacy as an ophthalmic vehicle by a nonivasive fluorometric technique. Fluorescein was used as the tracer, and its concentration in the anterior chamber was used as the principal measure of bioavailability. The gel afforded a twofold increase in penetration of fluorescein compared with an isotonic buffer solution; this increase is slightly more than can be obtained with simple viscous vehicles. The increase in penetration caused by Gelrite was confirmed by measurements of the contact time of fluorescein in the tear film with the cornea. Earlier experiments with scintigraphy suggested a considerably greater contact time of fluorescein with the cornea when Gelrite was used. However, this increased contact time may be because the technique also measures radioactive tracer that had dried out on the lid margins. Accordingly, significant quantities of fluorescein could be eluted from the lids after the penetration experiments were completed.     

Skin oxygenation after topical application of liposome-entrapped benzyl nicotinate as measured by EPR oximetry in vivo: Influence of composition and size

New and improved drug delivery systems are the important subject of much scientific research. The development of formulations that increase skin oxygenation and of methods for measuring oxygen levels in skin are important for dealing with healing processes affected by the level of oxygen. We have use EPR oximetry in vivo to compare the influence of liposomal formulations of different size and composition with that of hydrogel with respect to the action of the entrapped benzyl nicotinate (BN). Following the topical application of BN onto the skin of mice, pO₂ increase was measured by low-frequency EPR as a function of time. The effect of BN was evaluated by 3 different parameters: lag-time, time needed for maximum pO₂ increase, and overall effectiveness expressed by the area under the response-time curve. An increase in skin oxygenation was observed after BN application. The results show that the effect of BN incorporated in liposomes is achieved more rapidly than the effect from hydrophilic gel. The composition of the liposomes significantly affects the time at which BN starts to act and, to a lesser extent, the maximum increase of pO₂ in skin and the effectiveness of BN action. However, the size of the liposomes influences both the effectiveness of BN action and the time at which BN starts to act. After repeated application of liposomes, the pO₂ baseline increased and the response of the skin tissue was faster. Our results demonstrate that EPR oximetry is a useful method for evaluating oxygen changes after drug application and for following the time course of their action.