Radiosynthesis and in vivo evaluation of two imidazopyridineacetamides, [(11)C]CB184 and [ (11)C]CB190, as a PET tracer for 18 kDa translocator protein: direct comparison with [ (11)C](R)-PK11195.

OBJECTIVE: We report synthesis of two carbon-11 labeled imidazopyridines TSPO ligands, [(11)C]CB184 and [(11)C]CB190, for PET imaging of inflammatory process along with neurodegeneration, ischemia or brain tumor. Biodistribution of these compounds was compared with that of [(11)C]CB148 and [(11)C](R)-PK11195. METHODS: Both [(11)C]CB184 and [(11)C]CB190 having (11)C-methoxyl group on an aromatic ring were readily prepared using [(11)C]methyl triflate. Biodistribution and metabolism of the compounds were examined with normal mice. An animal PET study using 6-hydroxydopamine treated rats as a model of neurodegeneration was pursued for proper estimation of feasibility of the radioligands to determine neuroinflammation process. RESULTS: [(11)C]CB184 and [(11)C]CB190 were obtained via O-methylation of corresponding desmethyl precursor using [(11)C]methyl triflate in radiochemical yield of 73 % (decay-corrected). In vivo validation as a TSPO radioligand was carried out using normal mice and lesioned rats. In mice, [(11)C]CB184 showed more uptake and specific binding than [(11)C]CB190. Metabolism studies showed that 36 % and 25 % of radioactivity in plasma remained unchanged 30 min after intravenous injection of [(11)C]CB184 and [(11)C]CB190, respectively. In the PET study using rats, lesioned side of the brain showed significantly higher uptake than contralateral side after i.v. injection of either [(11)C]CB184 or [(11)C](R)-PK11195. Indirect Logan plot analysis revealed distribution volume ratio (DVR) between the two sides which might indicate lesion-related elevation of TSPO binding. The DVR was 1.15 ± 0.10 for [(11)C](R)-PK11195 and was 1.15 ± 0.09 for [(11)C]CB184. CONCLUSION: The sensitivity to detect neuroinflammation activity was similar for [(11)C]CB184 and [(11)C](R)-PK11195.

Amphiphilic inulin-D-α-tocopherol succinate (INVITE) bioconjugates for biomedical applications.

Herein is reported the synthesis and characterization of innovative inulin (INU)-vitamin E succinate (VITE) bioconjugates (INVITE). The obtained amphiphilic INU-based polymers, self-assembling in nanostructures, have been thought as new drug delivery systems (DDS) for the therapy of urinary tract infections (UTI). The synthesis of INVITE bioconjugates was carried out in bulk, without isolation of intermediate products, to reduce the amount of solvents used in the purification steps and to prevent possible VITE oxidation during work up. Six different INVITE conjugates (INVITE 1-6) have been synthesized by varying both the relative amount of VITE with respect to INU repetitive units and the reaction temperature. Afterwards, the ability of the new conjugates to form micelle systems, by applying two different established methods for critical aggregation concentration (CAC) evaluation, has been verified. Both methods produced similar CAC values ranging from 2.5 × 10(-3)mM to 2.4 × 10(-2)mM in agreement with the different degrees of derivatization shown by the INVITE 1-6 conjugates. The mean diameter of prepared INVITE micelles, resulted in the range 24-60 nm. The size of the obtained INVITE micelles did not change as measured at different time points up to 12 days, so confirming their stability upon storage.

Translocator protein (TSPO) ligand-Ara-C (cytarabine) conjugates as a strategy to deliver antineoplastic drugs and to enhance drug clinical potential.

The aim of this work was to evaluate TSPO ligand-Ara-C conjugation as an approach for the selective delivery of the antineoplastic agent to brain tumors as well as for overcome P-gp resistance induction observed for the majority of cytotoxic agents, enhancing the drug clinical potential. To this end, the novel N-imidazopyridinacetyl-Ara-C conjugates 3a-c, 10 and 15 have been prepared and evaluated for their cytotoxicity against glioma cell lines. In contrast to that observed for 3a-c and 10, the conjugate 15 resulted stable in both phosphate buffer and physiological medium. In all cases, the release of free Ara-C from hydrolyzed conjugates was checked by HPLC and ESI-MS analysis. Conjugates 10 and 15 displayed very high in vitro TSPO affinity and selectivity, and, hence, they may possess potential for targeted brain delivery. Due to the favorable features displayed by the conjugate 15, it was further evaluated on glioma cell lines, expressing high levels of TSPO, in the presence and in the absence of specific nucleoside transport (NT) inhibitors. In contrast to that observed for the free Ara-C, the presence of NT inhibitors did not reduce the cytotoxic activity of 15. Moreover, conjugate 15, as N(4)-acyl derivative of Ara-C, should be resistant to inactivation by cytidine deaminase, and it may possess enhanced propensity to target brain tumor cells characterized by a reduced expression of NTs. In addition, this conjugate behaves as a clear P-gp modulator and thereby may be useful to reverse MDR. Transport studies across the MDCKII-MDR1 monolayer indicated that conjugate 15 should overcome the BBB by transcellular pathway. All these features may be useful for enhancing the clinical potential of the nucleoside drug Ara-C.

PEG-PE micelles loaded with paclitaxel and surface-modified by a PBR-ligand: synergistic anticancer effect.

Selective ligands to the peripheral benzodiazepine receptor (PBR) may induce apoptosis and cell cycle arrest. An overexpression of PBR in certain cancers allowed us to consider the use of highly selective ligands to PBR for receptor-mediated drug targeting to tumors. With this in mind, we prepared PBR-targeted nanoparticulate drug delivery systems (PEG-PE micelles) loaded with the anticancer drug paclitaxel (PCL) to test possible synergistic anticancer effects. PEG2k-PE-based polymeric micelles with and without PCL were prepared in HBS, pH 7.5, and conjugated with a PBR-ligand (CB86) in 0.45% of DMSO. The cytotoxic effect of such micelles against the LN 18 human glioblastoma cell line was studied in cell culture. The micelles maintained their size and size distribution and remained intact without drug release after the PBR-ligand conjugation. The PCL-loaded PBR-targeted micelles showed a significantly enhanced toxicity against human glioblastoma LN 18 cancer cells in vitro. Thus, PBR-targeted nanopreparations may potentially serve as a new nanomedicine for targeted cancer therapy.

Peripheral benzodiazepine receptor ligand-PLGA polymer conjugates potentially useful as delivery systems of apoptotic agents.

Poly(d,l-lactic-co-glycolic acid) (PLGA) polymers having different average molecular weights were chemically conjugated to two imidazopyridinacetamides (1 and 2), chosen as model Peripheral Benzodiazepine Receptor (PBR) ligands, via an ester or amide linkage. It is in order to evaluate these conjugates as delivery systems of PBR ligands endowed with apoptosis inducing activity. Various coupling reaction conditions were tested to optimize the conjugation process. After purification by extensive dialysis procedures, the macromolecular conjugates were characterized by FT-IR, UV, (1)H NMR spectroscopy, DSC and the average molecular weights of synthesized conjugates were determined by GPC. PBR ligand released from these conjugates occurred in human serum and in 0.1 N HCl solution at a faster rate than that observed in phosphate buffer, pH 7.4. Moreover, the macromolecular conjugates displayed high affinity and selectivity for PBR. Cytotoxicity studies demonstrated that PBR ligand-PLGA polymer conjugates induce survival inhibition in rat C6 glioma cell line. Fluorescence microscopy studies evidenced the cellular uptake of FITC-conjugated probes 10 and 11 and moreover, the mitochondrial morphology modification induced by compounds 1 and 4a. Therefore, this study demonstrates that this PBR ligand-PLGA combination may provide a new mitochondrial targeted approach useful for improved cancer chemotherapy.

2-Phenyl-imidazo[1,2-a]pyridine compounds containing hydrophilic groups as potent and selective ligands for peripheral benzodiazepine receptors: synthesis, binding affinity and electrophysiological studies.

A series of imidazopyridine acetamides were synthesized to evaluate the effects of structural changes at both central (CBRs) and peripheral benzodiazepine receptors (PBRs). These changes include the introduction of polar substituents or ionizable functional groups at the 2- and 8-position of the imidazopyridine skeleton. The results suggest that substituents endowed with hydrogen bonding acceptor and/or donor properties in the para position of the phenyl ring lead to high affinity for PBR. In electrophysiological studies, it was found that compounds 9, 12, 13, and 28 markedly enhanced GABA-evoked Cl (-) currents in Xenopus oocytes expressing alpha 1beta 2gamma 2 GABA A receptors. The capability of flumazenil to reduce the stimulatory effect exerted by compound 9 supports the conclusion that the modulatory effects of the examined compounds occur involving the CBR. The ability of compound 16 to increase GABA A receptor-mediated miniature inhibitory postsynaptic currents in CA1 pyramidal neurons is indicative of its ability to stimulate the local synthesis and secretion of neurosteroids.

Radiosynthesis and in vivo evaluation of N-[11C]methylated imidazopyridineacetamides as PET tracers for peripheral benzodiazepine receptors.

Imidazopyridineacetoamide 5-8, a series of novel and potentially selective peripheral benzodiazepine receptor (PBR) ligands with affinities comparable to those of known PBR ligands, was investigated. Radiosyntheses of [11C]5, 6, 7 or 8 was accomplished by N-methylation of the corresponding desmethyl precursors with [11C]methyl iodide in the presence of NaH in dimethylformamide (DMF), resulting in 25% to 77% radiochemical yield and specific activitiy of 20 to 150 MBq/nmol. Each of the labeled compounds was injected in ddY mice, and the radioactivity and weight of dissected peripheral organs and brain regions were measured. Organ distribution of [11C]7 was consistent with the known PBR distribution. Moreover, [11C]7 showed the best combination of brain uptake and PBR binding, leading to its high retention in the olfactory bulb and cerebellum, areas where PBR density is high in mouse brain. Coinjection of PK11195 or unlabeled 7 significantly reduced the brain uptake of [11C]7. These results suggest that [11C]7 could be a useful radioligand for positron emission tomography imaging of PBRs.

N-benzyl-2-(6,8-dichloro-2-(4-chlorophenyl)imidazo[1,2-a]pyridin-3-yl)-N-(6-(7-nitrobenzo[c][1,2,5]oxadiazol-4-ylamino)hexyl)acetamide as a new fluorescent probe for peripheral benzodiazepine receptor and microglial cell visualization.

The aim of this work was to develop new fluorescent probes for the localization and function of the peripheral benzodiazepine receptor (PBR). This receptor is primarily expressed on the mitochondria, and it is overexpressed in a variety of different states including glioma, breast cancer, Alzeheimer's disease, and activated microglia. For the mentioned purpose, imidazopyridine and imidazopyrimidine compounds 5-20 were synthesized, and their affinity for PBR was determined. Although some intrinsically fluorescent imidazopyrimidine compounds 12-20 possess good binding affinity, they cannot be used for visualizing PBR due to their unfavorable fluorescence characteristics. Among the imidazopyridine-7-nitrofurazan conjugates 5-11, compound 10 was the most active, and it was found to stain live Ra2 microglial cells effectively. An in vivo biodistribution study carried out on compound 10 showed that this imidazopyridine derivative, injected in the carotid artery, is able to penetrate to liver parenchyma, whereas fluorescein isothiocyanate labeled dextran (FITC-dextran), used as a control dye, hardly penetrated from blood vessels to tissues. On the other hand, as for the distribution to brain, the patterns of staining with 10 and FITC-dextran are similar, indicating that both of them hardly penetrate into the brain because of the existence of the blood-brain barrier. The obtained results indicate that compound 10 represents a new useful fluorescent probe for visualization of activated microglia and PBR.

Novel L-Dopa and dopamine prodrugs containing a 2-phenyl-imidazopyridine moiety.

PURPOSE: The aim of this study was to gain insight into the feasibility of enhancing the delivery of L-Dopa and dopamine to the brain by linking these neurotransmitters and L-Dopa ethyl ester to 2-phenyl-3-carboxymethyl-imidazopyridine compounds giving rise to the so-called Dopimid compounds. MATERIALS AND METHODS: A number of Dopimid compounds were synthesized and both stability and binding studies to dopaminergic and benzodiazepine receptors were performed. To evaluate whether Dopimid compounds are P-gp substrates, [(3)H]ritonavir uptake experiments and bi-directional transport studies on confluent MDCKII-MDR1 monolayers were carried out. The brain penetration properties of Dopimid compounds were estimated by the Clark's computational model and evaluated by investigation of their transport across BBMECs monolayers. The dopamine levels following the intraperitoneal administration of the selected Dopimid compounds were measured in vivo by using brain microdialysis in rat. RESULTS: Tested compounds were adequately stable in solution buffered at pH 7.4 but undergo faster cleavage in dilute rat serum at 37 degrees C. Receptor binding studies showed that Dopimid compounds are essentially devoid of affinity for dopaminergic and benzodiazepine receptors. [(3)H]ritonavir uptake experiments indicated that selected Dopimid compounds, like L-Dopa and dopamine hydrochloride, are not substrates of P-gp and it was also confirmed by bi-directional transport experiments across MDCKII-MDR1 monolayers. By Clark's model a significant brain penetration was deduced for L-Dopa ethyl ester and dopamine derivatives. Transport studies involving BBMECs monolayers indicated that some of these compounds should be able to cross the BBB. Interestingly, the rank order of apparent permeability (P (app)) values observed in these assays parallels that calculated by the computational approach. Brain microdialysis experiments in rat showed that intraperitoneal acute administration of some Dopimid compounds induced a dose-dependent increase in cortical dopamine output. CONCLUSION: Based on these results, it may be concluded that some Dopimid compounds can be proposed as novel L-Dopa and dopamine prodrugs.

Comparative effects of some hydrophilic excipients on the rate of gabapentin and baclofen lactamization in lyophilized formulations.

The aim of this study was to gain insights into the role played by some excipients on the stability of gabapentin 1 and baclofen 2 which can undergo degradation giving rise to the corresponding lactams 2-azaspiro[4.5]decan-3-one 3 and 4-(4-chlorophenyl)-2-pyrrolidone 4, respectively. A screening study was carried out on drug and drug-excipient freeze-dried mixtures at 50 degrees C and under three different humidity values by using a number of commonly available excipients. These include hydroxypropyl-beta-(HP-beta-CD), sulfobutyl-beta-cyclodextrin (SBE-beta-CD), lactose, raffinose, trehalose, PVP-K30 and mannitol. For most cases, it was found that the lactam formation can be satisfactory described by an apparent zero-order equation. Excipients shown to negatively impact gabapentin stability are HP-beta-CD, SBE-beta-CD, lactose and PVP K30 while only this last excipient had a significant effect on the degradation of baclofen. The results can be rationalized in terms of conformational factors favouring the intramolecular dehydration reaction. A positive effect of moisture on the lactamization process was observed under some circumstances. Water may provide a favourable environment for degradation. These findings, taken together, should be considered during the selection of excipients for a possible formulation of gabapentin and baclofen.

A rapid screening tool for estimating the potential of 2-hydroxypropyl-beta-cyclodextrin complexation for solubilization purposes.

Quantitative structure-property relationships (QSPRs) were developed for predicting the solubility enhancement (expressed as logS/S0) of compounds in 45% (w/v) aqueous solution of HP-beta-CD. A set of 25 structurally different drugs, whose logS/S0 values were taken from literature, was used as a training set for building the computational models. Thirteen molecular descriptors, including parameters for size, lipophilicity, cohesive energy density and hydrogen-bonding capacity, were calculated and together with the experimental melting point (MP), used in multivariate analysis. Eight pertinent variables were detected after looking at the results of principal component analysis (PCA) and cluster analysis, and two reliable four-descriptor models generated by multiple linear regression (MLR) and by the partial least squares-projection to latent structures (PLS) methods. In both cases, satisfactory coefficients of determination values were obtained (i.e., R2 equal to 0.793 or 0.763 for MLR and PLS, respectively). The models were validated using a test set of six compounds. The equations generated can predict the aqueous solubility increase of poorly soluble compounds by complexation in 45% (w/v) aqueous solution of HP-beta-CD with a reasonable accuracy. These equations can allow formulation scientists to rapidly estimate, at the early stage of drug development, the potential of HP-beta-CD in increasing solubility of poorly water-soluble drugs.

Structural modifications and antimicrobial activity of N-cycloalkenyl-2-acylalkylidene-2,3-dihydro-1,3-benzothiazoles.

A series of N-cycloalkenyl-2-acylalkylidene-2,3-dihydro-1,3-benzothiazoles 5a-j, N-cycloalkyl-2-acylalkylidene-2,3-dihydro-1,3-benzothiazoles 8a-e, and N-alkyl-2-acylalkylidene-2,3-dihydro-1,3-benzothiazoles 8f-h, were synthesized and tested for in vitro antibacterial and antifungal activities against four gram-positive and five gram-negative bacteria (Bacillus subtilis 6633, Enterococcus faecalis 29212, Staphylococcus aureus 6538, Staphylococcus aureus 25923, Escherichia coli 25922, Acinetobacter calcoaceticus a1, A. calcoaceticus a2, Pseudomonas aeruginosa 27835, Klebsiella oxytoca 49131), four yeast-like fungi and one fungus (Candida tropicalis 750, C. albicans 14053, C. albicans 10231, Cryptococcus laurentii 18803, and Saccharomyces cerevisiae). Microdilution broth and agar dilution methods were used for antimicrobial tests. The findings obtained showed that some of the tested compounds 5 and 8 were effective against some of the bacterial strains used, whereas, only compounds 8b-g exhibited a moderate antifungal activity against the yeast strains evaluated.

Structure-activity relationships and effects on neuroactive steroid synthesis in a series of 2-phenylimidazo[1,2-a]pyridineacetamide peripheral benzodiazepine receptors ligands.

A series of 36 imidazopyridineacetamides (2-37) were designed and synthesized to evaluate the effects of structural changes on the amide nitrogen at both central (CBRs) and peripheral benzodiazepine receptors (PBRs). These changes include variations in the length and number of the alkyl groups as well as introduction of different aromatic, heteroaromatic, and conformationally constrained groups. The affinities of these compounds for CBRs and PBRs were determined, and the results indicate that bulkiness of the substituents, their branching, and length beyond an optimal value may cause hindrance to the ligand in its interaction with the receptor. The presence of aromatic or conformationally constrained substituents on the carboxamide nitrogen can be conducive to high affinity and selectivity. Furthermore, the ability of a subset of the most active ligands to stimulate synthesis of neuroactive steroids in plasma and brain was evaluated in vivo and in vitro. Compound 3 exhibited very marked effects on the peripheral and central synthesis of neuroactive steroids, while 36 (potent at subnanomolar level) showed a slight ability to affect neuroactive steroid content in the cerebral cortex.

Frog intestinal sac: a new in vitro method for the assessment of intestinal permeability.

The aim of this study was to evaluate a new experimental protocol utilizing isolated frog intestinal sacs for the assessment of intestinal drug permeability in humans. Segments of approximately 5.0 cm in length were used for these experiments. The intestinal sacs were filled with a solution of the appropriate drug in frog Ringer (FR) and immersed in a vial containing fresh FR. The transport was monitored for a period ranging from 2 to 5 h by moving the intestinal sac at each time point to a new vial containing fresh medium (Method A). Alternatively, according to Method B, at predetermined times aliquots of receiving mixture were taken up without removing the intestinal sac, and replaced with fresh drug-free FR. In all cases, the samples were analyzed by HPLC. A series of 20 noncongeneric drugs, predominantly absorbed by passive diffusion mechanism, was examined. The results indicate that drugs completely absorbed in humans had Papp values greater than 1 x 10(-6) cm/s, while drugs absorbed <90% had Papp values lower than 1 x 10(-6) cm/s. By plotting Log Papp values against percent human absorption, an approximately sigmoidal relationship was obtained. The frog intestinal sac method was evaluated as a permeability model to classify the 20 studied drugs into the Biopharmaceutics Classification System (BCS). By comparing the predictions made by this new approach with those reported in literature, it can be concluded that a satisfactory biopharmaceutical classification may be based on the Papp values determined by the frog intestinal permeability. Molecular properties relevant to passive intestinal permeability were considered to evaluate the correlation with the frog intestinal permeability rates recorded. Good relationships were observed when hydrogen-bonding parameters were expressed as a function of the Log Papp values. It can be concluded that the in vitro permeability coefficient deduced from isolated frog intestinal experiments can be used for predicting peroral absorption in humans for passively absorbed compounds. Computational methods for prediction of frog intestinal permeability may be applied in a highly simplified manner.

Evaluation of new propofol aqueous solutions for intravenous anesthesia.

The aim of this study was to evaluate the potential of using three new aqueous formulations of propofol for intravenous (i.v.) anesthesia. The first formulation can be prepared by using hydroxypropyl-gamma-cyclodextrin (HP-gamma-CD) as a solubilizer. Phase-solubility analysis showed a linear increase in the solubility of propofol to a maximum of 16.6 mg/ml in 30% (w/v) HP-gamma-CD. Moreover, phase-solubility studies demonstrated that 18% (w/v) HP-beta-CD or SBE-beta-CD and 24% HP-gamma-CD solutions, respectively, are required to dissolve 10mg of propofol in 1 ml of the vehicle; the corresponding solutions, however, are slightly hypertonic. Autoclaving the 10 mg/ml CD-based formulations for 15 min at 121 degrees C caused a change in pH which was more evident for the HP-beta-CD-based formulation while, in any case, no detectable fall in propofol concentration was observed. The second formulation herein evaluated is a co-solvent mixture (i.e., propylene glycol:water (1:1), v/v) which is able to dissolve 10 mg/ml of the anesthetic agent. However, although it is simple to prepare, the stability of this formulation is limited. The third aqueous formulation can be prepared by using the prolinate ester of propofol and its water-soluble derivative dissolved in water at equimolar concentration. The efficacy of all these formulations as i.v. anesthetic agents was assessed using a pharmacodynamic measure (onset and duration of loss of the righting reflex, LORR), and compared with that of the commercial propofol formulation (Diprivan, 10 mg/ml) in rats. It was found that minimizing the amount of cyclodextrin in all CD-based formulations, anesthetic effects comparable to those of propofol in Diprivan were still observed. Moreover, the prolinate ester constituted an effective i.v. anesthetic formulation with the same duration of action but with a longer induction time than Diprivan.

Social isolation increases the response of peripheral benzodiazepine receptors in the rat.

Social isolation of rats for 30 days immediately after weaning reduces the cerebrocortical and plasma concentrations of progesterone, 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-TH PROG), and 3alpha,5alpha-tetrahydrodeoxycorticosterone (3alpha,5alpha-TH DOC). The percentage increases in the brain and plasma concentrations of these neuroactive steroids apparent 30 min after intraperitoneal injection of the peripheral benzodiazepine receptor (PBR) ligand CB 34 (25 mg/kg) have now been shown to be markedly greater in isolated rats than in group-housed controls. The CB 34-induced increase in the abundance of 3alpha,5alpha-TH PROG was more pronounced in the brain than in the plasma of isolated rats. Analysis of [3H]PK 11195 binding to membranes prepared from the cerebral cortex, adrenals, or testis revealed no significant difference in either the maximal number of binding sites for this PBR ligand or its dissociation constant between isolated and group-housed animals. Social isolation also induced a small but significant decrease in the plasma concentration of adrenocorticotropic hormone. Moreover, CB 34 increased the plasma concentration of this hormone to a greater extent in isolated rats than in group-housed animals. The persistent decrease in the concentrations of neuroactive steroids induced by social isolation might thus be due to an adaptive decrease in the activity either of the hypothalamic-pituitary-adrenal axis or of PBRs during the prolonged stress, reflecting a defense mechanism to limit glucocorticoid production. The larger increase in neuroactive steroid concentrations induced by CB 34 and the enhanced pituitary response to this compound in isolated rats indicate that this mild stressor increases the response of PBRs.

Encapsulation and release of the hypnotic agent zolpidem from biodegradable polymer microparticles containing hydroxypropyl-beta-cyclodextrin.

The goal of this study was to design a prolonged release system of the hypnotic agent zolpidem (ZP) useful for the treatment of insomnia. In this work, ZP alone or in the presence of HP-beta-CD was encapsulated in microparticles constituted by poly(DL-lactide) (PDLLA) and poly(DL-lactide-co-glycolide) (PLGA) and the drug release from these systems was evaluated. ZP alone-loaded microparticles were prepared by the classical O/W emulsion-solvent evaporation method. Conversely, ZP/HP-beta-CD containing microparticles were prepared by the W/O/W emulsion-solvent evaporation method following two different procedures (i.e. A and B). Following procedure A, the previously produced ZP/HP-beta-CD solid complex was added to the water phase of primary emulsion. In the procedure B, HP-beta-CD was added to the aqueous phase and ZP to the organic phase. The resulting microparticles were characterized about morphology, size, encapsulation efficiency and release rates. FT-IR, X-ray, and DSC results suggest the drug is in an essentially amorphous state within the microparticles. The release profiles of ZP from microparticles were in general biphasic, being characterized by an initial burst effect and a subsequent slow ZP release. It resulted that co-encapsulating ZP with or without HP-beta-CD in PDLLA and PLGA the drug release from the corresponding microparticles was protracted. Moreover, in a preliminary pharmacological screening, the ataxic activity in rats was investigated and it was found that intragastric administration of the ZP/HP-beta-CD/PLGA microparticles prepared according to procedure B produced the same ataxic induction time as the one induced by the currently used formulation Stilnox. Interestingly moreover, there was a longer ataxic lasting and a lower intensity of ataxia produced by the ZP/HP-beta-CD/PLGA-B-formulation already after 60 min following the administration. However, a need for further pharmacokinetic and pharmacodynamic studies resulted to fully evaluate the utility of this last formulation for the sustained delivery of ZP.

Highly water-soluble derivatives of the anesthetic agent propofol: in vitro and in vivo evaluation of cyclic amino acid esters.

Cyclic amino acid esters of propofol were synthesized in an attempt to develop new water-soluble anesthetic agents. Their solubility and stability in aqueous solution, and their ability to release propofol in vitro under physiological conditions were determined. L-Proline (6a) and racemic nipecotic acid (6c) esters were found to be highly soluble in water. Sufficiently stable at physiological pH (half-lives >6 h), the alpha-amino acid esters, 6a and 6b, were found to be quantitatively hydrolyzed in plasma and liver esterase solutions within a few minutes, showing prodrug behavior. The in vitro activity of the esters, determined either by the [(35)S]tert-butylbicyclophosphorothionate ([(35)S]TBPS) binding assay or electrophysiological measurements of the action at cloned human receptors, proved to be a mechanism involving allosteric modulation of GABA(A) receptors. Indeed, L-proline (6a), and racemic pipecolinate (6b) and nipecotate (6c), like propofol, reduced [(35)S]TBPS binding, whereas isonipecotate (6d) showed bicuculline-like behavior, increasing [(35)S]TBPS binding. A nonlinear relation between GABA(A) receptor binding affinity and lipophilicity, as assessed by reversed-phase high-performance liquid chromatography, emerged as a trend. The in vivo anticonvulsant and anesthetic activities of prolinate 6a, intraperitoneally administered in water solution, showed that is a water-soluble propofol prodrug candidate for developing formulations useful for parenteral administration.

Peripheral benzodiazepine receptor ligand-melphalan conjugates for potential selective drug delivery to brain tumors.

To gain insight into the strategy to target PBR ligand-drug conjugates to brain tumors, novel N-imidazopyridinacetyl-melphalan conjugates and the corresponding ethyl esters have been prepared and evaluated for their cytotoxicity in melphalan-sensitive human (SF126, SF188) and rat (RG-2) glioma cell lines. These conjugates exhibited PBR binding affinity with IC(50) values ranging from 57 and 2614 nM. By a computational approach it can be predicted that these conjugates possess significant brain penetration. The stability of the conjugates in 0.05 M phosphate buffer at pH 7.4 and, in some cases, in dilute human serum solution was determined. All the ethyl ester derivatives were stable in 0.05 M phosphate buffer at pH 7.4 and their half-lives exceeded 28 h. Conversely, under the same conditions, the corresponding acids were found to undergo a fast cleavage within a few minutes. HPLC-MS analysis of the mixture from degradation in buffer and physiological medium of the representative cases allowed the identification of their main degradation products. A plausible degradation pathway accounting for the available experimental data is presented.

Alpidem analogues containing a GABA or glycine moiety as new anticonvulsant agents.

Alpidem analogues containing a GABA (1-3) or glycine (4-6) moiety were synthesized and their interaction with the GABA/benzodiazepine receptor complex at central (CBR) and peripheral (PBR) level was evaluated. In particular, their ability to modulate the specific binding of [3H]-GABA to washed membrane preparations from the rat cerebral cortex, as well as their effects on human recombinant GABA(A) receptors in Xenopus laevis oocytes, were assessed. Results from these in vitro assays showed that the most active compounds were 1 and 4. Intraperitoneal administration of compound 1 at a dose of 150 mg/kg significantly antagonized pentylenetetrazole-induced seizures in rats and the protective effects were evident for 90 min. However, compound 4 failed to interact with strychnine-sensitive Gly-binding sites. Consistent with these binding results, intraperitoneal administration of compound 4 at 150 mg/kg showed no effect against convulsions induced by strychnine, except for a prolonged time of the latency of convulsions. The results obtained suggest that compound 1 possesses interesting anticonvulsant activity and deserves further investigation as a novel lipophilic GABA derivative.