Iztok Grabnar

Preparation in high-shear mixer of sustained-release pellets by melt pelletisation.

The preparation of sustained-release pellets by melt pelletisation was investigated in a 10-l high shear mixer and ternary mixtures containing stearic acid as a melting binder, anhydrous lactose as a filler and theophylline as a model drug. A translated Doehlert matrix was applied for the optimisation of process variables and quality control of pellets characteristics. After determination of size distribution, the pellets were characterised with scanning electron microscopy, X-ray photoelectron spectroscopy and porosimetric analysis. Finally, the in vitro release from every single size fraction was evaluated and the release mechanism was analysed. Since the drug release rate decreased when enhancing the pellet size fraction, the 2000-microm fraction, exhibiting a substantially zero-order release, was selected for further in vivo biovailability studies. These data demonstrated that pellets based on the combination of stearic acid and lactose can be used to formulate sustained release pellets for theophylline.

The Role of Reactive Species in Epileptogenesis and Influence of Antiepileptic Drug Therapy on Oxidative Stress

Epilepsy is considered one of the most common neurological disorders. The focus of this review is the acquired form of epilepsy, with the development process consisting of three major phases, the acute injury phase, the latency epileptogenesis phase, and the phase of spontaneous recurrent seizures. Nowadays, an increasing attention is paid to the possible interrelationship between oxidative stress resulting in disturbance of physiological signalling roles of calcium and free radicals in neuronal cells and mitochondrial dysfunction, cell damage, and epilepsy. The positive stimulation of mitochondrial calcium signals by reactive oxygen species and increased reactive oxygen species generation resulting from increased mitochondrial calcium can lead to a positive feedback loop. We propose that calcium can pose both, physiological and pathological effects of mitochondrial function, which can lead in neuronal cell death and consequent epileptic seizures. Various antiepileptic drugs may impair the endogenous antioxidative ability to prevent oxidative stress. Therefore, some antiepileptic drugs, especially from the older generation, may trigger oxygen-dependent tissue injury. The prooxidative effects of these antiepileptic drugs might lead to enhancement of seizure activity, resulting in loss of their efficacy or apparent functional tolerance and undesired adverse effects. Additionally, various reactive metabolites of antiepileptic drugs are capable of covalent binding to macromolecules which may lead to deterioration of the epileptic seizures and systemic toxicity. Since neuronal loss seems to be one of the major neurobiological abnormalities in the epileptic brain, the ability of antioxidants to attenuate seizure generation and the accompanying changes in oxidative burden, further support an important role of antioxidants as having a putative antiepileptic potential.

Ivermectin pharmacokinetics in lactating sheep.

Ivermectin (IVM) concentrations in plasma and milk were studied in six Istrian Pramenka dairy sheep after a single subcutaneous dose of 0.2 mg/kg b.w. of IVM in the early lactation period to describe IVM disposition in milk and to evaluate the transfer of IVM residues via milk to suckling lambs. Large inter-animal in concentration variability of IVM in both matrices was observed. The highest overall concentration was found in the same animal: 21.7 microg/l of H(2)B(1a) in plasma on the second day and 44.9 microg/kg of H(2)B(1a) in milk on the first day after the drug was administered. The mean time in which IVM concentrations fell below the limit of detection for the whole ewe group was 22 and 23 days for plasma and milk, respectively. Time course of IVM concentration in milk was following the time course of IVM concentration in plasma, with an overall mean+/-S.D. of milk/plasma ratio of 1.67+/-0.50 for the first 7 days of the experiment. A mean of 0.7% of the dose was excreted through milk. Individual pharmacokinetic parameters were determined by fitting a one-compartment model to the milk and plasma concentration-time profiles. Mean t(max), c(max), t(1/2k(e)) and AUC values for plasma data were: 1.70+/-0.65 days, 11.88+/-6.96 microg/l, 2.85+/-1.97 days and 63.99+/-28.34 microg day/l, respectively, and for milk: 1.28+/-1.07 days, 22.67+/-18.27 microg/l, 3.56+/-2.01 days and 114.60+/-60.41 microg day/l, respectively. The highest level of concentration in suckling lamb plasma, 0.36 microg/l of H(2)B(1a), was slightly above the limit of determination. The mean lamb to ewe ratio of areas under the plasma concentration-time curve for the first 5 days was 0.02. On the basis of obtained results, it can therefore be claimed that indirect IVM exposure of the suckling lambs via milk was negligible.

Oral bioavailability of silymarin phytocomplex formulated as self-emulsifying pellets.

The objective of this study was to develop new solid self-emulsifying pellets to deliver milk thistle extract (silymarin). These pellets were prepared via extrusion/spheronisation procedure, using a self-emulsifying system or SES (Akoline MCM®, Miglyol®, Tween 80®, soy lecithin and propylene glycol), microcrystalline cellulose and lactose monohydrate. To select the most suitable formulations for extrusion and spheronisation, an experimental design of experiences was adopted. The screening amongst formulations (13 different blends) was performed preparing pellets and evaluating extrusion profiles and quality of the spheronised extrudates. The pellets were characterised for size and shape, density, force required to crush them. Although more than one type of pellets demonstrated adequate morphological and technological characteristics, pellets prepared from formulation 7 revealed the best properties and were selected for further biopharmaceutical investigations, including in vitro dissolution and in vivo trials on rats to study serum and lymph levels after oral administration of the pellets. These preliminary technological and pharmacokinetic data demonstrated that extrusion/spheronisation is a viable technology to produce self-emulsifying pellets of good quality and able to improve in vivo oral bioavailability of main components of a phytotherapeutic extract of more than 100 times by enhancing the lymphatic route of absorption.

Influence of chitosan and polycarbophil on permeation of a model hydrophilic drug into the urinary bladder wall.

Influence of dispersions of mucoadhesive polymers chitosan and polycarbophil on permeability properties of urinary bladder was investigated in vitro on isolated porcine urinary bladder. Pipemidic acid as a model hydrophilic drug was used. Its distribution in the bladder wall was determined from actual tissue concentrations by a method based on sectioning of frozen tissue and extraction of tissue slices. Pipemidic acid tissue concentration versus tissue depth profiles were evaluated by a diffusion model assuming constant diffusion coefficient. Increase in bladder wall permeability was observed in the presence of both polymers. Apparent permeability (mean+/-S.D.) of urinary bladder wall was increased 2.7+/-2.9 and 2.8+/-2.0 times for chitosan, and 2.3+/-2.0 and 4.3+/-4.2 times for polycarbophil at 0.5 and 1.0%, w/v polymer concentration, respectively. This increase is a consequence of the increased permeability of urothelium. These findings support investigations on application of chitosan and polycarbophil in development of mucoadhesive intravesical drug delivery systems. Experimental model may be applied to evaluate the results of experiments with drugs used in intravesical therapy.

MDR1 gene polymorphisms and response to acute risperidone treatment

Polymorphic multidrug resistant protein 1 (MDR1) transports drugs against a concentration gradient across the blood-brain barrier and reduces their accumulation in the brain. MDR1 may therefore influence antipsychotic brain availability contributing to inter-individual differences in treatment response and adverse effects, regardless of plasma concentrations. In the present study we investigated the influence of two common MDR1 polymorphisms on the improvement of psychopathological symptoms and occurrence of extrapyramidal side effects (EPS) in Slovenian schizophrenia patients acutely treated with risperidone. A total of 59 clinically well defined patients with first episode schizophrenia spectrum disorders or after tapering their maintenance treatment were genotyped for MDR1 C3435T and G2677T/A. Steady-state plasma concentrations of risperidone active moiety (sum of risperidone and the 9-hydroxyrisperidone) were determined. G2677T/A and C3435T genotypes were not associated to psychopathological symptoms, efficacy of treatment and risk for parkinsonism. Marginal associations with akathisia (p=0.039 and p=0.042, respectively) and dystonia (p=0.013 and p=0.034, respectively) were observed for both G2677T/A and C3435T genotypes. However, higher AIMS and BARS scores were observed only in heterozygous carriers of G2677T/A and C3435T polymorphisms and there was no tendency of gene-dose effect. The present study does not suggest a major influence of MDR1 G2677T/A and C3435T polymorphisms on treatment response during short-term risperidone therapy in patients with schizophrenia or schizoaffective disorder.

The study of drug release from microspheres adhered on pig vesical mucosa

The object of our work is the preparation of a mucoadhesive drug delivery system intended for intravesical application. In the present work, microspheres with Eudragit RS matrix polymer and different mucoadhesive polymers, i.e. chitosan hydrochloride (Ch), sodium salt of carboxymethyl cellulose (CMC) and polycarbophil (PC) were prepared to evaluate their influence on the mucoadhesive properties of microspheres. Different parameters were determined and their influence on pipemidic acid release from microspheres adhered on intact and damaged pig vesical mucosa was evaluated: swelling of polymers, mucoadhesion strength of polymeric films and drug dissolution according to USP XXIV method. The dissolution rate from microspheres containing different mucoadhesive polymers decreases as follows: PC>Ch>CMC. PC swelled to the largest volume among all polymers and as a result the fastest release of the drug from PC microspheres was obtained. The release rate of pipemidic acid from microspheres adhered on intact mucosa followed the order PC>CMC>Ch. These results show that both drug dissolution and mucoadhesion strength strongly influence drug release from adhered microspheres. The slowest release from Ch microspheres could be interpreted by the largest mucoadhesion strength of Ch polymeric films. The release rate of pipemidic acid from microspheres adhered on damaged mucosa followed the order PC=Ch>CMC. The results obtained on pathologically changed mucosa model support the indication of the role of glycosaminoglycans and polymer charge in the mucoadhesion process on vesical mucosa. Analysis of release data shows that the drug dissolution profiles follow the Higuchi kinetics better than the release profiles from adhered microspheres and different kinetics might be a consequence of different release mechanisms.

Thermoreversible in situ gelling poloxamer-based systems with chitosan nanocomplexes for prolonged subcutaneous delivery of heparin: design and in vitro evaluation.

In situ forming systems including thermoreversible hydrogels, which undergo sol-gel transition upon an increase in temperature have been used for various biomedical applications. Heparins are the standard of anticoagulation in the prophylaxis and treatment of deep vein thrombosis and pulmonary embolism. Both conditions require long-lasting treatment with frequent subcutaneous administrations of heparin. The objective of this study was to prepare and evaluate in situ forming gel systems designed by combination of two poloxamers (P407 and P188) and hydroxypropylmethylcellulose (HPMC) for prolonged release of heparin. Thermoreversible hydrogels were prepared with heparin solution and dispersion of heparin/chitosan nanocomplexes. Nanocomplexes formed by self-assembly of heparin with chitosan at various mass ratios were thoroughly characterized. A heparin/chitosan mass ratio of 1:1 with pH 5.20 was the most appropriate for preparation of small, homogenous and stable nanocomplexes (mean diameter 123 nm; polydispersity index 0.22 and zeta potential+35.5 mV). Thermoreversible hydrogels were evaluated by gelation temperature, viscosity over the temperature range 20-40 °C, rate of hydrogel dissolution, and heparin release in vitro. The addition of P188 to P407 gel formulations resulted in an increase in gelation temperature, decrease in viscosity at room temperature and faster gel dissolution. The opposite effects were observed with formulations containing HPMC which demonstrated 18-day-long gel dissolution and complete heparin release in 9days from gels containing heparin solution. Considerable prolongation of heparin release was achieved with incorporation of heparin/chitosan nanocomplexes into the gelling systems. It may be concluded that with poloxamer mixtures at specific concentrations, addition of HPMC and use of heparin/chitosan nanocomplexes dispersions, thermoreversible formulations for prolonged subcutaneous release of heparin are feasible.

Investigation of the Influence of CYP1A2 and CYP2C19 Genetic Polymorphism on 2-Cyano-3-hydroxy-N-(4-(trifluoromethyl)phenyl)- 2-butenamide (A77 1726) Pharmacokinetics in Leflunomide- Treated Patients with Rheumatoid Arthritis

Leflunomide is a disease-modifying antirheumatic drug used for the treatment of rheumatoid arthritis (RA). Cytochromes P450, mainly CYP1A2 and CYP2C19, may be involved in the transformation of leflunomide to leflunomide metabolite (A77 1726, 2-cyano-3-hydroxy-N-[4-(trifluoromethyl)phenyl]-2-butenamide). The aim of this study was to investigate whether genetic polymorphisms in CYP1A2 and CYP2C19 influence leflunomide pharmacokinetics, treatment response, and the occurrence of adverse drug reactions (ADRs). The study included 67 patients with RA and 4 patients with polyarthritis resembling RA and psoriasis treated with leflunomide. A77 1726 steady-state plasma concentrations were determined by validated high-performance liquid chromatography with UV detection. A population pharmacokinetic model was developed to estimate the oral clearance (CL/F) and volume of distribution (V/F). A genotyping approach was used to determine C-163A, C-729T, and T-739G in the CYP1A2 gene as well as single nucleotide polymorphisms that characterize CYP2C19*2, *3, *4, and *17 alleles. A large interindividual variability in trough A77 1726 steady-state plasma concentrations was observed (from 1.9 to 156.9 mg/l). A77 1726 CL/F was 71% higher in carriers of the CYP2C19*2 allele compared with noncarriers. The A77 1726 average steady-state plasma concentration was associated with the treatment response. Patients with a greater decrease in C-reactive protein (CRP) had higher average steady-state plasma A77 1726 concentrations: 49.7 ± 39.0 mg/l in patients with ΔCRP of more than 8.5 mg/l compared with 24.8 ± 13.7 mg/l in patients with ΔCRP of ≤8.5 mg/l (p = 0.015). No association of A77 1726 steady-state plasma concentrations with the occurrence of ADRs was observed. Our results suggest that genetic variability in leflunomide-metabolizing enzymes influences leflunomide metabolite concentrations that are associated with the treatment response but not with leflunomide-induced toxicity.

Antioxidants as a Preventive Treatment for Epileptic Process: A Review of the Current Status

Epilepsy is known as one of the most frequent neurological diseases, characterized by an enduring predisposition to generate epileptic seizures. Oxidative stress is believed to directly participate in pathways leading to neurodegeneration, which serves as the most important propagating factor, leading to the epileptic condition and cognitive decline. Moreover, there is also a growing body of evidence showing the disturbance of antioxidant system balance and consequently increased production of reactive species in patients with epilepsy. A meta-analysis, conducted in the present review confirms an association between epilepsy and increased lipid peroxidation. Furthermore, it was also shown that some of the antiepileptic drugs could potentially be responsible for additionally increased lipid peroxidation. Therefore, it is reasonable to propose that during the epileptic process neuroprotective treatment with antioxidants could lead to less sever structural damages, reduced epileptogenesis and milder cognitive deterioration. To evaluate this hypothesis studies investigating the neuroprotective therapeutic potential of various antioxidants in cells, animal seizure models and patients with epilepsy have been reviewed. Numerous beneficial effects of antioxidants on oxidative stress markers and in some cases also neuroprotective effects were observed in animal seizure models. However, despite these encouraging results, till now only a few antioxidants have been further applied to patients with epilepsy as an add-on therapy. Based on the several positive findings in animal models, a strong need for more carefully planned, randomized, double-blind, cross-over, placebo-controlled clinical trials for the evaluation of antioxidants efficacy in patients with epilepsy is warranted.