Showing papers in "Journal of pharmacy & pharmaceutical sciences : a publication of the Canadian Society for Pharmaceutical Sciences in 2003"

Pharmaceutical approaches to colon targeted drug delivery systems.

Abstract: Purpose. Although oral delivery has become a widely accepted route of administration of therapeutic drugs, the gastrointestinal tract presents several formidable barriers to drug delivery. Colonic drug delivery has gained increased importance not just for the delivery of the drugs for the treatment of local diseases associated with the colon but also for its potential for the delivery of proteins and therapeutic peptides. To achieve successful colonic delivery, a drug needs to be protected from absorption and /or the environment of the upper gastrointestinal tract (GIT) and then be abruptly released into the proximal colon, which is considered the optimum site for colon-tar- geted delivery of drugs. Colon targeting is naturally of value for the topical treatment of diseases of colon such as Chron's diseases, ulcerative colitis, colorectal cancer and amebiasis. Peptides, proteins, oligonucleotides and vac- cines pose potential candidature for colon targeted drug delivery. Methods. The various strategies for targeting orally administered drugs to the colon include covalent linkage of a drug with a carrier, coating with pH-sensitive polymers, formulation of timed released systems, exploita- tion of carriers that are degraded specifically by colonic bacteria, bioadhesive systems and osmotic controlled drug delivery systems. Various prodrugs (sulfasalazine, ipsala- zine, balsalazine and olsalazine) have been developed that are aimed to deliver 5-amino salicylic acid (5-ASA) for localized chemotherapy of inflammatory bowl disease (IBD). Microbially degradable polymers especially azo crosslinked polymers have been investigated for use in tar- geting of drugs to colon. Certain plant polysaccharides such as amylose, inulin, pectin and guar gum remains unaf- fected in the presence of gastrointestinal enzymes and pave the way for the formulation of colon targeted drug delivery systems. The concept of using pH as a rigger to release a drug in the colon is based on the pH conditions that vary continuously down the gastrointestinal tract. Times dependent drug delivery systems have been devel- oped that are based on the principle to prevent release of drug until 3-4 h after leaving the stomach. Redox sensitive polymers and bioadhesive systems have also been exploited to deliver the drugs into the colon. Results. The approach that is based on the formation of prodrug involves covalent linkage between drug and carrier. The type of linkage that is formed between drug and carrier would decide the triggering mechanism for the release of drug in colon. The presence of azo reductase enzymes play pivotal role in the release of drug from azo bond prodrugs while glycosidase activity of the colonic microflora is responsible for liberation of drugs from glycosidic pro- drugs. Release of drugs from azo polymer coated dosage forms is supposed to take place after reduction and thus cleavage of the azo bonds by the azoreductase enzymes present in the colonic microflora. Natural polysaccharides have been used as tools to deliver the drugs specifically to the colon. These polysaccharides remain intact in the phys- iological environment of stomach and small intestine but once the dosage form enters into colon, it is acted upon by polysaccharidases, which degrades the polysaccharide and releases the drug into the vicinity of bioenvironment of colon. However, they should be protected while gaining entry into stomach and small intestine due to enormous swelling and hydrophilic properties of polysaccharides. This has been achieved either by chemical crosslinking or by addition of a protective coat. Formulation coated with enteric polymers releases drug when pH move towards alkaline range while as the multicoated formulation passes the stomach, the drug is released after a lag time of 3-5 h that is equivalent to small intestinal transit time. Drug coated with a bioadhesive polymer that selectively provides adhesion to the colonic mucosa may release drug in the colon. Conclusions. Improved drug delivery systems are required for drugs currently in use to treat localized dis- eases of the colon. The advantages of targeting drugs spe- cifically to the diseased colon are reduced incidence of systemic side effects, lower dose of drug, supply of the drug to the biophase only when it is required and mainte- nance of the drug in its intact form as close as possible to the target site.

Drug delivery to the central nervous system: a review.

Abstract: The brain is a delicate organ, and evolution built very efficient ways to protect it. Unfortunately, the same mechanisms that protect it against intrusive chemicals can also frustrate therapeutic interventions. Many existing pharmaceuticals are rendered ineffective in the treatment of cerebral diseases due to our inability to effectively deliver and sustain them within the brain. General methods that can enhance drug delivery to the brain are, therefore, of great interest. Despite aggressive research, patients suffering from fatal and/or debilitating central nervous system (CNS) diseases, such as brain tumors, HIV encephalopathy, epilepsy, cerebrovascular diseases and neurodegenerative disorders, far outnumber those dying of all types of systemic cancer or heart disease. The clinical failure of much potentially effective therapeutics is often not due to a lack of drug potency but rather to shortcomings in the method by which the drug is delivered. Treating CNS diseases is particularly challenging because a variety of formidable obstacles often impede drug delivery to the brain and spinal cord. By localizing drugs at their desired site of action one can reduce toxicity and increase treatment efficiency. In response to the insufficiency in conventional delivery mechanisms, aggressive research efforts have recently focused on the development of new strategies to more effectively deliver drug molecules to the CNS. This review intends to detail the recent advances in the field of brain-targeting, rational drug design approach and drug delivery to CNS. To illustrate the complexity of the problems that have to be overcome for successful brain targeting, a brief intercellular characterization of the blood-brain barrier (BBB) is also included.

Comparative evaluation of plastic, hydrophobic and hydrophilic polymers as matrices for controlled-release drug delivery.

Abstract: Purpose: The present study was under- taken to investigate the effect of plastic, hydrophilic and hydrophobic types of polymers and their content level on the release profile of drug from matrix systems. As the physico-chemical nature of the active ingredients influence the drug retarding ability of these polymers, three different drugs were used to evaluate their comparative release char- acteristics in similar matrices. Methods: Matrix tablets of theophylline, diclofenac sodium and diltiazem HCl using Kollidon SR, Carnauba wax and Hydroxypropyl methylcel- lulose (HPMC-15cps) were prepared separately by direct compression process. The USP Basket method was selected to perform the dissolution test carried out in 250 ml 0.1N HCl for first two hours and 1000 ml phosphate buffer of pH 6.8 for ten hours. Results: Statistically signif- icant differences were found among the drug release pro- file from different classes of polymeric matrices. The release kinetics was found to be governed by the type and content of polymer in the matrix system. Higher polymeric content (75%) in the matrix decreased the release rate of drug because of increased tortuosity and decreased poros- ity. At lower polymeric level (25%), the rate and extent of drug release was elevated. Carnauba wax was found to cause the strongest retardation of drug. On the otherhand, highest drug release was from HPMC matrices while Kolli- don SR gave an intermediate release profile between these two polymers. Release rate was also found to be the func- tion of physico-chemical nature of drug molecule. Theo- phylline and diltiazem HCl, being soluble in nature, released faster than diclofenac sodium from all matrix sys- tems. The release mechanism was explored and explained with biexponential equation. Release profile showed a ten- dency to follow zero-order kinetics from HPMC matrix systems whereas Fickian (Case I) transport was predomi- nant mechanism of drug release from Kollidon SR matrix system. The mean dissolution time (MDT) was calculated for all the formulations and the highest MDT value was obtained with Carnauba wax for all the drugs under inves- tigation. Conclusions: The results generated in this study showed that the profile and kinetics of drug release were functions of polymer type, polymer level and physico- chemical nature of drug. A controlled plasma level profile of drug can be obtained by judicious combination of poly- mers and modulation of polymer content in the matrix system.

Poly n-butylcyanoacrylate (PNBCA) nanocapsules as a carrier for NSAIDs: in vitro release and in vivo skin penetration.

Abstract: PURPOSE The aim of this work was to prepare poly n-butylcyanoacrylate (PNBCA) nanocapsules loaded with indomethacin and to evaluate the ability of this carrier system to deliver the drug systemically after its topical application. METHODS Poly n-butylcyanoacrylate (PNBCA) nanocapsules of indomethacin were prepared by interfacial polymerization. The physicochemical characterization of the PNBCA nanocapsules was performed by measuring the drug content by HPLC and analyzing the particle size using scanning electron microscopy. The in vitro permeation of indomethacin through excised rat skin and an artificial membrane was determined for PNBCA nanocapsules in pH 7.4 phosphate buffer (I), and in PLF-127 gel (II) and were compared against indomethacin incorporated into 25%w/w PLF-127 gel alone (III). The in vivo percutaneous absorption of indomethacin following the application of the PNBCA nanocapsules and a 25%w/w Pluronic F-127 (PLF-127) gel (III) was monitored by the determination of drug plasma levels in rats. RESULTS The drug loading results indicated that approximately 76.6% of indomethacin was loaded onto the PNBCA nanocapsules; the average particle size was 188 nm. The in vitro results indicated a rank order for the three formulations (I, II and III) in both the flux at steady state and the cumulative amounts permeated at 8 hrs. The higher drug plasma levels over 6 hrs of indomethacin PNBCA nanocapsules are in agreement with the determined in vitro permeation results. CONCLUSION The presented data show that indomethacin loaded PNBCA nanocapsules can improve the transdermal delivery of indomethacin compared to a conventional gel formulation using Pluronic F-127. This might be due to their ultra fine particle size and their hydrophilic and hydrophobic surface characteristics.

Development of liposomal polyene antibiotics: an historical perspective.

Abstract: Purpose: The purpose of this review article is to review the development of a number of liposomal polyene antibiotics. Background: In the past thirty years, the increase in life-threatening pre-systemic and systemic fungal infections within cancer, diabetic and AIDS patients have reached alarming proportions. A number of antifungal agents have been developed to combat this problem. In particular, polyene antibiotics such as Ampho- tericin B (AmB) and Nystatin (Nys) have remained the most effective and widely used agents in the treatment of these infections. However, their administration is limited by dose-dependent toxicities. One such dose-limiting tox- icity is renal toxicity. Polyene antibiotic-induced renal tox- icity is believed to be mediated by the drug anchoring to cholesterol within the mammalian cell membrane, result- ing in pore formation, abnormal electrolyte flux, decrease in adenosine triphosphate (ATP), and eventually a loss of cell viability. Conclusion: In the 1980s and 90s a number of promising lipid-based AmB and Nys formulations were developed to overcome these toxicities. This article will review the development of these liposomal polyene antibi- otics.

Protection of pancreatic beta-cell by the potential antioxidant bis-o-hydroxycinnamoyl methane, analogue of natural curcuminoid in experimental diabetes.

Abstract: PURPOSE. To evaluate the antioxidant defense by bis-o-hydroxycinnamoylmethane, analogue of the naturally occurring curcuminoid bis-demethox- ycurcumin in streptozotocin induced diabetes in male Wistar rats and its possible protection of pancreatic β- cell against gradual loss under diabetic condition. METHODS. Male wistar rats were divided into five groups. Group1 served as control rats. Group2 was control rats treated intragastrically with bis-o-hydroxy- cinnamoyl methane at a dose of 15mg/kg body weight for 45 days. Group3, 4 and 5 rats were injected with 40mg /kg body weight of streptozotocin to induce dia- betes. Group4 rats were treated with the drug similar to group2 and group5 rats treated with the reference drug glibenclamide intragastrically for a similar period. After 45 days, the levels of plasma glucose, glycated hemoglobin, enzymic antioxidants (SOD, CAT) and non-enzymic antioxidants Vit C, Vit E was deter- mined. Histopathological sections of the pancreas were examined. RESULTS. The levels of plasma glucose and glycated hemoglobin which were elevated in group3 diabetic rats were reduced after treatment with the drug. The antioxidant levels showed an increase in the case of treated diabetic rats as compared to group3 dia- betic rats. The islets were shrunken in group3 diabetic rats in comparison to normal rats. In the treated dia- betic rats there was expansion of islets. CONCLU- SIONS. The experimental drug bis-o- hydroxycinnamoylmethane enhances the antioxidant defense against reactive oxygen species produced under hyperglycemic conditions and thus protects the pan- creatic β-cell against loss and exhibits antidiabetic prop- erty.

Pharmacokinetics of intravenously administered liposomal all-trans-retinoic acid (ATRA) and orally administered ATRA in healthy volunteers.

Abstract: PURPOSE To determine single-and multiple-dose pharmacokinetics of liposomal-all- trans -retinoic acid (Atragen) following intravenous and oral ATRA (Vesanoid) administration in healthy volunteers. METHODS This was a randomized, prospective, open-label, parallel pharmacokinetic study in which 29 subjects were given 90 mg/m (2) i.v. liposomal (L)-ATRA (16 subjects) every other day or 45 mg/m (2) oral ATRA (13 subjects) daily over 15 days. Pharmacokinetic parameters were assessed on days 1, 9, and 15. RESULTS Twenty-two subjects (11 in each group) completed the study and were evaluated. Area under the plasma concentration-time curve [AUC(0, infinity)] and maximum plasma concentration (C(max) ) of ATRA did not decrease during the 15 days of L-ATRA treatment. However, the oral ATRA regimen resulted in a significant decrease in the AUC(0, infinity) and C(max) of 45.3% and 31.8%, respectively, on day 9 as compared with that to day 1 (p< 0.05). In addition, the mean AUC(0, infinity) was 13- and 22-fold greater for L-ATRA than for oral ATRA on days 1 and 9, respectively. Despite the significantly higher plasma concentrations after L-ATRA treatment, the side effects of each formulation were similar, except for dermal exfoliation, which was seen in 31% of the subjects after L-ATRA dosing, and abnormal liver function tests that were seen in 23% of the subjects after oral ATRA administration. CONCLUSIONS These findings suggest that i.v. administration of L-ATRA maintains higher and stable plasma ATRA concentrations than oral ATRA in healthy subjects after repetitive administration. L-ATRA with a favorable pharmacokinetic profile may have potential advantages over oral ATRA and may be more efficacious in the treatment of acute promyelocytic leukemia or other retinoid-responsive cancers.

Poloxamer 407-mediated alterations in the activities of enzymes regulating lipid metabolism in rats.

Abstract: Purpose: Recently, the P-407-treated mouse was established as a useful animal model of hyper- lipidemia and atherosclerosis. The present study was aimed to determine whether P-407-induced hyperlipidemia in the rat is associated with alterations in the activities of enzymes responsible for lipid metabolism. Methods and Results: Rats were made hyperlipidemic by i.p. injection of 1.0 g/kg P-407 and blood samples collected 24 h after administration of P-407. Plasma from P-407-treated rats demonstrated 7- and 13-fold increases in cholesterol and triglycerides, respectively (p 50% in the presence of TP2, a monoclonal anti-CETP antibody (27.03 ± 3.16 vs. 10.87 ± 3.23; p < 0.05). The plasma CETP protein levels were also increased by 5-6-fold in P-407-treated animals (control 0.35 ± 0.17 vs. P-407 treated 1.87 ± 0.35 ug/ml, p < 0.05). However, the plasma hepatic lipase (HL) (control 49.2 ± 3.1 vs. P-407-treated 2.0 ± 0.38 umol/ml/h; p < 0.001) and lipoprotein lipase (LPL) (control 45.9 +/- 0.09 vs. P-407-treated 2.03 ± 0.38 µmol/ml/hr; p<0.001) activ- ities in these animals were significantly inhibited. Conclu- sions: In summary, P-407-induced hyperlipidemia in rats is associated with alterations in plasma LCAT, CETP, HL and LPL activities.

Attenuation of benzodiazepine dependence in mice by a tri-substituted benzoflavone moiety of Passiflora incarnata Linneaus: a non-habit forming anxiolytic.

Abstract: PURPOSE: A tri-substituted benzoflavone moiety (BZF) recently isolated from the methanol extract of aerial parts of the plant Passiflora incarnata Linneaus had exhibited encouraging results in countering the dependence produced by addiction-prone substances like morphine, nicotine, cannabinoids and ethyl alcohol, during the studies performed by the authors. Since the BZF moiety had exhibited significant anxiolytic properties at 10 mg/kg p.o. dose in mice, therefore, it was desirable to evaluate this potential phyto-moiety (BZF) for its own dependence-liabilities It was also deemed viable to evaluate BZF moiety for its possible usefulness in countering the dependence-liabilities associated with the chronic use of benzodiazepines keeping in light their tremendous clinical use in the management of anxiety and insomnia. METHODS: Different groups of mice were administered BZF alone (10, 50 or 100 mg/kg, p.o.), and concomitantly with diazepam (20 mg/kg, p.o.) in a 21-days treatment regimen, followed by no treatments for the next 72-hours. The withdrawal effects in the form of ambulatory behavior of the treated animals were recorded on the 25th day using an Actophotometer. RESULTS: The BZF-alone (three doses) treated mice exhibited a normal ambulatory behavior on 25th day. Mice groups receiving co-treatments, i.e., BZF-diazepam concomitantly, also exhibited a normal ambulatory behavior in a dose-dependent manner, i.e., the higher dose of BZF (100 mg/kg) being more effective in countering the withdrawal effects of chronically administered diazepam than the lower doses (10 or 50 mg/kg). CONCLUSIONS: The studies revealed that the chronic administration of the BZF moiety (three doses), did not exhibit any dependence-liability of its own, even upon an abrupt cessation. Additionally, the BZF co-treatments with diazepam also prevented the incurrence of diazepam-dependence, which might be because of the aromatase enzyme inhibiting properties associated with the BZF moiety.

Drug release characteristics of lipid based benzoporphyrin derivative.

Abstract: PURPOSE. The purpose of this study was to examine the transfer of verteporfin (BPDMA) from its lipid based formulation to serum proteins. METHODS. As a result of BPDMA being confined to the lipid phase, it was found that fluorescence from the photosensitizer was highly concentration quenched. This phenomenon was used to demonstrate rapid transfer of lipid-based drug to various plasma components such as albumin and lipopro- teins. Gel electrophoresis was used to show transfer of drug to lipoproteins. RESULTS. Loss of fluorescence quenching showed rapid transfer of the drug from its lipid based formulation to serum proteins. Gel electrophoresis showed that both the drug and phospholipid components were transferred to the lipoprotein fraction concurrently. The electrophoretic mobility of plasma lipoproteins was increased as a result of their interaction with lipid-based BPDMA. It was also shown that the lipid-based structures were readily destabilized in the presence of relatively low concentrations of plasma, and that liposomes of this lipid composition were highly unlikely to be found intact in the circulation following intravenous injection. CONCLU- SIONS. Verteporfin is rapidly transferred from its lipid based formulation to serum proteins. This rapid transfer, particularly to lipoproteins, provides a mechanism for its rapid delivery to cells.

Chemotherapy induced gastrointestinal toxicity in rats: involvement of mitochondrial DNA, gastrointestinal permeability and cyclooxygenase -2.

Abstract: Purpose: The gastrointestinal damage induced by xenobiotics is occurring more frequently and with greater toxicological significance than previously thought Although there are some pre-liminary clinical studies and reports, there does not appear to be an extensive examina- tion of gastrointestinal toxicity of various chemotherapeu- tic agents in the rat This study was undertaken to examine the suitability of a rat model to detect the gastrointestinal damage after administration of various anti-neoplastic agents including etoposide, teniposide, melphalan, 5-fluo- rouracil, methotrexate and cisplatin Methods: Acute toxic doses of indomethacin and chemotherapeutic agents were administered to rats The urinary excretion of orally administered sucrose and 51 Cr-EDTA were measured as markers of gastroduodenal and intestinal permeability, respectively Cyclooxygenase-2 messenger RNA and mito- chondrial DNA damage were measured as toxicological endpoints Results: Each anti-neoplastic agent examined induced appreciable and significant dose-dependent increase in gastrointestinal permeability that correlated with gross toxicological and pathological changes to the gastrointestinal tract including ulceration and bleeding COX-2 mRNA was upregulated > 2 fold in intestinal mucosa with enteropathy and dose-dependent mitochon- drial oxidative damage was apparent in gastric and intesti- nal mucosa After administration of each drug, the rats presented with histological evidence of drug-induced gas- troenteropathy, ulceration and increased cecal hemoglobin Conclusions: The rat appears to be a suitable model to study gastrointestinal toxicity of chemotherapeutic agents and non-steroidal anti-inflammatory drugs Damage to mitochondrial DNA occurs in both the gastric and intesti- nal epithelium after the administration of these agents and may be an important factor in the pathogenesis and resolu- tion of gastrointestinal toxicity

Co-encapsulation of two plasmids in chitosan microspheres as a non-viral gene delivery vehicle.

Abstract: Purpose. The aims of this study are to encapsu- late two different plasmid DNAs (pGL2 and pMK3) in the same microsphere structure and to investigate in vivo trans- fection characteristics of chitosan microspheres. Further- more, the effect of formulation factors, such as chitosan concentration and plasmid DNA amount on in vitro prop- erties of microspheres were studied. Methods. Double plasmid-loaded chitosan microspheres were prepared by complex coacervation. Release studies were done in phos- phate buffered saline at 37° C and released plasmid DNA was determined spectrophotometrically. Integrity of plas- mid DNAs was checked by agarose gel electrophoresis. For in vivo transfection studies, microspheres were injected into the muscle of the mice and expression of proteins (β-galactosidase and luciferase) was measured. Results. High encapsulation efficiency was obtained with chitosan microspheres (90%). The size of particles was about 1.15 - 1.28 µm. No dependence was observed between the size and formulation variables (chitosan concentration and the amount of plasmid). After encapsulation process, integrity of two plasmids did not change. Plasmid DNAs were con- tinuously released from chitosan microspheres. Chitosan concentrations and plasmid amounts affected in vitro release properties. After intramuscular injection of double plasmids loaded microspheres into muscle of the mice, co- expression was obtained. High β-galactosidase and luciferase productions were determined with these micro- spheres after a long post-transfection period (12 weeks). Conclusions. Our results showed that two plasmids could be encapsulated in chitosan microspheres without affect- ing their structural and functional integrity. Thus, sustained and high protein production was obtained with these

HPLC determination of amoxicillin comparative bioavailability in healthy volunteers after a single dose administration.

Abstract: Purpose An accurate, precise and sensitive HPLC assay was developed for the determination of amoxicillin in human plasma samples, to compare the bioavailability of two amoxicillin capsule (500mg) formulations (Amoxicilina from Brazil, as a test formulation and Amoxil from SmithKline Beecham Laboratories Ltda., Brazil, as a reference formulations) in 24 volunteers of both sexes. Methods Amoxicillin concentrations were analyzed by combined reversed phase liquid chromatography and UV detection (lambda=229 nm). Amoxicillin and cefadroxil (internal standard) were extracted from the plasma by addition of cold methanol. The separation was achieved using the Lichrosorb 10 microm, C18 reversed phase column at room temperature. The mobile phase consisted of a 95% phosphate buffer (0.01 mol/L), pH=4.8 and 5% acetonitrile mixture. The study was conducted using an open randomized 2-period crossover balanced design with a 1-week washout period between the doses. Plasma samples were obtained over an 8-hour period. The bioequivalence between the two formulations was assessed by calculating individual peak plasma concentrations (C(max) ) and area under the curve (AUC(0-8h) ) ratios (test/reference). The statistical interval proposed was 80-125%, as established by the US Food and drug administration Agency. Results The internal standard and amoxicillin eluted about 4.2 and 5.2 min, respectively at a flow rate of 1.3ml/min. The mean absolute recovery of AMO in plasma was 90.0% at 3 microg/ml, 98.6% at 25 microg/ml and 95.3 at 50 microg/ml. The assay showed excellent relationships between peak height ratios and plasma concentrations (r(2)>or= 0.999). The limit of quantification was 1g/ml, based on 200l of plasma. The geometric mean of Amoxicilina/Amoxil 500 mg capsules individual percentage ratio was 101.4% for AUC(0-8h), and 99.9% for C(max). The 90% confidence intervals were 98.3-104.4% and 95.7-103.9%, respectively. Conclusion This simple, rapid and selective method is suitable for pharmacokinetic, bioavailability and bioequivalence studies. Since the 90% CI for both C(max )and AUC(0-8h) lies within the 80-125% interval proposed by the Food and Drug Administration, it was concluded that Amoxicilina 500 mg capsules was bioequivalent to Amoxil capsules 500 mg, in terms of both the rate and extent of absorption.

A comparison of gastrointestinal permeability induced by diclofenac-phospholipid complex with diclofenac acid and its sodium salt.

Abstract: Purpose: Gastrointestinal (GI) side effects of the nonsteroidal anti-inflammatory drug (NSAID) diclofenac may be reduced if it is adminis- tered as a complex with phospholipid. The upper and lower GI permeability induced by a diclofenac-dipalm- itoyl phosphatidyl choline (DPPC) complex were compared with those of diclofenac acid and its sodium salt in rats. Methods: Pharmacokinetic studies were carried out to assess bioavailability of diclofenac prepa- rations. Adult male Sprague-Dawley rats were dosed orally (equivalent to 15 mg/kg diclofenac sodium) as the acid or its sodium salt as well as diclofenac-DPPC complex. Upper and lower GI permeability, as surro- gate markers of toxicity were determined using sucrose and 51 Cr-EDTA, respectively. Results: At 1 h post- dose only diclofenac sodium induced a significant increased upper GI permeability. Three h post-dose all formulations significantly increased upper GI perme- ability although the diclofenac acid had the least effect. In the lower GI tract, the induced increase in perme- ability was significant at 1 and 3 h post-dose for all for- mulations with no significant differences between them. Conclusion: The induced upper and lower GI toxicity of diclofenac was formulation and time depen- dent. The lack of effect of diclofenac acid was due to the decreased availability of the drug. In the upper GI tract, up to 1 h post-dose, the diclofenac-DPPC com- plex demonstrated reduced upper gastroduodenal per- meability as measured by sucrose. However, the protective effect of DPPC did not last and was not extended to the lower GI tract due to the systemic effect, contribution from the enterohepatic recircula- tion and/or dissociation of the complex. In assessing diclofenac GI toxicity, the effect of the different for- mulations on the entire GI tract at various times after drug administration must be considered.

Experimental estimation of the role of P-Glycoprotein in the pharmacokinetic behaviour of telithromycin, a novel ketolide, in comparison with roxithromycin and other macrolides using the Caco-2 cell model.

Abstract: PURPOSE: The aim of this study was to examine the transport mechanism of telithromycin in comparison with erythromycin, azithromycin, clarithromy- cin and roxithromycin. METHODS: These antibiotics were examined in Caco-2 cell monolayers in order to dem- onstrate the potential involvement of P-GP in the absorp- tion process, using verapamil as a P-GP competitor. A model using concentration equilibrium conditions was developed to delineate passive and active permeability components of telithromycin and roxithromycin transport in order to predict absorption in humans. RESULTS: Comparison of telithromycin Papp(AB)/Papp(BA) ratios with those of the other antibiotics indicated that an efflux pump was involved which limited the transport of the macrolides to a greater extent than that of telithromycin. Modulation of Caco-2 transport of these antibiotics by verapamil and their reciprocal effect upon verapamil trans- port confirmed the involvement of P-GP and demon- strated that two substrates of P-GP may increase the transport of each other. Under concentration equilibrium conditions, both roxithromycin and telithromycin exhib- ited high mean Papp values for passive diffusion which extrapolated to 88% and 77% predicted human absorption respectively, if the involvement of P-GP was ignored. Both Km and Vm values suggested that saturation of P-GP by telithromycin may occur at a lower dose level in humans than with roxithromycin (Km= 9.8 µM, Vm= 0.3 µM and Km= 45 µM, Vm= 1.1 µM, respectively). At 4.10 -5 M of either telithromycin or roxithromycin the passive flux was respectively 48% and 16% greater than the active efflux. CONCLUSIONS: The high absorption potential of telithromycin combined with the low Km and Vm val- ues and the high dose level suggest that in humans the efflux pump may not limit ketolide absorption and that the interaction with other P-GP substrates may not signifi- cantly increase its oral absorption.

A preliminary investigation of chitosan film as dressing for punch biopsy wounds in rats.

Abstract: Purpose. To investigate the wound healing effi- cacy of two chitosan films, Chit-AA and Chit-LA, in com- parison with a commercial preparation, Omiderm®, using punch biopsy wounds in rats. Methods. The punch biopsy wounds were created in the abdominal region of male Wistar rats. The films were evaluated in terms of transpar- ency, flexibility, adherence property, ease of removal from wounds without damaging underlying tissues and fluid accumulation. In addition, the wounds were examined for dryness, exudation, contraction, period of epithelialization and scar formation. Results. Chit-AA, Chit-LA and Omi- derm® films were comparable in terms of transparency, flexibility, adherence property, ease of film removal from wounds without damaging underlying tissues and fluid accumulation. Although there was no statistically signifi- cant difference in wound dryness and exudation between the film treated wounds and untreated wounds (Control), a significant difference was obtained in complete wound clo- sure (t 100% ), period of epithelialization and scar formation. Conclusions. Both Chit-LA and Chit-AA were able to promote wound healing with minimal scar formation.

Influence of storage conditions and type of plasticizers on ethylcellulose and acrylate films formed from aqueous dispersions.

Abstract: PURPOSE. To investigate the influence of storage conditions and types of plasticizers on the properties and stability of ethylcellulose and poly- methacrylate films and to elucidate the mechanism for the changes observed. METHODS. Films were pre- pared from Surelease, Aquacoat and Eudragit L 30D dispersions by the casting method. The effects of differ- ent plasticizers on the morphology, transparency, mechanical property and water vapour permeability of the prepared films were studied. The film samples were exposed to storage conditions of 30° C and 50 or 75 %RH. Samples were removed at pre-determined time intervals for mechanical testing and analysis of plasti- cizer content in the films. RESULTS. It was found that films prepared from aqueous ethylcellulose disper- sions were relatively weaker and more brittle than acrylate films. Acrylate films did not show any signifi- cant change in mechanical property when stored at high humidity. However, the properties of ethylcellu- lose films stored at high humidity varied depending on the type of plasticizers present. CONCLUSIONS. The changes in mechanical property of ethylcellulose films on storage were mainly attributed to the loss of plasticizers during storage, causing further coalescence of ethylcellulose films and to a smaller extent, reduc- tion in moisture content of the film.

Effect of cannulation surgery and restraint stress on the plasma corticosterone concentration in the rat: application of an improved corticosterone HPLC assay.

Abstract: PURPOSE. Stress increases plasma corticos- terone concentration in rats. We studied the time-course of plasma corticosterone post jugular vein cannulation, a potential stress producing process, and after 1 h restraint stress using an improved assay. Current HPLC methods for corticosterone analysis require long extraction times with large volumes of solvent. METHODS. Adult male, Sprague-Dawley rats were used. Tail vein blood samples were collected from uncannulated rats (n=4). Other rats were cannulated in the right jugular vein, allowed to recover overnight and assigned to days 1, 2, 3 or 4 groups (n=4-6/group). Blood was sampled before and after expo- sure to 1 h of restraint stress. Plasma was separated and extracted with 5 mL ethyl acetate (betamethasone as inter- nal standard), and then the extract was washed with sodium hydroxide (0.1 M) and water. After evaporation of ethyl acetate, the residue was dissolved in mobile phase (acetonitrile-water-acetic acid-TEA, 22:78:0.1:0.03, v/v) and injected into an isocratic HPLC consisting of a 10 cm C18 column, and UV detector at 254 nm. RESULTS. The minimum quantifiable concentration was 10 ng/mL of corticosterone based on 0.5 mL plasma. The standard curve was linear over the concentration range of 10-500 ng/mL. The extraction efficiency was 84-87%. The coeffi- cient of variation for intra-day and inter-day precision was <10% with <8% error. Plasma corticosterone before can- nulation (74 ± 17 ng/mL) and those measured daily before restraint stress were not significantly different from one day to another (day 1, 60 ± 21; day 2, 59 ± 39, day 3, 45 ± 23, and day 4, 41 ± 8 ng/mL). Exposure to restraint stress elevated corticosterone (day 1, 122 ± 33; day 2, 82 ± 23; day 3, 148 ± 33; and day 4, 134 ± 20 ng/mL). Except on day 2, all concentrations were significantly elevated as compared to the pre-stress levels. CONCLUSIONS. Cor- ticosterone concentrations were stable after jugular vein cannulation, and were increased by restraint stress. The present assay is a more convenient method as compared to those previously reported.

Preparation and in vitro evaluation of chitosan matrices for colonic controlled drug delivery.

Abstract: Purpose. The work was aimed at studying in vitro the release of 5-aminosalicylic acid (5-ASA) or diclofenac sodium (DS) from matrices based on chitosan (Ch) or Ch hydrochloride (Ch-HCl), destined to be intro- duced into enteric-coated capsules for controlled release to the colon. Methods. Matrices (diameter, 6 mm; weight, 50 mg) were prepared by compression of Ch or Ch-HCl microparticles mixed with 20 % 5-ASA or DS powder. Drug release from matrices to isotonic neutral buffers of different molarity was studie d in vitro. In some cases, matrix incubation in rat cecal contents preceded the release test. Results. The matrices, especially the Ch-HCl- based ones, swelled in the dissolution medium without dis- integrating. Drug release was diffusion-controlled and fol- lowed square-root-time kinetics. Release depended on the pH-dependent aqueous solubility of the drug. The internal pH of the swollen Ch-HCl-based matrix was acidic, so 5- ASA solubility and release were influenced by penetration of salts from the external buffer. In the Ch-HCl-based matrix DS was converted into the scarcely soluble diclofenac free acid, which prolonged the time for release of 50 % dose excessively (t 50 =11.26 h). The enzymatic action of rat cecal microflora accelerated drug release from the Ch-HCl-based matrix. On the other hand, neither such a microflora nor the external medium hydrodynamics sig- nificantly affected drug release from the Ch-based matrix. Conclusions. The Ch-based matrix was a reliable colonic controlled-release system for 5-ASA (t 50 =1.97 h) or DS (t 50 =3.58 h). For in vivo application, a number of matrices adequate to make up the therapeutic drug dose should be introduced into enteric-coated size 00 capsules.

Preparation and in vitro transfection efficiency of chitosan microspheres containing plasmid DNA:poly(L-lysine) complexes.

Abstract: Purpose. Studies on DNA complexes with cationic polymers are prompted by the search for non- viral DNA carriers for gene therapy. Among them, poly(L-Lysine) (PLL) has been extensively studied. On the other hand, these systems deliver DNA as a bolus without long-term release. The aims of this study were to encapsulate plasmid DNA:poly(L-lysine) (pDNA:PLL) complexes into chitosan microspheres as an alternative to the PLL based gene delivery and investigate its in vitro release and transfection charac- teristics as well as plasmid DNA integrity and stability against serum and DNase I challange. Methods. pUC18 plasmid DNA that encoded β-galactosidase was used as a model. The microspheres were prepared by complex coacervation method and the release and in vitro transfection properties were investigated. pDNA:PLL complexes were prepared at two different mass ratios. In vitro release studies were performed at 37 ± 0.5 0 C and drug release was monitored both spec- trophotometrically and fluorometrically. Structural integrity of the pDNA:PLL complexes were deter- mined by Southern blotting analysis. Protective effect of encapsulation of pDNA:PLL complexes against DNase I and serum treatment were also studied. In vitro transfection studies were performed by using 3T3 cell line. Results. According to our in vitro release data, the mass ratio of pDNA:PLL significantly affected the release of pDNA:PLL complexes from chi- tosan microspheres, and the structure of the plasmid DNA did not change during the experiments. pDNA:PLL-loaded chitosan microspheres indicated high stability against fetal bovine serum and DNase I treatment for a week. In vitro transfection data showed that pDNA:PLL-loaded chitosan microspheres could be effectively transfected 3T3 cells in vitro. Conclu- sion. As a conclusion, pDNA:PLL complexes could be encapsulated into chitosan microspheres with main-

Correlation of photosensitizer delivery to lipoproteins and efficacy in tumor and arthritis mouse models; comparison of lipid-based and Pluronic P123 formulations.

Abstract: PURPOSE. The purpose of this study was the use of animal models to demonstrate the importance of drug delivery (verteporfin) to plasma lipopropteins in order to attain efficacy of photodynamic therapy (PDT) in vivo. METHODS. Photosensitzers appropriately formu- lated in various vehicles such as pluronics and lipid-based systems were compared to delivery of the drug in DMSO in two in vivo systems. The first was a tumor model using male DBA/2 mice inoculated intradermally with M1 rhab- domyosarcoma cells and in the second, arthritis in the MRL-lpr mouse strain was enhanced by two intradermal injections of complete Freunds adjunct. RESULTS. Those formulations in which the drug was in a monomeric form were better able to transfer drug to lipoproteins, which in turn led to superior PDT in vivo. CONCLU- SIONS. The ability to introduce drug in monomeric form into the circulation correlates well with efficacy of photo- sensitizer formulations in mouse arthritis and tumor mod- els.

Farnesol for aerosol inhalation: nebulization and activity against human lung cancer cells.

Abstract: Purpose: A nebulized aerosol formulation of the anti-cancer agent farnesol is developed and shown to induce cell death of human lung cancer cells in vitro. Meth- ods: A nebulized farnesol formulation containing polysor- bate 80 (Tween 80) is developed. The measurements of the aerosol properties during nebulization were used as input for a mathematical model of airway surface liquid in the lung of an average adult, to estimate the airway surface liq- uid drug concentration of the deposited farnesol. Cytotox- icity of the formulations was measured in vitro on non- small cell lung cancer cells (H460 and A549). Results: As much as 100% of lung cancer cytotoxicity can be achieved by using Pari LC Star and LC Plus nebulizers. The esti- mated airway surface liquid concentrations of the depos- ited farnesol reveal that the IC50 of the nebulized farnesol can be achieved over the entire tracheobronchial region, using the above Pari nebulizers with a volume fill of 5 ml. Conclusions: Drug concentrations higher than IC50 in the airway surface liquid are predicted with our methods, suggesting in vivo trials of a formulation may be warranted with these particular nebulizers.Introduction

Therapeutic and hemolytic evaluation of in-situ liposomal preparation containing amphotericin - beta complexed with different chemically modified beta - cyclodextrins.

Abstract: PURPOSE The objective of this study was to evaluate therapeutic and haemolytic effects of liposomal preparation derived from proliposome entrapping inclusion complex of amphotericin B (AmB) with the chemically modified beta-cyclodextrin (beta-CD). METHODS a series of liposomal AmB formulations with varying beta-CD i.e. Hydroxy propyl beta-CD (HPBCD) and Sulfo butyl ether beta-CD (SBEBCD) having similar AmB content (0.5 mg/kg) were prepared and their effect compared with conventional liposomal amphotericin B (L-AmB) and free AmB on erythrocyte lysis and antifungals activity in experimental aspergillosis- and Cryptococcosis- mice model in-vivo. RESULTS the liposomal AmB - HPBCD and AmB - SBEBCD found to be 6 times less toxic than free AmB or conventional liposomal AmB. Experimental findings indicate that infected animals treated with L-AmB entrapped inclusion complexes significantly reduced CFU values (fungal counts), whereas infected animals treated with conventional liposome or free AmB showed insignificant reduction in CFU. A marked increase in the percent survival was observed in the case of animals treated with liposomal AmB formulation (HPBCD/SBEBCD). Furthermore, the in-vitro toxicity (haemolysis) of the proliposome-based liposomal vesicles (PBLV) entrapped AmB-SBEBCD/HPBCD at 37 degrees C was approx. 50% at maximum of the conventional liposomal AmB at a dose of 118 microg/ml as measured after 1 hr. incubation. CONCLUSIONS the results of these experiments permitted us to conclude that the stabilization of liposome derived from proliposome entrapping inclusion complex of amphotericin B (AmB) with beta-CD could serve an alternative approach to enhance the therapeutic window of AmB in clinical medicine.

Formulation dependent pharmacokinetics, bioavailability and renal toxicity of a selective cyclooxygenase-1 inhibitor SC-560 in the rat.

Abstract: Purpose: To delineate formulation depen- dent pharmacokinetics and bioavailability of SC-560, a rel- atively new cycloooxygenase-1 (COX-1) specific inhibitor, in the rat and examine its influence on the renal tubular enzyme, N-acetyl-beta-D-glucosaminidase (NAG), and urinary electrolytes. Methods: The pharmacokinetics of SC-560 was studied in Sprague-Dawley rats (n = 5 per group) after a single intravenous (iv) and oral dose (10 mg/ kg) in polyethylene glycol (PEG) 600 and a single oral dose (10 mg/kg) in 1% methylcellulose (MC). Serial blood sam- ples were collected via a catheter inserted in the right jugu- lar vein and serum samples were analysed for SC-560 using reverse phase HPLC. After oral administration of SC-560 in PEG, urine was also collected for 24 h and analysed for urinary sodium, chloride, and potassium as well as NAG. Results: After an iv dose (10 mg/kg) of SC-560, serum AUC, t 1/2, CL and V d were 9704 ± 4038 ng h/mL, 5.4 ± 0.8 h, 1.15 ± 0.46 L/h/kg and 9.1 ± 4.6 L/kg (mean +/ - SD, n = 5), respectively. Oral administration of 10 mg/kg SC-560-PEG and MC (n=5 rats) yielded serum AUC, C max , t max and t 1/2 of 1203.4 ± 130.3 and 523 ± 208 ng h/ mL, 218.5 ± 86.9 and 119.8 ± 15.5 ng/mL, 1.00 ± 1.8 and 2.0± 0 h, 3.7 ± 1.6 and 2.7 ± 1.7 h (mean +/- SD, n = 5), respectively. A single oral dose 10 mg/kg of SC-560 in PEG resulted in an increase in NAG excretion in urine and a reduction in 0-24 h urinary sodium, potassium, and chloride excretion. Conclusions: SC-560 extensively dis- tributes into rat tissues, and has a CL approaching hepatic plasma flow. The drug displays low <15% and formulation dependent bioavailability after oral administration and demonstrates kidney toxicity.

Measurement of nitric oxide in murine Hepatoma Hepa1c1c7 cells by reversed phase HPLC with fluorescence detection.

Abstract: PURPOSE. Nitric oxide (NO) is produced by various cell types in picomolar to nanomolar range and has important roles in a variety of biological functions. The aim of the present study was to investigate a sensitive and reproducible fluorometric HPLC method for measur- ing nitrite, one of the stable oxidation products of nitric oxide, in murine hepatoma Hepa 1c1c7 cells. METH- ODS. Hepa 1c1c7 cells were incubated with vehicle or recombinant murine tumor necrosis factor-α (TNF-α, 10 ng/ml) in Hanks' balanced salt solution (HBSS) for 10 hours. Thereafter, the HBSS medium was collected for nitrite analysis. NO production was examined by measur- ing the conversion of 2, 3-diaminonaphthalene (DAN) to its fluorescent product, 2, 3-naphthotriazole (NAT). NAT was analyzed after elution with 60% of 15 mM sodium phosphate buffer (pH = 7.5) and 40% methanol through a 10-µm reversed-phase C18 column (250 x 4.00 mm, I.D.) at a flow rate of 1 ml /min. Fluorescence was monitored with excitation at 375 nm and emission at 415 nm. RESULTS. NAT appeared in approximately 16 min with no interference peaks. The assay yielded linear response within the examined range of 10 - 200 pM (r 2 >0.99) with an intra and inter-day variability of 90%. The detection limit for nitrite was 10 pM. NO pro- duction by TNF-α treated Hepa 1c1c7 cells is estimated to be approximately 2 folds more than untreated cells. CONCLUSION. This fluorometric HPLC assay offers a sensitive and reliable measurement of NO production in cell culture medium.

Permeation prediction of M100240 using the parallel artificial membrane permeability assay.

Abstract: Purpose: Kansy et al (5) first introduced the Parallel artificial membrane permeation assay (PAMPA) in 1998. In this system, the permeability through a membrane formed by a mixture of lecithin and an inert organic solvent on a filter support is assessed. PAMPA shows definite trends in the ability of molecules to permeate membranes by transcellular passive diffusion. Its simplicity, low cost, high throughput, and wide pH range make it very attractive in modern drug discovery. Based on this concept, Whohnsland et al(1), Sugano et al(12) and Zhu et al (9) modified the assay and used it to screen compound per- meability. We used PAMPA for the permeation pre- diction of M100240, which was unable to be determined by cell-based assays due to compound instability. Methods: In this study, 92 commercially available agents provided the structural diversity used to generate a mathematical prediction model for human fraction absorbed, M100240 — an acetate thioester of MDL 100,173. Permeation of M100240 and MDL 100,173 was evaluated using the parallel arti- ficial membrane permeability assay (PAMPA). The donor and recipient solutions consisted of 0.5N HCl (pH 1.5) or phosphate-buffered saline (pH 5.5 or 7.4) with 2% dimethyl sulfoxide. The donor solution also contained 200 mM M100240 or MDL 100,173. Results: M100240 had a medium permeation at pH 5.5 (2.99%), corresponding to a high predicted Fa in humans (92%). Permeation of MDL 100,173 was low at this pH (0.72%), corresponding to a medium-to-low predicted Fa (46%). At pH 7.4, the permeation of M100240 was low (~ 1%) and no permeation was apparent for MDL 100,173. Conclusions: We pre- dicted M100240 is likely to be well absorbed via pas- sive diffusion across the human gastrointestinal tract following oral administration.

The antinociceptive activities of 1-(4-aryl-2-thiazolyl)-3,5-disubstituted-2 pyrazolines in mouse writhing test.

Abstract: PURPOSE. Sympathetic nervous system stimulation, which releases noradrenalin, influences the nociceptive activity that develops after tissue injury. The α 2 -adrenergic agonist, clonidine, produces analgesia through a central mechanism but also inhib- its noradrenalin release at terminal nerve fiber endings. METHODS. In this study the effects of some 1-(4- aryl-2-thiazolyl)-3,5-disubstituted-2-pyrazolines (com- pounds 1a-1e) as analogues of clonidine with some modifications were studied by writhing test, a visceral pain model in mice. RESULTS. Compounds 1a and 1c induced significant reduction in writhing response when compared to control. Regarding the dose- response relationship of compounds 1a and 1c, it is evi- dent that the activity of these compounds is in direct proportion to their dose and all compounds show activities comparable to clonidine. In addition com- pounds 1a and 1c, having methoxy and nitro groups respectively at para position of 4-aryl showed better antinociception than compounds having similar groups in meta position namely 1b and 1e. This might be due to better interaction of these compounds with the α 2 -receptors. CONCLUSIONS. The antinocicep- tive properties of these newly synthesized compounds is comparable to clonidine. Further studies are needed to explore the differences in the efficacy and safety of synthesized compounds.

Cost-savings from subsidized pro-active pharmacist interventions.

Abstract: PURPOSE. This paper evaluates a pilot project to determine the desirability of implementing a reimburse- ment model for pro-active interventions. A drug plan administration conducted an experiment in which a phar- macist could recommend to physicians the substitution of lower-cost therapies with equivalent health outcomes. The pharmacist shared any cost savings with the insurer. METHODS. Drug plan costs without the intervention were estimated using time-series forecasting models and compared to actual costs with the intervention. RESULTS. Over the course of this experiment, there were some cost savings generated by reactive pharmacist interventions but pro-active interventions, intended to influence subsequent physician behaviour, appear to have had no significant effect on the profile of drug expendi- tures. CONCLUSIONS. The evidence does not lend extensive support for full implementation of this type of reimbursement model.

Application of a new high performance liquid chromatography method to the pharmacokinetics of dibudipine in rats.

Abstract: PURPOSE. To develop a HPLC method for assay of dibudipine in biological fluids and to study its pharmacokinetics in the rat. METHODS. HPLC: 2 µl (20 µg/ml) mebudipine as internal standard, 0.2 ml NaOH 1 M and 2 ml ethyl acetate were added to 0.2 ml of rat plasma. The mixture was shaken for 10 min, centrifuged, and the supernatant was dried under nitrogen. The dissolved residue was injected to a C 18 analytical column. Mobile phase flowed at 1 ml/min with a composition of methanol--water-acetonitrile (70-25-5). The eluent was monitored at 238 nm. Phar- macokinetic study: plasma samples were collected peri- odically after intravenous (0.5 mg/kg) or oral (10 mg/ kg) administration of dibudipine to rats (n = 4/group). In addition, separate groups of animals were adminis- tered 0.5 mg/kg doses of the drug for serial collection of brain, heart, kidney and liver (n = 4/time). The concentration of the drug in tissue or plasma was assayed using the above HPLC method. RESULTS. Calibration curves were linear over a concentration of 10-1000 ng/ml and CV was less than 10%. Dibudipine showed a bi-exponential decline after IV injection in the rats with a t 1/2 beta of 2.5 ± 0.5(mean ± SE) hr. Oral bioavailability was low. Distribution of dibu- dipine to the examined tissues was rapid, and with the exception of the brain, the concentrations of the drug in all tissues were higher than the plasma levels CON- CLUSIONS. The HPLC method was simple and con- venient. Moreover, it could be applied to investigations of the pharmacokinetics of dibudipine in the rat.