Novel gastroretentive controlled-release drug delivery system for amoxicillin therapy in veterinary medicine.

Beta-lactam antimicrobials, commonly used in both veterinary and human medicine, generally present short biologic half-lives, whereas their activity is enhanced as pathogen exposure is prolonged. These properties necessitate multiple-dose regimens of standard dosage forms, thereby hampering pet owner adherence, frequently resulting in therapeutic failure. This study presents a novel controlled-release gastroretentive oral drug delivery system for beta-lactams with which single-dose administration provides an effective antimicrobial course, optimizing pharmacokinetic (PK)-pharmacodynamic (PD) profiles, minimizing adverse effects and emergence of antimicrobial resistance and facilitating adherence. Our prototype sustained-delivery swelling-tablet (SDST), based on a degradable hydrophilic polymeric matrix, was designed to enable continuous input of these drugs to their absorption sites over several days. Several SDST formulations of the beta-lactam amoxicillin were evaluated in in vitro dissolution studies. Two formulations were selected for further in vivo canine studies, for determination of gastric retention and PK-PD profiling. Prolonged gastric retention times maintaining allowed for maintained effective drug concentrations against many clinically relevant pathogens for more than 48 h for one formulation and more than 5 days for the other. Both SDST formulations offer significant advantages over standard immediate-release therapy in achieving PK-PD goals and enhancing adherence. The prototypical formulations represent a novel platform which may be modified to meet various clinical requirements.

A novel subcutaneous controlled-release amoxicillin degradable implant for extended-interval administration in veterinary medicine.

Dosage forms of antimicrobials play a critical role in facilitating the attainment of pharmacokinetic-pharmacodynamic (PK-PD) targets as well as adherence in both veterinary and human medicine. The purpose of this study was to develop and evaluate a controlled-release subcutaneous amoxicillin implant for single-dose therapy of large ruminants such as goats, sheep, and deer. The degradable implant, designed to attain PK-PD targets following single administration, was evaluated for amoxicillin release rate and time-concentration profile. In vitro release studies demonstrated constant-rate release of approximately 40% of amoxicillin content within 96 h. In an in vivo study in goats, serving as a model for target animals, a serum concentration of approximately 0.4 mg/L was achieved within 8 h of implant insertion and maintained for >6 days. In comparison, in control goats given a standard single intramuscular amoxicillin dose of 15 mg/kg, amoxicillin peaked at 1.2 mg/L after 1 h, rapidly dropping to below detection level at 8 h. These results suggest that the proposed implant offers a unique modality for animal caregivers to conveniently administer a full antimicrobial course following a single dose of an efficient PK-PD-optimized dosage form. Furthermore, modifications of implant composition may allow for tailoring of its characteristics to various PK, PD, microbiological, and clinical requirements.

Culturing of diagnostic muscle biopsies as spheroid-like structures: a pilot study of morphology and viability.

OBJECTIVE: The aim of this study was to establish three-dimensional cultures originating from muscle biopsies and evaluate the viability and morphology. METHOD: Muscle biopsies from patients with suspected neuromuscular disorders were obtained and established as primary muscle tissue cultures. Tissue pieces, 1-2 mm of diameters, were placed in culture medium and subjected to sporadic stirring to prevent attachment and outgrowth as monolayer cells. Morphology and ability to attach to the surface were investigated by light microscopy. Viability was evaluated by (99m)Tc-tetrofosmin uptake. After 1 month, histology was evaluated by light microscopy and immunocytochemistry. The findings of a healthy muscle and a dystrophic muscle were compared. RESULTS: Initially, the tissue pieces were unshaped but formed spheroid-like structures during the culture period. For dystrophic muscle, attachment capacity to the surface was initially potent and decreased during the culture period, whereas control muscle showed weak attachment from the start that increased during the culture period. The uptake of (99m)Tc-tetrofosmin increased in control muscle, while it decreased in dystrophic muscle, during the culture period. The histological investigation demonstrated larger destruction of myofiber, weaker satellite cell activation and reduced myofiber regeneration in the dystrophic muscle as compared to the control muscle. CONCLUSION: The cellular components of the muscle tissue can survive and proliferate as spheroid-like primary cultures. The cellular composition resembles the in vivo condition, which allows studies of degeneration of the original fibers, and activation and proliferation of the satellite cells. The culture system may provide better understanding of the degeneration and regeneration processes in different muscle disorders and allow investigations of pharmacological interventions.

Effects of inhibitors of the arachidonic acid cascade on primary muscle culture from a Duchenne muscular dystrophy patient.

The aim of this study was to elucidate the mechanisms of action for potential targets of therapeutic intervention related to the arachidonic acid cascade in muscular dystrophy. Primary cultures from a Duchenne patient were used to study the expression of dystrophin-1, utrophin, desmin, neonatal myosin heavy chain (MHCn) and Bcl-2 during inhibition of phospholipase A2 (PLA2), cyclooxygenase (COX) and lipoxygenase (LOX). Hypo-osmotic treatment was applied in order to trigger Ca2+ influx and PLA2 activity. Inhibition of PLA2 and LOX with prednisolone and nordihydroguaiaretic acid (NDGA) caused a semi-quantitative increase of utrophin and Bcl-2-, and a dose-dependent, quantitative increase of desmin expression, an effect that was augmented by hypo-osmotic treatment. Our results indicate that LOX inhibitors, similarly to corticosteroids, can be beneficial in the treatment of muscular dystrophies.

Intracytoplasmic sperm injection with motile and immotile frozen-thawed testicular spermatozoa (the Hungarian experience).

The authors summarize their experience in 75 in vitro fertilization cycles, where frozen-thawed testicular spermatozoa were used for intracytoplasmic sperm injection. In 32 cases, motile spermatozoa could be observed in the frozen-thawed sample. In 34 cases, motility could be induced by pentoxifylline and in nine cases immotile spermatozoa, selected with hypoosmotic swelling test, were used for fertilization. The fertilization rates obtained with motile and immotile spermatozoa (66.1% versus 52.3%) were not significantly different. Our data demonstrate that freezing of testicular spermatozoa opened new possibilities for the treatment of azoospermic men. The clinical pregnancy rate per embryo transfer (ET) (21.87%) was comparable with previous results use of fresh testicular spermatozoa (27.7%). The quality and number of transferred embryos had the most significant impact on the pregnancy rate. The fertilization rate and frequency distribution of good-quality embryos were lower in the case of immotile spermatozoa, and pregnancies were only achieved when motile spermatozoa had been used.

Endothelial nitric oxide synthase gene interactions and the risk of ischaemic stroke.

OBJECTIVE: Endothelial nitric oxide synthase (eNOS), which produces NO, plays an important role in the endothelial function under a wide range of physiological conditions. eNOS exon 7 polymorphism (Glu298Asp, G894T) has been considered to influence the risk of coronary artery disease. Alone, however, it has not been shown to be a genetic risk factor for ischaemic stroke. With the assumption of additive interactions, we examined whether the eNOS G894T or eNOS 894TT genotypes in combination with the methylenetetrahydrofolate reductase 677TT (MTHFR 677TT) or angiotensin-converting enzyme (ACE) D/D genotype could contribute to acute ischaemic stroke. MATERIAL AND METHODS: The data on 407 consecutive patients with acute ischaemic stroke who had never suffered a previous stroke event were analysed. As a control group, 295 stroke and neuroimaging alteration-free Caucasian subjects were examined. With the use of the PCR technique, the eNOS G894T, eNOS 894TT, MTHFR 677TT and ACE D/D mutations, as unfavourable common genotypes were determined in the participants. Logistic regression models were used to evaluate the roles of the genotypes and their combinations in the development of ischaemic stroke. RESULTS: The MTHFR C677TT genotype combined with the eNOS G894T or eNOS 894TT genotypes occurred significantly more frequently in the subjects with ischaemic stroke (7.1%; P < 0.025) than in the control group (3.1%). The co-occurrence of the ACE D/D genotype and eNOS G894T or eNOS 894TT was calculated to be more frequent in the ischaemic stroke group (20.9%, P < 0.0001) than in the control group (5.4%). CONCLUSION: The eNOS G894T or eNOS 894TT genotypes in combination with the MTHFR 677TT or ACE D/D genotype increases the risk of ischaemic stroke.

Novel PDGFbetaR antisense encapsulated in polymeric nanospheres for the treatment of restenosis.

Nanospheres composed of the biocompatible and biodegradable polymer, poly-DL-lactide/glycolide and containing platelet-derived growth factor beta-receptor antisense (PDGFbetaR-AS) have been formulated and examined in vitro and in vivo in balloon-injured rat restenosis model. The nanospheres (approximately 300 nm) of homogenous size distribution exhibited high encapsulation efficiency (81%), and a sustained release of PDGFbetaR-AS (phosphorothioated). Cell internalization was visualized, and the inhibitory effect on SMC was observed. Partially phosphorothioated antisense sequences were found to be more specific than the fully phosphorothioated analogs. A significant antirestenotic effect of the naked AS sequence and the AS-NP (nanoparticles) was observed in the rat carotid in vivo model. The extent of mean neointimal formation 14 days after injection of AS-NP, measured as a percentage of luminal stenosis, was 32.21 +/- 4.75% in comparison to 54.89 +/- 8.84 and 53.84 +/- 5.58% in the blank-NP and SC-NP groups, respectively. It is concluded that PLGA nanospheres containing phosphorothioated oligodeoxynucleotide antisense could serve as an effective gene delivery systems for the treatment of restenosis.

Five years experiences with microinjection of testicular spermatozoa into oocytes in Hungary.

The aim of the study was to summarize our five years experience (1996-2000) of testicular spermatozoa for intracytoplasmic sperm injection in Hungary. The influence of sperm count, maternal age, number of transferred embryos, and application of assisted hatching on outcome was investigated. Testicular spermatozoa were retrieved by microsurgical testicular sperm extraction. Samples were classified depending on the number of spermatozoa. Indication for testicular sperm extraction in conjunction with intracytoplasmic sperm injection was severe azoospermia or azoospermia combined with tubal origin infertility. Ovarian stimulation was carried out using an ultrashort protocol with GnRH agonist and gonadotrophin. Intracytoplasmic sperm injection was performed without PVP. Embryos were cultured for 48 or 72 h before embryo transfer. Indications for assisted hatching included elevated maternal age, increased zona thickness or at least two previous unsuccessful IVF cycles. Testicular spermatozoa were successfully retrieved in 218 out of 273 cases. Extreme low sperm count was found more frequently in cases of nonobstructive azoospermia. No significant differences were observed in fertilization rate (61.1% vs. 51.7%) or clinical pregnancy rate (29.0% vs. 26.7%) between patients with obstructive or nonobstructive azoospermia. Maternal age, number of transferred embryos and application of assisted hatching had a significant effect on outcome. A total of 55 clinical pregnancies were achieved, including 14 sets of twins, three sets of triplets and two sets of quadruplets. It is concluded that testicular sperm extraction is an efficient way of obtaining testicular spermatozoa, allowing not only successful fertilization by ICSI, but also freezing of testicular spermatozoa for use in subsequent cycles.

Changes in the reducing power of synovial fluid from temporomandibular joints with "anchored disc phenomenon".

PURPOSE: In this study, we examined the hypothesis that uncontrolled oxidative stress causes collapse of the lubrication system, which is considered a major initiator of temporomandibular joint (TMJ) dysfunction. The oxidative stress was evaluated by measuring the overall antioxidant capacity of the low-molecular-weight antioxidants in the TMJ, using cyclic voltammetry (CV), in synovial fluid from normal and anchored disc phenomenon (ADP) TMJs. MATERIALS AND METHODS: Synovial fluids samples were taken from 13 normally functioning and 33 ADP TMJs. The samples were frozen initially on collection and analyzed in CV to measure their overall reducing power. RESULTS: CV measurements of the fluids collected from 90% of the healthy joints showed an anodic waves at peak potential [Ep(a)1/2] of 290 +/- 30 mV. Of the samples 56% showed another wave at 650 +/- 100 mV. These waves were generally absent in the fluid collected from ADP TMJs, but 2 new waves at 465 +/- 90 mV and greater than 750 mV were detected in 68% and 87% of the patients, respectively. CONCLUSIONS: The results show more anodic waves, most of which of higher potentials (greater than 750 mV) in ADP TMJs, indicating that the capacity to cope with oxidative stress is lower in these joints. It is not clear whether this is due to absence of low-molecular-weight antioxidants or their consumption by uncontrolled production of reactive oxygen species, which might be the initial step in the collapse of the lubrication system.

[Kennedy disease in a patient with progressive speech disorder]. / Kennedy-betegség egy progresszív beszédzavarban szenvedó férfiben.

Kennedy disease is an adult onset neuromuscular disease characterized by slowly progressive proximal and bulbar muscle weakness. The disease associates with gynecomastia, adult onset infertility and sensory neuropathy, and caused by pathologic expansion of CAG repeats at the N-terminal region of the androgen-receptor gene at Xq11-q12. We report on a patient presenting with slowly progressive muscle weakness of the lower extremities, progressive dysartry and swallowing difficulties. The clinical symptoms were not fully specific for the disease. Moreover the family history was suggestive for an autosomal dominant trait meaning a diagnostic pitfall at the original examination. Finally the firm diagnosis of the Kennedy disease was established by a polimerase chain reaction based method.

Formulation and delivery mode affect disposition and activity of tyrphostin-loaded nanoparticles in the rat carotid model.

Poor drug residence in the arterial wall hinders clinical implementation of local drug delivery strategies for the treatment of restenosis. A rat carotid model of vascular injury and intraluminal delivery of tyrphostin-containing polylactic acid (PLA) nanoparticles (NPs) were used to determine the relationship between residence properties and biological activity of different formulations and administration modes. The effects of delivery modes (denudation and delivery time) and formulation variables (adsorbed vs encapsulated drug, and NP size) on arterial drug/NP retention were examined. Antirestenotic effects of large (160 nm) and small (90 nm) tyrphostin-containing NPs, surface-absorbed tyrphostin, and systemic treatment were compared. Fluorescent NPs were used to study the spatial distribution of the carrier in the arterial wall. The decrease in arterial tyrphostin level over time fitted a biexponential model. Delivery time and pressure, endothelium integrity, particle size, and drug-polymer association affected local pharmacokinetics and the antirestenotic results after 14 days. The PLA-based tyrphostin NP formulation ensured a prolonged drug residence at the angioplasty site after single intraluminal application. Several readily adjustable formulation and procedural factors considerably modified arterial ingress of the drug-loaded NPs and governed their subsequent redistribution, tissue binding, elimination, and ensuing antirestenotic effect.

Heat shock protein 60 in corpora amylacea.

Heat shock protein 60 representation in the corpora amylacea of the brain was investigated in five different neurological diseases. In the cases with cerebral infarct, amyotrophic lateral sclerosis, multiple sclerosis, acute disseminated encephalomyelitis and primary tumors of the nervous system the corpora amylacea showed similar appearance with strong HSP-60 positivity in all investigated disorders at the predilection sites. In the inflammatory diseases, besides corpora amylacea, several cellular elements exhibited HSP-60 immunostaining too. In these cases, the widespread HSP-60 immunoreactivity associated with relative moderate corpora amylacea production as compared to other diseases. From this contradiction we concluded the corpora amylacea participate in the cellular stress reaction but stress protein synthesis certainly is not the primary event in corpora amylacea formation. In the development of the corpora amylacea the incipient process is most probably degenerative in nature, which later on is accompanied by stress protein synthesis and slow growing of these round structures designated for a protective role in the brain. However, the role of the stress protein synthesis in the corpora amylacea formation and growth was not unequivocally answered in this study. It is necessary to perform further comparative investigations of the stress protein representation and corpora amylacea formation in different diseases which may help in discovering useful pathogenetic data and the biological role of this degenerative structure.

Lead accumulation in human ovarian follicular fluid, and in vitro effect of lead on progesterone production by cultured human ovarian granulosa cells.

Lead content of ovarian follicular fluid obtained from 23 women was determined by atomic absorption spectrophotometry. In an in vitro experiment the direct effect of lead on the morphology and on progesterone (P) production by cultured granulosa cells of six women was investigated. Follicular fluid and granulosa cells were obtained from follicular aspirates of women undergoing in vitro fertilization (IVF) and embryo transfer (ET). Granulosa cells were cultured for 48 h to form monolayers in the presence or absence of lead acetate (100-1,600 microM). The effect of the metal proved to be concentration dependent. While 100-400 microM lead had no effect on the integrity of the monolayer, concentrations as high as 800 microM or higher inhibited cell adhesion and induced detachment of cells. The lead levels found in follicular fluid were 11.29 +/- 1.38 microg/L (0.056 +/- 0.007 microM). With lead in vitro at 1,600 microM (331.5 mg/L) there resulted a significant decrease in P production by granulosa cells. This concentration is very much higher than that measured in follicular fluid of IVF/ET patients, specifically nonexposed to lead, and even higher than mean blood levels reported by others in high exposure groups. In conclusion, lead seems not to exert a specific effect on the steroidogenesis by cultured human granulosa cells. Therefore, the lead levels measured in the ovarian follicular fluid seem not to pose a hazard with respect to progesterone secretion by the ovary.

Fluorescence in situ hybridization of chorionic interphase cells for prenatal screening of Down syndrome.

OBJECTIVE: Our purpose was to determine the usefulness and reliability of fluorescence in situ hybridization on interphase chorionic villi cells in the prenatal diagnosis of Down syndrome. METHODS: A total of 336 samples of chorionic villi were analysed by direct chromosome preparation and FISH with a DNA probe specific to chromosome 21. The samples were obtained as part of the routine obstetric investigation and management. RESULTS: The sampling and direct karyotyping was successful in all cases. At least 50 cells were valuable by FISH in 331 of 336 samples. Both methods showed Down syndrome in 12 cases. The follow-up investigations showed that there was no false-negative or false-positive result following these procedures. CONCLUSION: Based on these results and the fact that it is possible to analyse by interphase FISH at least ten times more cells than by conventional cytogenetic methods, and these cells originate from different tissues of chorionic villi, it is concluded that FISH increases the reliability of the diagnosis. Nevertheless, more data are needed for correct statistical analysis. Since this method is cheaper and gives diagnosis earlier than cell culture, the combination of direct chromosome preparation and FISH on chorionic villi is offered for prenatal Down syndrome screening.

Neurofibromatosis and glioblastoma in a case of multiple sclerosis.

The case of a 37-year-old woman is presented. Cutaneous neurofibromatosis was associated with a progressive course of multiple sclerosis. Unexpectedly, autopsy revealed a right hemispheric glioblastoma which was silent during her life.

A peptide prodrug approach for improving bisphosphonate oral absorption.

This work was aimed at improving the absorption of bisphosphonates by targeting carrier systems in the intestine and the intestinal peptide carrier system (hPEPT1), in particular. (14)C-Labeled pamidronate and alendronate as well as radiolabeled and "cold" peptidyl-bisphosphonates, Pro-[(3)H]Phe-[(14)C]pamidronate, and Pro-[(3)H]Phe-[(14)C]alendronate were synthesized. In situ single-pass perfusion studies revealed competitive inhibition of transport by Pro-Phe, suggesting peptide carrier-mediated transport. Prodrug transport in the Caco-2 cell line was significantly better than that of the parent drugs, and the prodrugs exhibited high affinity to the intestinal tissue. Oral administration of the dipeptidyl prodrugs resulted in a 3-fold increase in drug absorption following oral administration in rats, and the bioavailability of Pro-Phe-alendronate was 3.3 (F(TIBIA)) and 1.9 (F(URINE)) times higher than that of the parent drug. The results indicate that the oral absorption of bisphosphonates can be improved by peptidyl prodrugs via the hPEPT1; however, other transporters may also be involved.

Skin low molecular weight antioxidants and their role in aging and in oxidative stress.

The overall skin low molecular weight antioxidant (LMWA) capacity was evaluated during the aging process and following exposure to oxidative stress. Several invasive and non-invasive techniques were developed for evaluating total antioxidant activity. It was found that the skin possesses an extremely efficient and unique antioxidant activity that is better than other tissues. During the aging process a significant decrease in the levels and activity of the water-soluble LMWA was detected while no change and even a slight increase was recorded for the lipophilic LMWA. Similar results were obtained following exposure to oxidative stress. A significant decrease in the water soluble LMWA was recorded in all the stress induced procedures indicating a common mechanism of response. It has also been shown that along with the reduction in total water soluble antioxidant activity there is an accumulation of oxidized adducts. This was observed both on the surface of the skin and in deeper layers. It has been found that skin releases LMWA from its surface. This secretion phenomenon was found to be age dependent. Following exposure to oxidative stress of various kinds, the release of LMWA from the skin was significantly enhanced. This may suggest a physiological mechanism of the skin to cope with oxidative stress, which would open new possibilities for intervention.

Quantification of the overall reactive oxygen species scavenging capacity of biological fluids and tissues.

A method has been developed for measuring and evaluating the overall antioxidant activity derived from the low-molecular weight antioxidants (scavengers). The principle governing this method is based on a common chemical characteristic of the scavengers, their reducing properties. It was hypothesized and then demonstrated that an evaluation of the overall reducing power of a biological sample correlates with the overall scavenging activity of the sample. In order to quantify the total reducing power, the cyclic voltammetry methodology was applied. The resulting measurements correlated with the antioxidant activity of both hydrophilic and lipophilic scavengers. The method is suitable for use in biological fluids and in tissue homogenates, and can supply information concerning the type of antioxidants and their total concentration without having to determine specific compounds. A noninvasive procedure for determining skin overall scavenging activity is also described. This method is based on a well containing an extraction solution that is attached to the skin's surface. Following incubation time the extraction solution is analyzed using the cyclic voltammeter instrument and other methods. We have found these methods suitable for evaluating the reducing capacity status in various clinical conditions such as diabetes, ionizing and nonionizing irradiation, brain degenerative diseases, head trauma, and inflammatory bowel diseases. This method is also an efficient tool for evaluating the overall antioxidant capacity of mixtures of antioxidant preparations in vitro. The measurements themselves are simple and rapid. Furthermore, they do not require manipulation of the samples.

Nanoparticulate delivery system of a tyrphostin for the treatment of restenosis.

Restenosis, the principal complication of percutaneous transluminal coronary angioplasty is responsible for the 35-40% long-term failure rate following coronary revascularization. The neointimal formation, a morphological substrate of restenosis, is dependent on smooth muscle cells (SMC) proliferation and migration. Signal transduction through the platelet-derived growth factor (PDGF)/PDGF receptors system is involved in the process of post-angioplasty restenosis. The unsuccessful attempts to control restenosis by systemic pharmacological interventions have prompted many researchers to look for more promising therapeutic approaches such as local drug delivery. Tyrphostins are low molecular weight inhibitors of protein tyrosine kinases. We assessed the release kinetics and in vivo effects of nanoparticles containing PDGF-Receptor beta (PDGFRbeta) tyrphostin inhibitor, AG-1295. AG-1295-loaded poly(DL-lactide) (PLA) nanoparticles were prepared by spontaneous emulsification/solvent displacement technique. In vitro release rate and the impact of drug/polymer ratio on the nanoparticle size were determined. The degree of tyrosine phosphorylation was assessed by Western blot with phosphotyrosine-specific antibody in rat SMC extracts. Several bands characteristic of PDGF BB-stimulated SMC disappeared or weakened following tyrphostin treatment. Local intraluminal delivery of AG-1295-loaded PLA nanoparticles to the injured rat carotid artery had no effect on proliferative activity in medial and neointimal compartments of angioplastisized arteries, indicating a primary antimigration effect of AG-1295 on medial SMC.

Orienting response reinstatement and dishabituation: effects of substituting, adding, and deleting components of nonsignificant stimuli.

The prediction that orienting response (OR) reinstatement is negatively related to the measure of common features, shared by the stimulus input and representations of preceding events, and positively related to the measure of their distinctive features, was examined. A nonsignificant test stimulus (TS) was presented after nine repetitions of a standard stimulus (SS), followed by two additional repetitions of SS. TS was created by either substituting 0, 1, or 2 components of SS (Experiment 1), or by either adding or deleting 0, 1, or 2 components of SS (Experiment 2). Skin conductance changes to TS (OR reinstatement) and the subsequent SS (dishabituation) were used as dependent measures. The results of Experiment 1 supported the prediction that substituting components of neutral stimuli affects OR reinstatement, with a larger effect for between-categories than within-categories substitution. Experiment 2 demonstrated that adding and deleting components similarly affects OR reinstatement.