Percutaneous delivery of carbamazepine and selected N-alkyl and N-hydroxyalkyl analogues.

Advantages associated with the transdermal delivery route are well documented, but in the past scientists have concentrated primarily on means of decreasing the barrier function of the skin for improved permeability. Pro-drugs, which possess more favourable physicochemical properties for improved transdermal permeability may have considerable potential. These have been considered in the past but recent information concerning structure activity relationships in dermal penetration has prompted increased interest. During this study, N-methyl (2), N-ethyl (3) and N-(2-hydroxyethyl) carbamazepine (4) analogues were synthesised for transdermal evaluation.

Predicting the dermal absorption of thalidomide and its derivatives.

Concern has been expressed about the ability of simple algorithms to predict skin permeability and hence skin flux. For a series of thalidomide analogues, a number of software packages have been used to predict octanol water partition coefficients. These, in conjunction with molecular weight, have then been used to calculate skin permeability coefficients. These compare favourably with experimental values. Some of the software packages also predict aqueous solubilities, which can be subsequently used to calculate maximum skin flux. The predicted and measured solubilities have been compared together with the maximum fluxes. The results show that software can be used to predict octanol water partition coefficients and aqueous solubilities (more accurately if the melting point of the compound is known) and hence to obtain very reasonable estimates of skin permeation parameters. These are useful in predicting which analogue has the most appropriate properties for dermal delivery; in the case of the thalidomide analogues, it is the methyl-substituted compound that is best.

Hepatic osteodystrophy in rats results mainly from portasystemic shunting.

BACKGROUND AND AIMS: In chronic liver disease, bone disease frequently develops. The contributions of the different features of liver disease such as parenchymal inflammation, portal hypertension, and portasystemic shunting on bone metabolism have not been systematically studied. The aim of this study was to identify the features of liver disease contributing to bone disease using rat models. METHODS: Parenchymal liver disease was induced by carbon tetrachloride administration, portal hypertension by partial portal vein ligation, and portasystemic shunting by end to side anastomosis of the portal vein to the inferior vena cava. Normal and sham operated surgical animals served as controls. Serum calcium, 25-hydroxy vitamin D (25-OH vit D), and osteocalcin levels, and urinary deoxypyridinoline excretion were analysed. Testosterone and oestradiol levels were determined in male and female rats, respectively. Interleukin 1, interleukin 6, and tumour necrosis factor alpha (TNF-alpha) were determined in serum. Bone density was measured in all groups and in addition, in the surgical groups, histomorphometry was performed on undecalcified specimens of the proximal tibia. The calcium content of the femurs, removed at termination and ashed, was determined. RESULTS: Early parenchymal disease and portal hypertension did not affect bone metabolism or body mass. Portasystemic shunting increased bone resorption, decreased bone formation, bone density, and trabecular bone volume which were commensurate with a reduction in body mass. TNF-alpha levels were elevated and testosterone levels were low in male portasystemic shunted rats. CONCLUSIONS: Portasystemic shunting in the rat adversely affects bone metabolism as part of a generalised catabolic state where high TNF-alpha and low testosterone and 25-OH vit D levels may play a role.

Evaluation of a novel heminested PCR assay based on the phosphoglucosamine mutase gene for detection of Helicobacter pylori in saliva and dental plaque.

A novel heminested PCR protocol was developed for the specific detection of Helicobacter pylori at low copy numbers. A set of primers specific for the phosphoglucosamine mutase gene (glmM) of H. pylori produced a 765-bp fragment that was used as template for the heminested primer pair delineating a 496-bp fragment. By using agarose gel electrophoresis for detection of the heminested PCR-amplified products, amplification of H. pylori genomic DNA was achieved at concentrations as low as 0.1 pg, equivalent to 5 x 10(2) bacteria. A study was subsequently undertaken to evaluate the heminested PCR for detection of H. pylori in dental plaque and saliva. Specimens collected from 58 individuals were cultured, and PCR was subsequently performed on the oral cultures. Identification of H. pylori in the same series of saliva and dental plaque specimens was carried out with PCR using a primer pair specific for the H. pylori urease B gene and by the heminested PCR assay. The identity of the amplified products was confirmed by DNA sequencing. Our results demonstrate that the heminested PCR assay was specific for detection of H. pylori, yielding no false-positive results, and that H. pylori had a low prevalence (approximately 3%) in specimens obtained from the oral cavity.

A comparative study of an in situ adapted diffusion cell and an in vitro Franz diffusion cell method for transdermal absorption of doxylamine.

In order to determine whether a drug shows the potential for percutaneous absorption, both in situ and in vitro studies are used. In vitro studies are good indicators of transdermal drug delivery, but the possibility exists that anatomical changes in excised skin can influence drug delivery. The aim of this study was to compare the in vitro Franz diffusion cell method with an in situ adapted diffusion cell method. A saturated aqueous solution of doxylamine succinate was used as model drug and the receptor phase was an isotonic Sörensen buffered solution. The in vitro permeation studies were conducted using vertical Franz diffusion cells with nude mice skin. For in situ studies, a diffusion cell was implanted under the dorsal skin of a nude mouse, simulating the in vitro method. Both in situ and in vitro experiments were conducted over a period of 12 h during which samples were collected every 90 min. The mean steady-state flux from Franz diffusion cells was 0.164+/-0.045 microg/cm2/h and flux determined by the in situ method was 0.113+/-0.034 microg/cm2/h. A statistical significant difference existed between the permeation results of the in vitro and in situ experimental methods. A subjective, semi-quantitative assessment of histological changes to excised nude mouse skin was done using light microscopy. This showed that excised skin undergoes sub-lethal injury (necrosis) during in vitro experiments, which may lead to increased permeability of the drug. It was noticed that in vitro and in situ permeation results showed very close correlation until approximately 4.5 h after commencement of experiments, after which, the permeation through excised skin increased. It was assumed that cell necrosis occurred to such an extent after approximately 4.5 h, that the barrier function of the stratum corneum decreased and permeation of the drug increased.

Intranasal toxicity of selected absorption enhancers.

The intranasal toxicity of selected absorption enhancers (LPC, DM beta CD, N-trimethyl chitosan chloride (TMC) and chitosan hydrochloride) were determined in vivo by investigating the acute microscopic toxic potential on the morphology of rat nasal epithelium with transmission electron microscopy (TEM) and in vitro by measurement of the ciliary beat frequency (CBF), of human ciliated nasal epithelium. TEM evaluations showed that LPC (1% w/v) caused severe epithelial damage and pyknosis. No damage to the rat nasal epithelium was caused by the other absorption enhancers. CBF measurements showed that LPC resulted in total loss of ciliated cells while DM beta CD, TMC and chitosan hydrochloride did not cause any major changes in CBF.

The selection of non-steroidal anti-inflammatory agents for dermal delivery.

An analysis has been conducted to show how the penetration of a selection of non-steroidal anti-inflammatory agents (NSAIDs) through the skin may be predicted. The calculations are based on physicochemical parameters that can be predicted using commercially available software. Where available the predictions compare favourably with the literature values. The bio-effectiveness of the NSAID will be a function of both its penetration through the skin and its potency. The variation in potency has also been considered. Most NSAIDs are carboxylic acids, therefore the pK(a) will be an important determinant in ionisation and hence permeation. pH partition behaviour into the skin has been considered together with the relative impact of decreased permeation but increased solubility with degree of ionisation.

The influence of the physicochemical characteristics and pharmacokinetic properties of selected NSAID's on their transdermal absorption.

The purpose of this study was to determine the plasma concentrations of selected NSAIDs after topical gel administration and to determine the influence of the physicochemical characteristics of these drugs on transdermal absorption. Plasma concentrations of the drugs were determined using high performance liquid chromatography. The logP values obtained from literature for piroxicam, ketoprofen, naproxen, ibuprofen and indomethacin, (1.8, 0.97, 3.22, 3.6 and 3.8, respectively) correlated with the area under the plasma-time curve (AUC) values. The AUC values determined were 527.00 (piroxicam) 269. 45 (ketoprofen) 258.65 (naproxen) 243.22 (indomethacin) and 88.09 (ibuprofen) microg/ml per h. It was concluded that the most reliable parameter for transdermal absorption was the lipophilic character of a drug (logP value). The molecular mass, solubility constraint and percentage unionized moiety can only be used in combination with other properties in the prediction of possible transdermal drug delivery.

Morphine and naltrexone modulate D2 but not D1 receptor induced motor behavior in MPTP-lesioned monkeys.

Interactions at the behavioral level between dopamine (DA) and opioid receptors in the mammalian brain have been amply demonstrated. Considering the pivotal role for DA receptors in the pharmacotherapy of Parkinson's disease (PD), these interactions might be clinically relevant. Therefore, in the present study the effects of the opioid antagonist naltrexone and agonist morphine on D1 and D2 receptor induced stimulation of motor behavior in the unilateral MPTP monkey model (n = 5) of PD were investigated. The results show that both naltrexone and morphine [0.1-1.0 mg/kg; intramuscular injection (IM)] inhibited D2 receptor stimulated contralateral rotational behavior and hand use induced by administration of quinpirole (LY 171555; 0.01 mg/kg, IM) in a dose-related way. However, no effects of these opioid drugs were observed on D1 receptor stimulated contralateral rotational behavior and hand use induced by administration of SKF 81297 (0.3 mg/kg, IM). Interestingly, the action of the alleged preferential mu-receptor antagonist naltrexone was mimicked by the selective delta-opioid antagonist naltrindole (0.5 mg/kg, IM). From this study it is concluded that in a non-human primate model of PD, alteration of opioid tonus leads to modulation of D2 receptor but not D1 receptor controlled motor behavior. The possible underlying mechanisms and clinical relevance of these findings are discussed.

The dopamine D1 agonist SKF 81297 and the dopamine D2 agonist LY 171555 act synergistically to stimulate motor behavior of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned parkinsonian rhesus monkeys.

At present, the pharmacotherapy of Parkinson's disease (PD) consists mainly of L-dihydroxyphenylalanine (L-DOPA) and/or dopamine D2 receptor agonists. However, in general the clinical efficacy of D2 agonists is less than that of L-DOPA. Therefore, attention is being focussed on the role of the D1 receptor as a target for therapeutic intervention in PD. Recently, we reported that SKF 81297 is a selective D1 agonist that stimulates motor behavior of unilaterally MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-lesioned rhesus monkeys. Presently, we studied the effect of coadministration of SKF 81297 and the D2 agonist LY 171555 using the same model of PD. Coadministration of behaviorally active doses of SKF 81297 (0.3 mg/kg) and LY 171555 (0.01 mg/kg) resulted in a prolongation of the motor stimulation induced by either of the drugs alone. Neither administration of SKF 81297, in a dose of 0.03 mg/kg, nor of LY 171555, in a dose of 0.003 mg/kg, were behaviorally active, whereas the combined administration of these compounds induced a significant stimulation of motor behavior. These data suggest that (a) D1 receptor stimulation will prove to be useful in the treatment of PD and (b) better therapeutic results will be obtained by simultaneous stimulation of D1 and D2 receptors as compared with stimulation of both receptors alone.

No direct correlation between behaviorally active doses of the dopamine D2 agonist LY 171555 and displacement of [123I]IBZM as measured with SPECT in MPTP monkeys.

Almost no information is available concerning the link between clinical effects of dopamine D2 receptor agonists in the treatment of Parkinson's disease (PD) and the extent of D2 receptor occupancy in the brain. Therefore, we investigated the possible correlation between administration of behaviorally active doses of the selective D2 agonist LY 171555 and in vivo D2 receptor occupancy in the unilateral 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-lesioned rhesus monkey model of PD. Single photon emission computed tomography (SPECT) with the D2 receptor antagonist [123I]IBZM (iodobenzamide) as radioligand was used to estimate the receptor occupancy. The MPTP-lesioned monkeys consistently showed signs of unilateral parkinsonism. LY 171555 (0.01 or 0.3 mg/kg) significantly increased contralateral rotation (away from the lesion), being most effective at the lower dose. In the MPTP-lesioned monkeys [123I]IBZM activity in the left (lesioned) striatum was significantly higher as compared to that in the right striatum. Only upon administration of 0.3 mg/kg LY 171555 a significant amount of receptor occupancy by LY 171555, as measured with [123I]IBZM SPECT, at both lesioned and non-lesioned side, was detected. Using D2 receptor mediated inhibition of the evoked release of [3H]acetylcholine from rat striatal tissue as a functional model, we showed that the lack of effect with 0.01 mg/kg LY 171555 was not due to non-competitive interaction between LY 171555 and IBZM at the D2 receptor. We conclude that the D2 antagonist [123I]IBZM is not a suitable SPECT ligand to study the relationship between behavioral effects of the selective D2 agonist LY 171555 in unilaterally MPTP-lesioned monkeys and the D2 receptor occupancy in vivo in this animal model of PD.

Neuropeptides and alcohol addiction in monkeys.

Neuropeptides have been implicated in experimental drug addiction. Desglycinamide (Arg8) vasopressin (DGAVP) attenuates heroin and cocaine intake during initiation of drug self-administration in rats. beta-Endorphin is self-administered in rats and a role of endogenous opioids in cocaine reward has been proposed. The present studies deal with voluntary alcohol consumption in monkeys under free choice conditions. Monkeys initiated alcohol drinking within a few days and after a stable drinking pattern was acquired increased their ethanol consumption during a short period following interruption of the alcohol supply (relapse). The alcohol drinking behavior seems under the control of reinforcement principles. DGAVP reduced the acquisition of alcohol drinking in the majority of treated monkeys. Initiation of alcohol drinking induced modifications in neuroendocrine homeostasis e.g. an increased plasma beta-endorphin. Both the opioid antagonist naltrexone and the opioid agonist morphine dose-dependently decreased alcohol intake during continuous supply and after imposed abstinence. The monkeys were more sensitive to both drugs after imposed abstinence. The effects are interpreted in the context of the endorphin compensation hypothesis of addictive behavior. It is suggested that endorphins may be particularly implicated in craving for addictive drugs and in relapse of addictive behavior.

The selective dopamine D1 receptor agonist, SKF 81297, stimulates motor behaviour of MPTP-lesioned monkeys.

The alleged selective, high efficacy dopamine D1 receptor agonist, SKF 81297 (0.05-0.3 mg/kg i.m.), induced rotational behaviour away from the lesion and stimulated use of the dominant right hand in unilaterally (left side) 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned rhesus monkeys (Macaca mulatta). The effects of SKF 81297 were completely blocked by the dopamine D1 receptor antagonist, SCH 23390 (0.05 mg/kg), but not by the dopamine D2 receptor antagonist, remoxipride (1 mg/kg), and were similar to those induced by the selective dopamine D2 agonist, LY 171555 (0.01 mg/kg). These results suggest a functional stimulatory role for the dopamine D1 receptor on motor behaviour in a non-human primate model of Parkinson's disease when stimulated with a high efficacy selective dopamine D1 receptor agonist.

Endocrine profile during acquisition of free-choice alcohol drinking in rhesus monkeys; treatment with desglycinamide-(Arg8)-vasopressin.

This study concerns the effect of spontaneous acquisition of alcohol drinking in rhesus monkeys on plasma levels of beta-endorphin, ACTH, prolactin, cortisol and testosterone. Twelve monkeys had free-choice access to water and two ethanol/water solutions (1%, 2%, v/v) for 4 weeks. During the first 2 weeks, six monkeys were injected (i.m.) twice daily with 0.50 microgram/kg desglycinamide-(Arg8)-vasopressin (DGAVP), a neuropeptide, that has been postulated to interfere with central positive reinforcement processes. The other six were treated with a placebo. Hormonal plasma levels after the first 2 weeks and after another 2 weeks of alcohol drinking were compared to pre-alcohol hormonal levels (baseline). The placebo-treated subjects significantly increased, but the DGAVP-treated subjects significantly decreased ethanol intake over time. After 2 weeks of alcohol, significant increases were found in beta-endorphin and ACTH levels. After 4 weeks, prolactin was increased, cortisol decreased and particularly beta-endorphin remained significantly increased. No relationship was found between baseline hormonal levels and subsequent ethanol intake. No significant differences in plasma hormonal changes were observed between DGAVP- and placebo-treated subjects. Two placebo-treated subjects that showed the highest increase in ethanol intake over time, reacted differently, by reducing beta-endorphin and ACTH levels over time, showing the largest decreases in cortisol and hardly any prolactin reaction. It is concluded that spontaneous alcohol drinking by naïve subjects disturbs hormonal processes and that two animals deviated with respect to the acquisition in alcohol drinking and endocrine functioning.

Low doses of morphine reduce voluntary alcohol consumption in rhesus monkeys.

Experimental opioid modulation has been found to influence the consumption of alcohol in animals. Whereas it has generally been agreed upon that opiate antagonists reduce alcohol consumption, the results with opiate agonists are less consistent. The present study reports on the effect of low doses of morphine in 8 adult male rhesus monkeys that had a free choice in drinking water, a 16% and a 32% ethanol/water solution, (a) during continuous ad libitum access (Experiment I), and (b) after 2 days of alcohol abstinence (Experiment II). In both experiments each monkey received a single morphine injection (i.m.) in 5 different doses (0.03, 0.06, 0.17, 0.50, 1.50 mg.kg-1); each morphine injection (i.m.) was placebo-controlled in a cross-over design. Consumption was measured from 16.00 h in the afternoon (30 min after injection) to 08.30 h the next morning. In Experiment I after 0.50 and 1.50 mg.kg-1 of morphine ethanol intake and water consumption were both reduced during the first hours after injection; only ethanol intake remained reduced during the subsequent night. Effects lasted not longer than 24 h. In Experiment II, morphine administered 30 min before reintroduction of ethanol solutions reduced ethanol intake at doses of 0.17, 0.50 and 1.50 mg.kg-1; water consumption was unaffected. The reduction lasted for the subsequent night after the 2 highest doses. Records obtained of various spontaneous behavioural activities made it unlikely that the used dose range had induced some aspecific sedation; monkeys remained alert and active. The results are contradictory with studies in which low doses of morphine stimulated alcohol drinking in rats. The present results seem to support the hypothesis that at least in monkeys morphine can compensate for some effects of alcohol.

The effect of desglycinamide-(Arg8)-vasopressin (DGAVP) on the acquisition of free-choice alcohol drinking in rhesus monkeys.

The vasopressin analog desglycinamide-(Arg8)-vasopressin (DGAVP) has been reported to reduce the acquisition of heroin and cocaine self-injection behavior in rats. This led to the hypothesis that DGAVP can reduce the self-administration of psycho-active drugs (including ethanol) by attenuating central reinforcement processes. Under forced ingestion conditions, DGAVP has been reported, however, to enhance alcohol drinking in rats. We studied the effect of DGAVP on the acquisition of voluntary, free-choice alcohol drinking in naive rhesus monkeys, that had concurrent access to either 1% and 2% (n = 12) or to 4% and 8% (n = 8) ethanol/water solutions in addition to drinking water. Half of the monkeys were injected twice per day with 50 micrograms.kg-1 of DGAVP for 14 successive days, the other half received placebo. Subsequently, all subjects had access to the same solutions for another 14 days without treatment. DGAVP did not significantly affect concentration preference behavior. With regard to net ethanol ingestion in animals drinking 1% and 2% solutions, DGAVP decreased net ethanol intakes, having a time-dependent and long lasting effect; placebo-treated animals gradually increased net ethanol intakes over time. The placebo-treated animals in the 4% and 8% group, showed a different acquisition pattern; DGAVP reduced net ethanol intake in two animals in a similar way as above. Two animals behaved differently. It is concluded that in a free-choice condition DGAVP did not enhance the acquisition of alcohol drinking in monkeys, but rather inhibited ethanol self-administration in the majority of the subjects.

Effect of naltrexone on alcohol consumption during chronic alcohol drinking and after a period of imposed abstinence in free-choice drinking rhesus monkeys.

Relapse into problematic alcohol drinking is a serious problem in the treatment of alcoholism. Free-choice drinking rhesus monkeys show relapse-like behaviour after imposed abstinence of alcohol, by immediately reinitiating ethanol intake at an increased level. The relapse-like behaviour of the monkeys seems not induced by physical withdrawal, but rather argues for a resistance to extinction of ethanol-reinforced behaviour. It has been suggested that endogenous opioids play a role in the positive reinforcing effect of ethanol. In this study, the effect of the opiate antagonist naltrexone was investigated in eight adult male rhesus monkeys (Macaca mulatta) who had about 1 year experience with alcohol drinking, under two conditions: 1) (expt 1) during continuous and concurrent supply of drinking water and two ethanol/water solutions (16% and 32% (v/v], and 2) (expt 2) after 2 days of alcohol abstinence. In both experiments, each monkey received six doses of naltrexone (0.02, 0.06, 0.17, 0.5, 1.0, 1.5 mg.kg-1); each dose was paired with a placebo injection (im) in a cross-over design. Consumption was measured from 16.00 hours in the afternoon (30 min after injection) to 9.00 hours the next morning. In experiment 1 naltrexone reduced total net ethanol intake in a graded dose-dependent manner. The effect of naltrexone was apparent shortly after injection, and lasted until the following day. Consumption of drinking water was reduced only shortly after injection. In expt 2, reduction of net ethanol intake was largely restricted to the first few hours of reinitiation of alcohol drinking, i.e. the period in which the abstinence-induced increase was manifest. Consumption of drinking water was not affected by naltrexone. Naltrexone hardly influenced consumption of the non-preferred ethanol solution of 32%. It is postulated that the opioid modulation specifically interacted with positively reinforced behaviour. In expt 2 naltrexone reduced ethanol intake at a lower dose (0.17 mg.kg-1) compared to expt 1 (0.50 mg.kg-1), but net ethanol intakes however remained higher. It might be that alcohol abstinence resulted in altered opioid activity, leading to increased ethanol-seeking behaviour. The renewed presentation of ethanol solutions (also) might have stimulated reinitiation of alcohol drinking, representing conditioned incentive stimuli. The reported monkey model of relapse in alcohol drinking could be a useful tool to evaluate new hypotheses and experimental treatments with respect to human alcoholism.

Analysis of spontaneous alcohol drinking in rhesus monkeys.

This analysis aims at determining to what extent spontaneous alcohol drinking in adult male rhesus monkeys (Macaca mulatta) represents ethanol-directed behaviour. It is shown that in a condition of free access to an ethanol/water solution (2 percent v/v) and drinking water, alcohol drinking was initiated in all subjects (n = 4) within a few days, without any specific induction procedure. Relationship between drinking behaviour and ethanol concentrations was studied in 8 subjects by use of a concurrent 3-bottle-design. 2 bottles containing ethanol solution (concentrations 2.4; 4.8; 8.16; 16.32 percent v/v), 1 bottle contained drinking water. When ethanol concentrations in the solutions increased, consumption of ethanol solutions decreased, of drinking water increased, and of total water decreased. Net ethanol intake from a certain solution was influenced by its concentration and the concentration of the concurrently available solution. After an initial increase, total net ethanol intake remained relatively constant. Consumed amounts of ethanol (on the average 2-6 ml.kg-1 per day) could lead to notable blood ethanol levels. Drinking from ethanol solution was not just an alternative for ingesting water. The observed alcohol drinking is interpreted as resulting from a central reinforcement of ethanol intake and avoidance of negative, potentially harmful effects of ethanol.