Evaluation of mTHPC-loaded PLGA nanoparticles for in vitro photodynamic therapy on C6 glioma cell line.

Photodynamic therapy (PDT) is a very attractive strategy to complement or replace common cancer treatments such as radiotherapy, surgery, and chemotherapy. Some molecules have shown their efficiency as photosensitizers (PS), still many issues have to be solved such as the inherent cytotoxicity of the PS or its hydrophobic properties causing limitation in their solubility, leading to side effects. In this study, the encapsulation of an approved PS, the meso-tetra hydroxyphenylchlorine (mTHPC, Foscan®) within biocompatible and biodegradable poly(D, l-lactide-co-glycolide) acid (PLGA) NPs prepared by the nanoprecipitation method was studied. The mTHPC-loaded NPs (mTHPC ⊂ PLGA NPs) were analyzed by UV-vis spectroscopy to determine the efficiency of mTHPC encapsulation, and by dynamic light scattering (DLS) and atomic force microscopy (AFM) to determine mTHPC ⊂ PLGA NPs sizes, morphologies and surface charges. The longitudinal follow-up of mTHPC release from the NPs indicated that 50% of the encapsulated PS was retained within the NP matrix after a period of five days. Finally, the cytotoxicity and the phototoxicity of the mTHPC ⊂ PLGA NPs were determined in murine C6 glioma cell lines and compared to the ones of mTHPC alone. The studies showed a strong decrease of mTHPC cytotoxicity and an increase of mTHPC photo-cytotoxicity when mTHPC was encapsulated. In order to have a better insight of the underlying cellular mechanisms that governed cell death after mTHPC ⊂ PLGA NPs incubation and irradiation, annexin V staining tests were performed. The results indicated that apoptosis was the main cell death mechanism.

Influence of schedule of administration on methotrexate penetration in brain tumours.

The influence of the administration schedule (intravenous (i.v.) bolus versus i.v. infusion) on the pharmacokinetics of methotrexate (MTX) in plasma and extracellular fluid (ECF) of a brain C6-glioma was investigated in rats. MTX concentrations were determined by high performance liquid chromatography (HPLC)-ultraviolet radiation (UV). MTX (50 mg/kg) was administered by i.v. bolus or i.v. infusion (4 h). Concentration-time profiles were fitted to a two-compartment open model. Maximum MTX concentrations ranged between 178 and 294 microgram/ml (i.v. bolus), and between 11 and 24 microgram/ml (i.v. infusion) in plasma. MTX rapidly entered the tumour tissue although its concentrations in the ECF were much lower than those observed in plasma for both modes of administration. In spite of an important interindividual variability, AUC(ECF) was approximately 5-fold higher and mean MTX penetration in tumour ECF (AUC(ECF)/AUC(Plasma)) was approximately 3-fold higher after i.v. bolus than after i.v. infusion administration. These results indicate that i.v. bolus administration schedules promote MTX delivery in brain tumour tissue.

Distribution of gacyclidine enantiomers after experimental spinal cord injury in rats: possible involvement of an active transport system.

The pharmacokinetics of gacyclidine enantiomers, a noncompetitive N-methyl-D-aspartate (NMDA) antagonist, were studied in plasma and spinal cord extracellular fluid (ECF) after experimental spinal cord injury in rats. Spinal cord trauma was produced by introducing an inflatable balloon in the dorsal subdural space. Upon implantation of microdialysis probes in spinal cord (T9) and intravenous (iv) bolus administration of (+/-)-gacyclidine (2.5 mg/kg), concentrations in plasma and ECF were monitored over 5 h and analyzed by a stereospecific gas chromatography-mass spectrometry (GC-MS) assay. In plasma, concentrations of (+)-gacyclidine were approximately 25% higher than those of (-)-gacyclidine over the duration of the experiment and decayed in parallel (t(1/2 alpha) approximately 7 min; t(1/2 beta) approximately 90 min) with no significant difference between the two enantiomers. Clearance (CL) and volume of distribution (Vd) of (-)-gacyclidine were approximately 20% higher than those of its optical antipode (CL: 285 versus 236 mL. kg(-1). min(-1); Vd(beta): 39.3 versus 31.2 l/kg). Protein binding (approximately 91%) was not stereoselective. In spinal cord ECF, both enantiomers were quantifiable within 10 min after drug administration, and their concentration remained stable over the duration of the experiment in spite of changing blood concentrations. Repeated iv bolus injections of gacyclidine did not modify these profiles. Areas under the curves (AUCs) of concentration in ECF versus time were similar for both enantiomers and not correlated with AUCs in plasma. Penetration of (-)-gacyclidine was, however, significantly higher (approximately 30%) than that of (+)-gacyclidine. In summary, the disposition of gacyclidine enantiomers is stereoselective. Both enantiomers exhibit a high affinity for spinal cord tissue, and the drug exchange between plasma and spinal cord ECF involves an active transport system. These findings contribute to the explanation of the discrepancy between drug concentrations in plasma and spinal cord ECF.

Pharmacokinetics of gacyclidine enantiomers in plasma and spinal cord after single enantiomer administration in rats.

The purpose of this study was to determine the pharmacokinetics of gacyclidine, a non-competitive NMDA antagonist, in plasma and spinal cord extracellular fluid (ECF) after IV administration of single enantiomers in rats. After implantation of microdialysis probes in spinal cord, concentrations in plasma and ECF dialysates were determined by a chiral GC/MS assay over 5 h after administration of either (+)-gacyclidine or (-)-gacyclidine (1.25 mg/kg). Plasma protein binding was estimated in vitro by equilibrium dialysis. Plasma concentrations decayed in parallel in a biphasic manner (t(1/2)alpha approximately 9 min; t(1/2)beta approximately 90 min) with no significant difference between the two enantiomers. Clearance of (+)-gacyclidine and (-)-gacyclidine (291 versus 275 ml/min per kg, respectively), volume of distribution (Vdbeta: 38 versus 40 l/kg), and protein binding (90 versus 89%) were not stereoselective. Both gacyclidine enantiomers were quantifiable in spinal cord ECF 10 min after drug administration and their concentrations remained stable over the duration of the experiment in spite of changing blood concentrations. Penetration of the two enantiomers in spinal cord ECF was similar although highly variable between animals. Exposure of spinal cord ECF was comparable for both enantiomers, and not correlated with plasma AUCs. This study showed the absence of any pharmacokinetic difference between the two enantiomers when administered individually, and no enantiomeric inversion. Both gacyclidine enantiomers penetrate rapidly and extensively into spinal cord ECF, and their distribution may involve an active transport system.

Synthesis of several substituted phenylpiperazines behaving as mixed D2/5HT1A ligands.

Twenty-two different compounds have been synthesized with the aim of creating new, mixed D2/5HT1A ligands. For this purpose 1-substituted phenylpiperazines attached by the N-4 nitrogen to dopaminergic pharmacophores of the 2-(5-benzimidazole)ethyl-, 2-(5-benztriazole)ethyl-, 2-[5-(benzimidazole-2-thione)]ethyl- and 2-[6-(1,4-dihydroquinoxaline-2,3-dione)]ethyl-type were selected according to known structure-affinity requirements of 1-arylpiperazines. All the new compounds were evaluated for in-vitro binding affinity at the dopamine (D1 and D2) and 5-HT1A receptors. Synaptosomal membranes prepared from fresh bovine caudate nuclei and hippocampi were used as a source of dopamine and 5-hydroxytryptamine receptors, respectively. [3H]SCH 23390 (D1 selective), [3H]spiperone (D2 selective) and 8-OH-[3H]DPAT (5-HT1A selective) were employed as the radio-ligands. None of the compounds expressed affinity for binding at D1 dopamine receptors. Compounds 3b and 4b were inactive 8-OH-[3H]DPAT competitors whereas 1b, 2b and 4b were inactive in the [3H]spiperone-binding assay. The other compounds tested showed fair (1c, 1e, 1f, 2c, 2f, 3b, 3c and 4c) to high (1a, 1d, 2a, 1d, 3a, 3d-3f, 4a, and 4d) affinity in the [3H]spiperone-binding assay, the most potent representative being 4-[2-(5-benzimidazole-2-thione)ethyl]-1-(2-methoxyphenyl)piperazine, 3a (Ki = 1.7 nM). In the 8-OH-[3H]DPAT-displacement assay compounds 1b, 1d, 1f, 2b, 2f and 3f behaved as moderate competitors and 1a, 1c, 1e, 2a, 2c, 2d, 3a, 3c-3e, 4a, 4c, 4d and 4f as rather strong competitors; 4-[2-(5-benztriazole)ethyl]-1-(2-methoxyphenyl)piperazine, 2a had the highest binding affinity at the 5-HT1A receptors (Ki = 2.6 nM). Because many antipsychotic and anxiolytic agents behave as mixed dopaminergic and serotonergic ligands, the high affinity of several of these new ligands for binding at both D2 and 5-HT1A receptors make them promising candidates deserving further pharmacological evaluation as antipsychotic or anxiolytic pharmaceuticals.

[The (18)(q 22----qter) deletion in patients with complete clinical features of the De Grouchy syndrome]. / Delecija--(18) (q 22----qter) u pacijenta sa kompletnom klinickom slikom sindroma De Grouchy.

A case of a 4-year old boy with de Grouchy syndrome was reported. The patient showed generalised muscular hypotonia, marked mental retardation (RQ = 30), developmental milestones retarded, craniofacial dysmorphic features, congenital heart disease, abnormalities of the external genitalia and skeletal deformities. The karyotype analysis revealed a partial deletion of the distal bands of chromosome 18:48 48 xy del (18) (q 22----qter). Qualitative and quantitative characteristics analysis of digital and palmar dermatoglyphics supported the diagnosis.

[Colonization of cental venous catheter in the Intensive Care Units at the Institute of Cardiovascular Disease "Dedinje"--one year study].

OBJECTIVES: The application of Central Venous Catheters (CVC) is associated with increased risk of microbial colonization and infection. The aim of present study was to assess the frequency of pathogens colonizing CVC and to determine their susceptibility pattern to various antimicrobial agents. MATERIALS AND METHODS: A total of 253 samples of CVC from intensive care units (ICU) patients were received for culture during 2003. All microorganisms were identified by standard microbiological methods and the susceptibility to antimicrobial agents was determined according to NCCLS recommendations. RESULTS: A total of 184 (72.7%) cultures were positive and 223 pathogens were isolated. Coagulase negative staphylococci (CNS) were the dominant isolates (24.7%), followed by Enterobacter spp. (12.1%), Pseudomonas spp. (11.7%), Enterococcus spp. (9.9%), Klebsiella spp. (8.6%), Candida spp. (7.6%), Acinetobacter spp. (7.6%), other Gram negative nonfermentative bacilli (5.8%), Serratia spp. (4.5%), Staphylococcus aureus (2.6%), Proteus mirabilis (2.2%), E. coli (1.8%) and Citrobacter spp. (0.9%). Meropenem (84.5%) and vancomycin (100%) remain the most effective antimicrobial agents against Gram negative and Gram positive bacteria, respectively. CONCLUSION: Gram negative bacilli and CNS are the commonest microorganisms colonizing CVC from ICU patients. The increasing resistance of the bacteria to antimicrobial agents is the major problem in spite of restricted policy of using antimicrobial agents in ICU.

[Dr. Koloman (Kalman) Kalivoda, a physician from Senta and a participant in the first congress of Serbian physicians and naturalists in 1904]. / Dr Koloman (Kalman) Kalivoda, lekar iz Sente, ucesnik prvog kongresa Srpskih lekara i prirodnjaka 1904. godine.

INTRODUCTION: The first congress of Serbian physicians and naturalists was held in Belgrade from September 5-7, 1904. There were 433 active registered participants listed in the Collection of Congress Papers. A great number of physicians from Vojvodina took part in the work of Congress. There is an authentic list of 27 physicians participants from Vojvodina. Although they made only 6% of all participants, their attendance was of utmost importance due to delicate political situation of that time. However, there is no explanation why only one physician from Vojvodina read his paper at this meeting. It was Dr. Koloman Kalivoda from Senta and his paper was tittled "Alcoholism and People". CONGRESS REPORT: Dr. Koloman (Kalman) Kalivoda was probably born between 1870 and 1875. He moved to Senta in 1901 and in September 1901 started working as a regional physician in the third area. From 1908 he was a member of the Medical Board in Senta. He worked in Senta till October 31, 1918 when he submitted resignation from the post of a regional physician at the Assembly meeting. Then he moved from Senta and started civil service as a Hungarean Royal Resort physician. This paper presents Dr. Koloman's paper in complete, published in the Collection of Congress Papers. This paper received great attention and praises were found at several places such as: journal of "Czech Physicians", number 44 from 1904; "Serbian Literary Herald", volume XIII, number 3 and in journal called "Pozor" from Moravska, number 146 from 1904. Further on, authors comment on Dr. Koloman's attitudes to alcoholism and treatment of alcoholics. Some were estimated as drastic: "the role of alcohol in rase cleansing"; how to "exterminate" alcoholics; suggestions for "interning drunkards overcome by fury" and suggestions for passing a law which would forbid drunkards to get married. Such attitudes of Dr. Koloman Kalivoda towards alcoholism may be associated with the circumstances of that time in Senta, because there are reliable data according to which in 1894 there were 26,648 inhabitants in Senta, whereas one brandy taproom or tavern came on every 213 inhabitants. Contrary to these attitudes, authors think that Dr. Koloman had very progressive ideas in regard to prevention of alcoholism as well as some suggestions and measures that should be undertaken by the society from the aspect of public hygiene.

[Cerebellar infarct with complications]. / Infarkt malog mozga sa komplikacijama.

INTRODUCTION: Symptoms of circulatory disorders depend on the volume and localization of the cerebellar infarction. An isolated blood vessel occlusion may have various symptoms due to numerous anastomoses in the cerebellar hemispheres. Dominant symptoms are vertigo, vomiting, nausea and balance disorders. Due to a great number of anastomoses between the three cerebral arteries, if only one of them is occluded, expected symptoms rarely occur, so that vertebral angiography performed in the first 24 hours from the onset of clinical picture, points to a lesion of the cerebellar parenchyma on the basis of radiologic signs of occlusion. Further on, brain computerized tomography solves the diagnostic dilemma, but unfortunately, ischemic cerebellar lesions are often detected only in pathoanatomic findings. Development and course of cerebellar malformation are of great importance for occurrence of symptoms. Cerebellar symptoms often occur in cases of brain-stem lesions. In dorsal hemisection lesions the following symptoms occur: vertigo, vomiting, nystagmus, taste disorders and secretory trophic disorders in the gastrointestinal tract. In cases of spreading to the pyramidal neurons the following symptoms occur: a soft palate and vocal cord paresis, swallowing difficulties and disorders of phonological as well as hemiplegia and sensibility disorders. CASE REPORT: This is a case of a male patient, 60 years of age. He was admitted to the Neuropsychiatric Department because of high blood pressure, severe headache in the back of the head, vertigo and nausea, without vomiting. He complained about weakness in the left side of the body. The somatic status was normal. In regard to neurologic status he had a slow photomotoric reaction of pupils to light, swallowing difficulties, hoarse voice, decreased pharyngeal reflex, symmetrically fast reflexes, walk on wide basis, in Romberg position unstable, whereas other findings were normal. Psychically average. The reaction to therapy was good. Subjectively the patient felt-well, but swallowing difficulties persisted. On the seventh day from admission his status suddenly aggravated, with severe vertigo, vomiting on several occasions, weakness and high temperature, hypotension, tachycardia, left eyelid ptosis with horizontal nystagmus when looking to the left. Lung radiography revealed bilateral pneumonia; computerized tomography of the brain revealed a great ischemic zone in the left cerebellar hemisphere discretely dislocating the brain mass to contralateral side. Resistant hypotension persisted. Surgical findings of the rectum revealed a dark content. Gastroscopy revealed esophageal candidiasis; mucus bleeding was spontaneous and at touch, whereas in the lower third of esophagus there was a mucous lesion. Haematinised blood was found in the stomach and duodenum. The patient's state continually aggravated and after massive stool blood loss a surgery was performed. A great penetrating ulcer was detected at the back duodenal wall 30 mm of size, visualizing the pancreas. A final surgical procedure was performed at the Clinic for Abdominal Surgery in Novi Sad. The postoperative course was good, but on the 7th postoperative day unexpected fatal outcome occurred due to massive lung embolism. Pathoanatomic examination revealed a postencephalomalacic pseudocyst in the left cerebellar hemisphere. CONCLUSION: This is a case report of a patient with lateral medullar syndrome associated with complications which can only partly be explained by the basic disease. Pneumonia and a secretory trophic disorder of the gastrointestinal tract are frequent complications that can be life threatening is spite of intensive management.

Structure-affinity relationship studies on D-2/5-HT1A receptor ligands. 4-(2-Heteroarylethyl)-1-arylpiperazines.

With an aim of creating new, mixed D-2/5-HT1A ligands, sixteen different compounds were synthesized. For this purpose 1-arylpiperazines attached through N-4 nitrogen to the dopaminergic pharmacophores of 2-(5-benzimidazole)-ethyl-, 2-(5-benztriazole)-ethyl-, 2-[5-(benzimidazole-2-thione]-ethyl- and 2-[6-(1,4-dihydroquinoxaline-2,3-dione)]-ethyl-type were selected according to their structural diversity (phenyl, substituted phenyl, heteroaryl and naphthyl). All new compounds were checked for in vitro binding affinity at the dopamine (D-1 and D-2) and serotonin (5-HT1A) receptors. Synaptosomal membranes prepared from fresh bovine caudate nuclei and hippocampus were used as a source of dopamine and serotonin receptors, respectively, [3H]SCH 23390 (D-1 selective), [3H]spiperone (D-2 selective) and 8-OH-[3H]DPAT (5-HT1A selective) were employed as the radioligands. All compounds were inactive competitors of [3H]SCH 23390, 1c and 2b being inactive in the two other binding assays, as well. Derivatives of 1-(2-pyridyl)piperazine 1b and 2a and of 1-phenylpiperazine 1a expressed moderate to low affinity for both D-2 and 5-HT1A receptors, while 1-(2-pyridyl)-piperazines 3b and 4b and 4-(1-phenylethyl)-1-(1-naphthyl)-piperazine 10 were moderate [3H]spiperone, but potent 8-OH-[3H]DPAT competitors. Among them, derivatives of 1-(1-naphthyl)-piperazine 1e, 2d and 3e and of 1-(2,3-dimethylphenyl)-piperazine 1d, 2c and 3d were the strongest competitors at both D-2 and 5-HT1A receptors.

Synthesis and dopaminergic properties of 3- and 4-substituted 1-[2-[5-(1H-benzimidazole-2-thione)]ethyl]piperidines and related compounds.

With an aim of creating new, high affinity dopaminergic ligands, six different 3- and 4-substituted 1-[2-[5-(1H-benzimidazole-2-thione)] ethyl]piperidines and nine related heterocyclic congeners were synthesized and evaluated for in vitro binding affinity at D1 and D2 dopamine receptors. Synaptosomal membranes prepared from fresh bovine caudate nuclei were used as a source of the dopamine receptors. Only 4-[bis-(4-fluorophenyl)methylene]-piperidines, compounds 9e, 10d, and 11d, expressed moderate affinity for the D1 receptors, while all other compounds were inactive competitors of [3H]SCH 23390. Compounds 9c, 9d, 10c, 11a, and 11c were inactive in the D2 receptor binding assay, as well. Derivatives of 4-phenylpiperidine (9-11b) and 3-phenylpiperidine (10a) expressed a moderate to low affinity for the D2 receptors. However, racemic (+/-)-1-[2-[5-(1H-benzimidazole-2-thione)] ethyl]-3-phenylpiperidine 9a and its enantiomer (+)-9a behaved as selective, high affinity D2 receptor ligands, the latter being some four times more active than the racemate.

Synthesis of N-n-propyl-N-(2-arylethyl)-5-(1H-benzimidazol-2-thione)-eth ylamines and related compounds as potential dopaminergic ligands.

Twelve different N-n-propyl-N-(2-arylethyl)-5-(1H-benzimidazol-2-thione)-ethy lamines and 12 related heterocyclic congeners were synthesized with the aim of creating new high affinity dopaminergic ligands. Upon thorough chemical analysis they were evaluated for their affinity towards the D-1 and the D-2 dopamine receptors (DAR) by in vitro binding assay using synaptosomal membranes of the bovine caudate nuclei and [3H]SCH 23390 (R (+)-7-chloro-8-hydroxy-3-methyl-1-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride) (D-1 selective) and [3H]spiperone (D-2 selective) as the radioligands. None of the synthesized compounds expressed affinity for the binding to the D-1 DAR Compounds 11e, 11j, 11l, 12b, 12d, 13a, 13d, 14a, and 14d were also inactive competitors of [3H] spiperone binding to the D-2 DAR. However, 1H-benzimidazol-2-thione derivatives 11k, 11h, and 11f and 1, 4-di-hydroquinoxalin-2,3-dione derivative 12c (in this rank order of potencies) acted as strong competitors of [3H] spiperone binding to the D-2 receptors under conditions that prevented radioligand binding to serotonin 5HT(2) receptors. Relationship between the structure and affinities of the new ligands for the binding to the D-2 DAR as well as the demands of the receptor itself for the interaction with the new compounds are discussed.

[Indicators of hospital treatment and the organization and management of injuries and diseases in armed combat in 1991 and 1992]. / Neki pokazatelji bolnickog lecenja i organizacije zbrinjavanja povredenih i obolelih u oruzanim sukobima 1991/92. godine.

Efficiency of organisation of management of the injured and diseased in combat operations in 1991/2 and results of hospital treatment are studied. The questionnaire was established for collection of data about stationary treatment of the wounded and diseased in 7 military and 15 civilian medical institutions. Data about 9.243 injured of whom only 5.57% were civilians are especially analysed. In most cases injuries were inflicted by mine-explosive devices (48.77% in combat and 6.27% in non-combat operations), then those inflicted by bullets (38.58% in combat and 8.02% in non-combat operations). Self-inflicted injuries occurred in 5.79% in combat and 10.32% in non-combat operations. Injuries inflicted in non-combat operations were found in a high percentage (46.60). They mostly occurred in the open air (41.26%). Injuries of extremities are predominant (64.70%), then injuries of the head and neck (17.90%), thorax (9.90%), abdomen and pelvis (7.50%). Young men (20-24 years old) were commonly injured but the older ones (over 40 years) have been also frequently injured (18.30%). Evacuation of the wounded and diseased was performed mainly by ambulances (about 60%) or helicopters (16%), especially to the medical institutions far in the country (to M.M.A. up to 60%). The first aid was given within the first ten minutes after injury in 55.00% and within 30 minutes in 65.58% of cases. Mutual aid was most frequently applied (33.12%) and then first aid given by general practitioners (32.12%). Surgical treatment was provided one hour after injury to every fourth wounded (24.08%) and after six hours to more than one third of the wounded (31.88%).(ABSTRACT TRUNCATED AT 250 WORDS)

[Intrathecal synthesis of immunoglobulin G in patients of various ages with acute viral meningitis and meningoencephalitis]. / Intratekalna sinteza imunoglobulina G kod bolesnika razlicitog zivotnog doba u toku akutnih virusnih meningitisa i meningoencefalitisa.

Intrathecal antibody synthesis is a common local immunologic reaction in acute meningoencephalitis and encephalitis caused by Herpes group viruses, myxo and paramyxoviroses. The purpose of this study was to determine the quantity of de novo synthesized IgG during 24 hours in the liquor of patients of different ages suffering from acute viral meningitis and meningoencephalitis and to establish correlations between the dysfunction of blood-brain barrier and de novo IgG synthesis. We examined 73 patients of different ages divided into 5 groups according to their age. The state of blood-brain barrier was the most stable with high Qalb values of 225,596 for the youngest (from 2 to 6) and 193,190 for the somewhat older children (from 7 to 15). The most common damage of the blood-brain barrier was established in the oldest group of patients--over 40 years of age. The lowest intrathecal IgG production was established in the youngest group from 2 to 6 (6.631 mg/24 hours), and the highest (64.61 mg/24 hours) at the beginning of the disease in the group from 16 to 25 years of age. We established a correlation between damages of blood-brain barrier and de novo synthesis of IgG, especially in the youngest patients in the first days of the disease as well as 14 and more days later, when a low immunoglobulin production was established but a well preserved blood-brain barrier too. In the group of patients older than 40 years of age such a correlation was not found which points to a tissue synthesis of antibodies in the central nervous system.

Determination of free extracellular levels of methotrexate by microdialysis in muscle and solid tumor of the rabbit.

PURPOSE: To determine of the pharmacokinetic profile of methotrexate (MTX) in blood and extracellular fluid (ECF) of VX2 tumor and muscle in rabbits. METHODS: Microdialysis probes were inserted into VX2 tumor and in muscle tissue. Following intravenous administration of MTX (30 mg/kg), serial collection of arterial blood samples and dialysates of muscle and tumor ECF for 4 h was carried out. Quantitation of MTX and determination of free plasma concentrations was performed by fluorescence polarization immunoassay and ultrafiltration, respectively. Correlations were established between the unbound plasma and ECF MTX concentrations. RESULTS: Total and free plasma concentrations exhibited a parallel three exponential decay in both healthy and tumorigenic animals. Total clearance (8.9 vs 6.5 ml-1.min-1.kg-1) and volume of distribution (4.0 vs 2.9 l.kg-1), however, tended to decrease in the tumor-bearing group. The ECF/plasma AUC ratio equaled 14.2 +/- 8.8% in muscle and 23.9 +/- 15.9% in tumor. The concentration-time profile of muscle ECF MTX was parallel and highly correlated (r = 0.97) to that determined in plasma. In contrast, free MTX plasma levels were not correlated with tumor ECF concentrations (r = 0.564). CONCLUSIONS: In addition to the well-known pharmacological variability in the concentration-effect relationship, the important inter-individual variability in tumor exposure to MTX may partly explain that studies in patients with solid tumors have often failed to demonstrate firm correlations between MTX blood pharmacokinetics and the chemotherapeutic response.

Sensitive gas chromatographic-mass spectrometric method for the determination of gacyclidine in rat plasma and spinal cord dialyzates.

A sensitive gas chromatographic-mass spectrometric (GC-MS) procedure is described for the selective determination of gacyclidine (a non-competitive N-methyl-D-aspartate antagonist) in rat plasma and spinal cord dialyzates. It involves a single-step liquid-liquid extraction of plasma samples and dialyzates with hexane (pH 8.0) and the use of phencyclidine as an internal standard. The compounds were separated on a GC capillary column and specifically detected by MS in the selected-ion monitoring mode. Gacyclidine and its internal standard were monitored by using the fragment ions at m/z 206 and 200, respectively. The method was accurate and reproducible (intra- and inter-day reproducibility < 12%) with a limit of quantification of 1.6 ng ml-1 using 100 microliters plasma of dialyzate samples. The calibration curves for rat plasma and Ringer's solution were linear (r2 > 0.996) over a range from 1.6 to 200 ng ml-1. The extraction efficiency was close to 100%. This simple and rapid assay (total run time < 10 min) was validated for a pilot pharmacokinetic study in healthy rats after intravenous injection of a bolus dose of gacyclidine (2.5 mg kg-1).

Introduction of a methyl group in alpha- or beta-position of 1-heteroarylethyl-4-phenylpiperazines affects their dopaminergic/serotonergic properties.

1-(2-Heteroarylalkyl)-4-phenylpiperazines containing methyl group in either the alpha- or the beta-position of the side alkyl chain were synthesized as racemic mixtures. They were evaluated for in vitro binding affinity at the D1 and D2 dopamine and 5-HT1A serotonin receptors using synaptosomal membranes of the bovine caudate nucleus and hippocampus, respectively, as a source of the corresponding receptors. Tritiated SCH 23390 (D1 receptor-selective), spiperone (D2 receptor-selective), and 8-OH-DPAT (5-HT1A receptor-selective) were employed as the radioligands. None of the new compounds expressed significant affinity for the D1 receptor. Introduction of the methyl group into the beta-position of the parent molecules increased the affinity for the D2 receptor (10b-13b), and decreased the affinity for the 5-HT1A receptor with the exception of imidazole (11b) which was a rather efficient displacer of 8-OH-DPAT. Most potent of the newly synthesized compounds in [3H]spiperone assay were compounds (+/-)6-[1-methyl-2- (4-phenylpiperazin-1-yl)-ethyl]-1,4-dihydroquinoxaline-2,3-dione (10b), Kd = 6.0 nM and (+/-)5-[1-methyl-2-(4-phenylpiperazin-1-yl)-ethyl]-1,3-dihydrobenzoimidazol- 2-thione (13b), Kd = 5.3 nM. However, compounds containing methyl group in alpha-position (10a-13a) of the parent molecules expressed a decreased affinity for the D2 receptor, while the affinity for the 5-HT1A receptor remained in the same range of concentrations as that of closely related achiral parent compounds (14-17) run in the same binding assays as references.

Distribution of gacyclidine enantiomers in spinal cord extracellular fluid.

PURPOSE: Determination of the pharmacokinetics of gacyclidine enantiomers, a non-competitive NMDA antagonist, in plasma and spinal cord extracellular fluid (ECF) of rats. METHODS: Implantation of microdialysis probes in spinal cord (T9). Serial collection of plasma samples and ECF dialysates over 5 hours after IV bolus administration of (+/-)-gacyclidine (2.5 mg/kg). Plasma protein binding determined in vivo by equilibrium dialysis. Chiral GC/ MS assay. RESULTS: Plasma concentrations of (+)-gacyclidine were approximately 25% higher than those of (-)-gacyclidine over the duration of the experiment in all animals. Plasma concentrations decayed in parallel in a biphasic manner (t1/2alpha approximately 9 min; t1/2beta approximately 90 min) with no significant difference between enantiomers. Clearance and volume of distribution of (-)-gacyclidine were approximately 20% higher than those of its optical antipode (CL: 248 vs 197 ml.kg(-1)x min(-1); Vdbeta: 31.6 vs 23.5 1/kg). Protein binding (approximately 90%) was not stereoselective. Both gacyclidine enantiomers were quantifiable in spinal cord ECF 10 min after drug administration and remained stable over the duration of the experiment in spite of changing blood concentrations. Penetration of (-)-gacyclidine was significantly higher (approximately 40%) than that of (+)-gacyclidine in all animals. Yet, exposure of spinal cord ECF was similar for both enantiomers, and not correlated with plasma AUCs. CONCLUSIONS: The disposition of gacyclidine enantiomers is stereoselective. Both enantiomers exhibit a high affinity for spinal cord tissue and their distribution may involve a stereoselective and active transport system. This hypothesis could also explain the discrepancy between drug concentrations in plasma and spinal cord ECE

Pharmacokinetics of methotrexate in the extracellular fluid of brain C6-glioma after intravenous infusion in rats.

PURPOSE: Establishment of the pharmacokinetic profile of methotrexate (MTX) in the extracellular fluid (ECF) of a brain C6-glioma in rats. METHODS: Serial collection of plasma samples and ECF dialysates after i.v. infusion of MTX (50 or 100 mg/kg) for 4 h. HPLC assay. RESULTS: Histological studies revealed the presence of inflammation, edema, necrosis, and hemorrhage in most animals. In vivo recovery (reverse dialysis) was 10.8 +/- 5.3%. MTX concentrations in tumor ECF represented about 1-2% of the plasma concentrations. Rapid equilibration between MTX levels in brain tumor ECF and plasma. ECF concentrations almost reached steady-state by the end of the infusion (4 h), then decayed in parallel with those in plasma. Doubling of the dose did not modify MTX pharmacokinetic parameters (t1/2alpha, t1/2beta, MRT, fb, Vd, and CL(T)), except for a 1.7-fold increase of AUC(Plasma) and a 3.8-fold increase in AUC(ECF), which resulted in a 2.3-fold increase in penetration (AUC(ECF)/AUC(Plasma)). In spite of an important interindividual variability, a relationship between MTX concentrations in plasma and tumor ECF could be established from mean pharmacokinetic parameters. CONCLUSIONS: High plasma concentrations promote the penetration of MTX into brain tissue. However, free MTX concentrations in tumor ECF remain difficult to predict consistently.

Shear force near-field optical microscope based on Q-controlled bimorph sensor for biological imaging in liquid.

Shear force near-field microscopy on biological samples in their physiological environment loses considerable sensitivity and resolution as a result of liquid viscous damping. Using a bimorph-based cantilever sensor incorporating force feedback, as recently developed by us, gives an alternative force detection scheme for biological imaging in liquid. The dynamics and sensitivity of this sensor were theoretically and experimentally discussed. Driving the bimorph cantilever close to its resonance frequency with appropriate force feedback allows us to obtain a quality factor (Q-factor) of up to 10(3) in water, without changing its intrinsic resonance frequency and spring constant. Thus, the force detection sensitivity is improved. Shear force imaging on mouse brain sections and human skin tissues in liquid with an enhanced Q-factor of 410 have shown a high sensitivity and stability. A resolution of about 50 nm has been obtained. The experimental results suggest that the system is reliable and particularly suitable for biological cell imaging in a liquid environment.