In Vitro Evaluation of Microparticles and Polymer Gels for Use as Nasal Platforms for Protein Delivery

Purpose. Nasal delivery of protein therapeutics can be compromised by the brief residence time at this mucosal surface. Some bioadhesive polymers have been suggested to extend residence time and improve protein uptake across the nasal mucosa. We examined several potential polymer platforms for their in vitro protein release, relative bioadhesive properties and induction of cytokine release from respiratory epithelium. Methods. Starch, alginate, chitosan or Carbopol^® microparticles, containing the test protein bovine serum albumin (BSA), were prepared by spray-drying and characterized by laser diffraction and scanning electron microscopy. An open-membrane system was used to determine protein release profiles and confluent, polarized Calu-3 cell sheets were used to evaluate relative bioadhesion, enhancement of protein transport and induction of cytokine release in vitro. Results. All spray-dried microparticles averaged 2–4 m in diameter. Loaded BSA was not covalently aggregated or degraded. Starch and alginate microparticles released protein more rapidly but were less adhesive to polarized Calu-3 cells than chitosan and Carbopol^® microparticles. Protein transport across polarized Calu-3 cells was enhanced from Carbopol^® gels and chitosan microparticles. A mixture of chitosan microparticles with lysophosphatidylcholine increased protein transport further. Microparticles prepared from either chitosan or starch microparticles, applied apically, induced the basolateral release of IL-6 and IL-8 from polarized Calu-3 cells. Release of other cytokines, such as IL-l, TNF-, GM-CSF and TGF-, were not affected by an apical exposure to polymer formulations. Conclusions. We have described two systems for the in vitro assessment of potential nasal platforms for protein delivery. Based upon these assessments, Carbopol^® gels and chitosan microparticles provided the most desirable characteristics for protein therapeutic and protein antigen delivery, respectively, of the formulations examined.     

鹅不食草油鼻用微乳温敏凝胶释药系统鼻黏膜刺激性及主要脏器影响研究

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

Effects of Proteolytic Enzyme Inhibitors on the Nasal Absorption of Vasopressin and an Analogue

Proteolytic enzyme inhibitors were examined as absorption enhancers for the nasal delivery of vasopressin (AVP) and desmopressin (l-d-8-DAVP) in rats. Aprotinin, soybean trypsin inhibitor, and camostat mesilate were used as enzyme inhibitors. The nasal absorption of AVP and l-d-8-DAVP was evaluated by measuring its antidiuretic effect. Nasal administration of AVP (0.005 IU/kg) or l-d-8-DAVP alone (2.5 ng/kg) produced a small antidiuretic effect. Coadministration with aprotinin (1000 and 10000 KIU/kg) or soybean trypsin inhibitor (1.25 and 6.25 mM) did not change the antidiuretic effect. However, coadministration with camostat mesilate (1 to 50 mM) significantly increased the antidiuretic effect and, thus, the nasal absorption of AVP and l-d-8-DAVP. The activities of aminopeptidase, cathepsin-B, and trypsin in the nasal mucosal tissue of rats were 7 nmol/min/mg protein, 0.7 nmol/min/mg protein, and 4.6 pmol/min/mg protein, respectively. Aprotinin and soybean trypsin inhibitor inhibited only the trypsin activity, whereas camostat mesilate inhibited aminopeptidase and trypsin activities. Aprotinin (MW 6500) and soybean trypsin inhibitor (MW 8000), with relatively high molecular weights, may not permeate into the nasal mucosal tissue. In contrast, camostat mesilate is slowly absorbed (8%/hr) and could inhibit the proteolytic activity in the nasal mucosa, resulting in enhanced nasal absoprtion of AVP and l-d-8-DAVP.     

Preparation and In Vitro Characterization of Mucoadhesive Hydroxypropyl Guar Microspheres Containing Amlodipine Besylate for Nasal Administration

Amlodipine besylate microspheres for intranasal administration were prepared with an aim to avoid first-pass metabolism, to achieve controlled blood level profiles and to improve therapeutic efficacy. Hydroxypropyl Guar, a biodegradable polymer, was used in the preparation of microspheres by employing water in oil emulsification solvent evaporation technique. The formulation variables were drug concentration, emulsifier concentration, temperature, agitation speed and polymer concentration. All the formulations were evaluated for particle size, particle shape and surface morphology by scanning electron microscopy, percentage yield, drug entrapment efficiency, in vitro mucoadhesion test, degree of swelling and in vitro drug diffusion through sheep nasal mucosa. The microspheres obtained were free flowing, spherical and the particles ranged in size from 13.4±2.38 μm to 43.4±1.92 μm very much suitable for nasal delivery. Increasing polymer concentration resulted in increased drug entrapment efficiency and increased particle size. Amlodipine besylate was entrapped into the microspheres with an efficiency of 67.2±1.18 % to 81.8±0.64 %. The prepared microspheres showed good mucoadhesion properties, swellability and sustained the release of the drug over a period of 8 h. The data obtained were analysed by fitment into various kinetic models; it was observed that the drug release was matrix diffusion controlled and the release mechanism was found to be non-Fickian. Stability studies were carried out on selected formulations at 5±3°, 25±2°/60±5% RH and 40±2°/75±5% RH for 90 days. The drug content was observed to be within permissible limits and there were no significant deviations in the in vitro mucoadhesion and in vitro drug diffusion characteristics.     

Increased Intestinal Delivery of Viable Saccharomyces boulardii by Encapsulation in Microspheres

Purpose Although probiotics are of a major potential therapeutic interest, their efficacy is usually limited by poor bioavailability of viable microorganisms on site. The aim of this study was to protect the probiotic Saccharomyces boulardii from degradation in order to ensure a greater number of viable yeast in the colon. Methods Alginate microspheres coated with or not with chitosan were used to encapsulate the yeast by an extrusion method. The efficiency of encapsulation was assessed both in vitro and in vivo. Results In vitro, less than 1% of the non-encapsulated probiotic survived after 120 min at pH 1.1, whereas the majority of encapsulated yeast cells remained entrapped within both types of microspheres. Further exposure to a pH 6.8 allowed the release of about 35% of viable yeasts. In vivo, the percentage of viable yeast excreted over 96 h after a single oral dose of 2 × 10⁸ cfu/100 g in rats was 2.5% for non-encapsulated yeast and reached 13.3 and 9.0% of the dose administered for the uncoated and chitosan-coated microspheres, respectively. Conclusions Given the dose-dependent efficacy of S. boulardii and the efficiency of microencapsulation in protecting the yeast from degradation, alginate microspheres could be of great interest in therapeutic applications of the yeast.     

HPLC法测定鲑降钙素鼻用喷雾剂中有关物质

目的 建立测定鲑降钙素鼻用喷雾剂中有关物质的HPLC方法。方法 使用Macherey-Nagel Nucleosil 100-5 C₁₈AB色谱柱（250 mm×4.0 mm，5 μm）；以0.04 mol/L四甲基氢氧化铵溶液（用磷酸调pH 2.5）–乙腈（9∶1）为流动相A，以0.04 mol/L四甲基氢氧化铵溶液（用磷酸调pH 2.5）–乙腈（4∶6）为流动相B，梯度洗脱；检测波长为220 nm；体积流量为1.0 mL/min；进样量为100 μL；柱温为65 ℃；样品室温度：10 ℃。按加校正因子的主成分自身对照法计算降钙素C、N-乙酰半胱氨酰鲑降钙素（杂质A）、9-D-亮氨酸鲑降钙素（杂质B）、去-22-酪氨酸鲑降钙素（杂质C）。结果 降钙素C和鲑降钙素杂质A、B、C在0.20～7.50 μg/mL线性关系良好，平均回收率分别为98.1%、98.9%、100.5%、99.7%，RSD值分别为1.6%、1.5%、1.0%、1.1%。杂质A、B、C的校正因子均超出0.90～1.10。结论 方法简便、快速，可作为鲑降钙素鼻用喷雾剂中有关物质的检测方法。     

Monitoring the in Vivo Delivery of Proteins from Carbomer Hydrogels by X-Ray Fluorescence

Purpose. To measure the effect of protein size on their disappearance from subcutaneously implanted carbomer hydrogel matrices. Methods. A series of different molecular weight (MW) proteins were iodinated, incorporated into Carbopol hydrogels, injected subcutaneously into rats, and monitored using X-ray fluorescence (XRF). Results. A 10 mg/mL minimum concentration of Carbopol-940 was necessary before protein [50 mg/mL iodinated bovine serum albumin (I-BSA)] retention times increased with increasing hydrogel concentration. The decreasing protein signal was not caused by outward protein diffusion or iodoprotein hydrolysis. As the protein MW increased, protein retention times lengthened [e.g., 6.2 h for insulin (5.7 kDa) to 13.3 h for thyroglobulin (669 kDa)]. Protein disappearance was monophasic first-order for some proteins and biphasic first-order for others. The disappearance rate constant ranged from 0.093 ± 0.005 h^– _1/2 to 0.187 ± 0.057 h^– _1/2, indicating gel erosion rather than protein diffusion as the rate-limiting mechanism. Entrapped I-BSA in Carbopol-1342 NF, pH 7.4, and Carbopol 2001-ETD, pH 7.4, gel matrices yielded different disappearance rates and profiles than Carbopol-940. The overall 50% disappearance rate of I-BSA was greatest for Carbopol-1342 NF (41 ± 8 h), followed by Carbopol-2001 ETD (25 ± 2 h) and Carbopol-940 (10.5 ± 0.7 h). Conclusion. XRF is a noninvasive technique that can be used to follow the status of macromolecules in vivo.     

Efficiency of Cytoplasmic Delivery by pH-Sensitive Liposomes to Cells in Culture

The intracellular processing of pH-sensitive liposomes composed of cholesterylhemisuccinate (CHEMS) and dioleoylphosphatidylethanolamine (DOPE) by eukaryotic cell lines has been compared to non-pH-sensitive liposomes made of CHEMS and dioleoylphosphatidylcholine (DOPC). The pH-sensitive liposomes can deliver encapsulated fluorescent molecules [calcein, fluoresceinated dextran, fluoresceinated polypeptide, and diphtheria toxin A chain (DTA)] into the cytoplasm. Cytoplasmic delivery can be blocked in the presence of ammonium chloride or EDTA, indicating that the process requires a low-pH environment and the presence of divalent cations. Inhibition of cellular protein synthesis by DTA delivery from the pH-sensitive liposome is orders of magnitude greater than from the non-pH-sensitive liposome composition. The delivery of DTA into the cytoplasm by pH-sensitive liposomes is at least 0.01% of cell-associated liposomal DTA. There is no significant difference in the degradation rate of bovine serum albumin (BSA) or the rate of acidification of pH-sensitive dye, 8-hydroxy-l,3,6-pyrene-trisulfonate (HPTS), when delivered to cells in pH-sensitive and non-pH-sensitive liposomes. Thus the efficiency of cytoplasmic delivery is less than 10% of the cell-associated liposome contents, which is the smallest difference that can be detected by these two assays. Based upon the various assays used to measure liposome content disposition in the cell, we conclude that the efficiency of cytoplasmic delivery by the CHEMS/DOPE liposomes is greater than 0.01% and less than 10% of the cell-associated liposomal contents.     

Discriminative stimulus effects of intravenous l-nicotine and nicotine analogs or metabolites in squirrel monkeys

Squirrel monkeys were trained to emit one response after IV administration of l-nicotine (0.4 or 0.2 mol/kg) and a different response after IV administration of saline. After stable discriminative performances were established, subjects were tested with cumulative doses of l-nicotine (0.02–2.2 mol/kg), d-nicotine (0.02–19.7 mol/kg), l-nornicotine (0.2–12.0 mol/kg), l-cotinine (56.8–567.5 mol/kg), and dl-anabasine (0.6–19.7 mol/kg). All of the drugs produced dose-related increases in the percentage of drug-appropriate responses emitted, from predominately saline-appropriate responses after low doses, to predominately drug-appropriate responses at the highest doses studied. Relative potency comparisons indicated that l-nicotine was 28 times more potent than d-nicotine, 29 times more potent than l-nornicotine, and approximately 2000 times more potent than l-cotinine. Each of the drugs also produced decreases in rates of responding, with potency order similar to that obtained for the discriminative effects. The effects of l-cotinine may be attributed to trace amounts of l-nicotine, which existed within the l-cotinine. The effects of dl-anabasine were lethal in one subject and were consequently not studied in the other subjects.     

Modulation of allergen-induced nasal symptoms and mediator release by treatment with N-acetyl-aspartyl-glutamate (ZY15106)

The aim of this study was to evaluate the effects of the new anti-allergic drug, N-acetyl-aspartyl-glutamate (ZY15106), on allergen-induced nasal symptoms and mediator release. Fifteen outpatients suffering from seasonal allergic rhinitis due to grass pollen were included in the study. A nasal antigen challenge followed by evaluation of symptoms was performed in basal conditions. Ten of the 15 patients underwent sequential nasal lavages in order to evaluate allergen-induced mediator release. The study was performed in winter, when the patients were symptom free, and was a randomized single-blind crossover trial of a 6 % solution of ZY15106 (daily dosage: 48 mg) versus placebo (lactose). The drug and the placebo were administered intranasally q.i.d. for 1 week, with a 2-week interval between the two treatments. Treatment with ZY15106, but not with placebo, caused a significant reduction in nasal obstruction in the first 30 min after challenge and at 60 min and itching in the first 10 min after challenge, but did not reduce sneezing and rhinorrhoea. Moreover, ZY15106 significantly reduced the histamine release in 5 min postchallenge lavage (4.5 ng·ml^–1 after placebo administration vs 2.5 ng·ml^–1, after treatment with ZY15106). A reduction in immunoreactive LTC₄ release in the 5 and 10 min post-challenge lavages was observed after ZY15106 administration (placebo vs active treatment: at 5 min 2.9 ng·ml^–1 vs 1.4 ng·ml^–1; at 10 min: 2.25 ng·ml^–1 vs 0.9 ng·ml^–1). These results indicate that 1-week treatment with ZY15106 can reduce antigen-induced nasal obstruction and itching, and mediator release in human nasal airways. The clinical activity of ZY15106 in the treatment of allergic rhinitis may be related to its ability to inhibit mediator release.     

Pharmacokinetics and Metabolism of Transdermal Oxybutynin: In Vitro and in Vivo Performance of a Novel Delivery System

Purpose. The purpose of this work was to characterize in vitro/in vivo delivery and pharmacokinetics of oxybutynin (OXY) and its active metabolite, N-desethyloxybutynin (DEO), by a novel matrix transdermal system (TDS). Methods. Two in vivo, randomized, three-way crossover trials examined single/multiple OXY TDS doses. Abdomen, buttock, and hip application sites were compared and dose proportionality was evaluated. Model independent pharmacokinetics, elimination rate constants, and metabolite/drug ratios were derived from both plasma OXY and DEO concentrations. Results. Single/multiple applications of the OXY TDS to the abdomen yielded mean C _max OXY concentrations of 3.4 ± 1.1/6.6 ± 2.4 ng/mL and median t _max of 36/10 h, with steady state achieved during the second application. Plasma OXY and DEO concentrations decreased gradually after C _max until system removal. Buttock and hip applications resulted in bioequivalent OXY absorption. AUC ratios of DEO/OXY were 1.5 ± 0.4 (single dose) and 1.3 ± 0.3 (multiple dose). Mean in vitro OXY skin absorption (186 g/h) was comparable to the estimated in vivo delivery (163 g/h) over 96 h. Conclusions. Sustained delivery over 4 days and multiple sites allow a convenient, well-tolerated, twice-weekly OXY TDS dosing. A low incidence of anticholinergic side effects is expected during clinical use because of the avoidance of presystemic metabolism and low DEO plasma concentrations. The consistent delivery, absorption, and pharmacokinetics should result in an effective treatment of patients with overactive bladder.     

Gamma Sterilization of a Semi-Solid Poly(ortho ester) Designed for Controlled Drug Delivery—Validation and Radiation Effects

Radiation sterilization is becoming increasingly popular for the sterilization of many pharmaceutical products. Although this technique is not limited to the sterilization of polymers, it is probably the most suitable method for such materials. This method however suffers several drawbacks. The sterilization of a product must lead to a safety level of 10^–6, i.e. one chance in a million to find a contaminated sample. In many cases, this assurance of sterility can be achieved by using a uniform treatment dose of 2.5 Mrad, recommended by the pharmacopeia. We investigated the possibility of using doses of radiation inferior to 2.5 Mrad to sterilize a semi-solid poly(ortho ester) (POE) developed for use as carrier in controlled drug delivery. After determination of the initial bioburden, the polymer was intentionally contaminated with the bioindicator Bacillus pumilus E 601. Following exposure to gamma irradiation, the D₁₀ value of the radio resistant bioindicator was determined. Using the initial contamination value, the reduction factor D₁₀ and the safety level, it is possible to calculate an optimal sterilizing dose for POE. All polymers are affected by ionizing radiation and the amount of radiation which produces a significant change in properties may vary from one polymer to the other. A molecular weight and dynamic viscosity decrease resulting from backbone cleavage was observed for this POE at a dose lower than 2.0 Mrad. Evaluation of the structure using ¹H-NMR, ¹³C-NMR and IR analysis shows that for doses higher than 2.0 Mrad, another degradation process takes place. Formation of two isomeric esters of the triol used for the synthesis was identified by these methods. Cleavage of the monomer cycle is believed to be the main cause of the degradation observed. A radiation dose of not less than 7 times the D₁₀ value but less than 2.0 Mrad was used for this semi-solid biodegradable poly-(ortho ester) in order to ensure its sterility and avoid an excessive formation of degradation products.     

不同采收时期及干燥方法对栀子中栀子苷含量的影响

目的:评价不同采收期以及不同干燥方法对栀子苷的影响.方法:应用高效液相色谱法测定,色谱柱为Agilent Zorbax XDB-C18色谱柱(150 mm×4.6 mm,5 μm);流动相为乙腈-水(15:85),流速1.0 ml·min-1;柱温:30℃;检测波长238 nm.结果:栀子苷的线性范围为0.31～155.76 μg·ml-1(r=0.999 9),加样回收率为100.9%(RSD=1.2%);定量分析了6个采收时间、12种干燥方法的栀子中栀子苷含量.结论:不同采收时期、不同干燥方法对栀子中栀子苷的含量有一定的影响.     

Efficacy and Pharmacokinetics of Site-Specific Cefazolin Delivery Using Biodegradable Implants in the Prevention of Post-operative Wound Infections

Purpose. The study objective was to evaluate the efficacy and pharmacokinetics of cefazolin delivered locally as a glyceryl monostearate (GMS) based biocompatible implant for prevention of post-operative wound infection in Sprague Dawley rats subcutaneously inoculated with Staphylococcus aureus. Methods. For the efficacy and pharmacokinetic studies, 18 rats were subcutaneously inoculated with 4.5 x 10⁷ CPU of S. aureus on the dorsum (6 per rat), and randomly assigned into three group of 6 rats each: (1) the control group, in which rats did not receive antibiotics, (2) the intermittent IM treatment group, in which rats received IM injections of 10 mg/kg cefazolin every 4 hr (total of 180 mg/kg in 3 days), and (3) the implant treatment group, in which rats were implanted subcutaneously with four Cefazolin-GMS implants in the vicinity of the inoculations. The implants were designed to deliver 180 mg/kg cefazolin over a 3 day period. For efficacy evaluation, the rats were euthanized one week post-inoculation and abscess count, weight and size were determined. Results. Rats in the control group had developed 21 abscesses out of the 36 inoculations, indicating validity of the infection model. The local delivery of cefazolin resulted in complete eradication of the infection, since no abscesses formed in the rats in the implant group. In the IM treatment group, only one abscess was formed and no significant difference in efficacy between the two treatment groups was observed. The GMS implants sustained the release of cefazolin for a period of three days with only 3-fold fluctuations in plasma concentration (5.5–17.5 g/ml). However, plasma concentrations after the intermittent IM administration of cefazolin fluctuated 110-fold between 44-0.4 g/ml every 4 hr. The release rate of cefazolin from the implants was nearly zero order for the entire duration. Bioerosion of the implants was determined by examining the condition of the implants six weeks post-implantation. Two of the 12 implants had completely disappeared and the remaining implants were in a pasty form and had lost 20–80% of their weight. Absence of irritation or inflammation around the implants indicated biocompatibility of the GMS implants. Conclusions. Implantable system that provided a prolonged delivery of cefazolin was found to be effective against S. aureus infection, and demonstrated suitable pharmacokinetics and biocompatibility with significant bioerosion.     

In Vitro and in Vivo Investigations of Dihydropyridine-Based Chemical Delivery Systems for Anticonvulsants

A dihydropyridine-based chemical delivery system (CDS), intended to improve drug delivery to the brain, was investigated with a series of analogues of the anticonvulsant stiripentol. In vitro experiments demonstrated that the rates of hydrolysis of the corresponding pyridinium conjugates were influenced markedly by small changes in the structure of the drug moiety to be released. Thus, allylic esters were hydrolyzed rapidly to drug in all aqueous media, while the analogous saturated esters and an allylic amide derivative were almost totally stable. The mechanism of hydrolysis, which is particular to this series of CDS conjugates, appeared to occur via ionization to a resonance-stabilized carbocation intermediate. The same CDS compounds were investigated in vivo and compared to the corresponding drugs after intravenous administration. Only those CDS compounds that were found to hydrolyze in vitro released appreciable amounts of drug in vivo. Prolonged release of the drug from the CDS in the brain could be demonstrated for these compounds, but the gain in the ratio of brain-to-plasma AUC when the CDS was administered depended on the innate distribution characteristics of the drug. Thus, the drug D3, which had a high brain-to-plasma AUC ratio, did not show an improvement in this ratio when administered as CDS3. In contrast, stiripentol with a poor brain-to-plasma AUC ratio showed a two- to threefold increase in this ratio when administered as a CDS. These investigations highlight the need for a thorough understanding of the mechanism of drug release and the importance of the pharmacokinetic properties of the drug in designing a carrier system for delivery of drugs to the brain.     

Enhancement of muscle blood cell flux and pO2 by cromakalim (BRL 34915) and other compounds enhancing membrane K+ conductance,but not by Ca2+ antagonists or hydralazine,in an animal model of occlusive arterial disease

Summary The effect of Ca²⁺ antagonists, hydralazine and agents which enhance membrane K⁺ conductance (cromakalim, pinacidil and nicorandil) in smooth muscle cells, was compared on normal and hypoxic skeletal muscle blood cell flux and pO₂. The K⁺ conductance enhancers and verapamil, diltiazem and nifedipine increased blood cell flux in normally perfused muscle. At equieffective blood pressure lowering dosages, the Ca²⁺ antagonists produced greater increases than the K⁺ channel openers. Hydralazine did not elevate blood cell flux in the normal muscle. In hypoxic skeletal muscle, the K⁺ conductance enhancers produced a marked increase in blood cell flux and in tissue oxygen tension, indicating that they had increased the nutritive blood flow in the muscle. The Ca²⁺ antagonists and hydralazine either did not change hypoxic muscle blood cell flux and pO₂ or reduced them. The dissimilarity in the activity of the compounds may be due to differences in their site of action in the vascular bed. Ca²⁺ antagonists and hydralazine are known to reduce arteriolar vessel resistance and do not increase blood flow in hypoxic skeletal muscle. The positive effect of cromakalim, pinacidil and nicorandil may be due to relaxant activity on larger arterial blood vessels including collaterals. This effect could be related to their ability to enhance membrane K²⁺ conductance in vascular smooth muscle cells.Send offprint requests to: D. Angersbach     

Development of an in Vitro Rat Intestine Segmental Perfusion Model to Investigate Permeability and Predict Oral Fraction Absorbed

Purpose The aims of the study are to develop and evaluate an in vitro rat intestine segmental perfusion model for the prediction of the oral fraction absorbed of compounds and to assess the ability of the model to study intestinal metabolism. Methods The system consisted of a perfusion cell with a rat intestinal segment and three perfusion circulations (donor, receiver, and rinsing circulation). Lucifer yellow (LY) was applied as internal standard together with test compounds in the donor circulation. To validate the model, the permeability of eight noncongeneric passively absorbed drugs was determined. Intestinal N-demethylation of verapamil into norverapamil was followed in the donor and receiver circulations by high-performance liquid chromatography analysis. Results The in vitro model allowed ranking of the tested compounds according to their in vivo absorption potential. The Spearman's correlation coefficient between the oral fraction absorbed in humans and the ratio of permeation coefficient of test compound to the permeation coefficient of LY within the same experiment was 0.98 (P < 0.01). Moreover, intestinal N-demethylation of verapamil, its permeation, and the permeation of its metabolite norverapamil could be assessed in parallel. Conclusions Up to six permeation kinetics can be obtained per rat, and the method has shown to be a valuable tool to estimate human oral absorption.     

Direct analysis of the enantiomers of mexiletine and its metabolites in plasma and urine using an HPLC—CSP

A high-performance liquid chromatographic assay has been developed for the quantification of the enantiomers of mexiletine and its four major metabolites, in plasma and in urine. Mexiletine and all metabolites were determined, after derivatization of mexiletine and its hydroxymetabolites,p-hydroxymexiletine and hydroxymethylmexiletine, using a Chiralpak AD chiral stationary phase, based on a carbamoyl derivative of amylose.o-phthalaldehyde was chosen as derivatization reagent to increase the sensitivity of detection, to achieve separation of all compounds in one chromatographic system, and to avoid interferences.     

Effect of two weeks' treatment with chlorimipramine and nortriptyline,alone or in combination with alcohol,on learning and memory

Mature male Wistar rats were given d-amphetamine sulphate (200 mg/l in the drinking water for a period of three weeks. The drug was then withdrawn and the rats were killed 12, 24, 36 and 48 h later. Pronounced behavioural depression was observed 12 h after the withdrawal of amphetamine; 24 h after withdrawal, behaviour was substantially normal but depression recurred at 36 h. Recovery appeared to be complete after 48 h. Fluorimetric determinations showed that noradrenaline and 5-hydroxytryptamine concentrations were reduced by the chronic administration of amphetamine in the cortex, hippocampus, thalamus/hypothalamus and mid-brain. Noradrenaline concentrations were also reduced in the pons/medulla. Twelve and 36 h after withdrawal, there was a further reduction in noradrenaline concentrations in the cortex, hippocampus and mid-brain; noradrenaline concentrations in the striatum were also reduced. 5-Hydroxytryptamine concentrations in the cortex and striatum were lower 12 and 36 h after the withdrawal than during chronic amphetamine treatment; 36 h after withdrawal, concentrations in the hippocampus and the pons/medulla were also lower than during drug treatment. There were also changes in the concentrations of 5-hydroxyindoleacetic acid and normetanephrine.