Drug Delivery Studies in Caco-2 Monolayers. Synthesis,Hydrolysis, and Transport of O-Cyclopropane Carboxylic Acid Ester Prodrugs of Various β-Blocking Agents

A series of O-cyclopropane carboxylic acid ester prodrugs of various -blocking agents was synthesized. All prodrugs were hydrolyzed to give their parent compounds in aqueous phosphate buffer of pH 7.4 and in 80% human plasma. The half-lives in buffer solutions varied from 4 hours for the timolol prodrug to about 1 day for the prodrug of alprenolol. In human plasma the half-lives were shorter, ranging from 1 to 7 hours. The formation of the O-cyclopropane carboxylic acid ester derivatives significantly increased the lipophilicities of the -blockers as measured by the distribution coefficient between n-octanol and aqueous phosphate buffer of pH 7.4. To characterize the biomembrane permeability characteristics of the -blockers, transport properties across Caco-2 cell monolayers were investigated. An increase in lipophilicity resulted in a higher permeability of the prodrugs as compared to the parent compounds. Hence, acebutolol experienced an increment of a factor 17 on the apparent permeability coefficient, Papp, whereas Papp for the more lipophilic drug propranolol was increased by a factor of only 1.26. Some conversion of the prodrugs to their parent compounds was observed during the transport and appeared to be due to enzymatic intracellular metabolism.Deseaced.     

Selective inhibitors of glial GABA uptake: synthesis, absolute stereochemistry, and pharmacology of the enantiomers of 3-hydroxy-4-amino-4,5,6,7-tetrahydro-1,2-benzisoxazole (exo-THPO) and analogues

3-Methoxy-4,5,6,7-tetrahydro-1,2-benzisoxazol-4-one (20a), or the corresponding 3-ethoxy analogue (20b), and 3-chloro-4,5,6, 7-tetrahydro-1,2-benzisothiazol-4-one (51) were synthesized by regioselective chromic acid oxidation of the respective bicyclic tetrahydrobenzenes 19a,b and 50, and they were used as key intermediates for the syntheses of the target zwitterionic 3-isoxazolols 8-15 and 3-isothiazolols 16 and 17, respectively. These reaction sequences involved different reductive processes. Whereas (RS)-4-amino-3-hydroxy-4,5,6,7-tetrahydro-1,2-benzisoxazole (8, exo-THPO) was synthesized via aluminum amalgam reduction of oxime 22a or 22b, compounds 9, 11-13, and 15-17 were obtained via reductive aminations. Compound 10 was synthesized via N-ethylation of the N-Boc-protected primary amine 25. The enantiomers of 8 were obtained in high enantiomeric purities (ee >/= 99.1%) via the diastereomeric amides 32 and 33, synthesized from the primary amine 23b and (R)-alpha-methoxyphenylacetyl chloride and subsequent separation by preparative HPLC. The enantiomers of 9 were prepared analogously from the secondary amine 27. On the basis of X-ray crystallographic analyses, the configuration of oxime 22a was shown to be E and the absolute configurations of (-)-8 x HCl and (+)-9 x HBr were established to be R. The effects of the target compounds on GABA uptake mechanisms in vitro were measured using a rat brain synaptosomal preparation and primary cultures of mouse cortical neurons and glia cells (astrocytes). Whereas the classical GABA uptake inhibitor, (R)-nipecotic acid (2), nonselectively inhibits neuronal (IC(50) = 12 microM) and glial (IC(50) = 16 microM) GABA uptake and 4,5,6,7-tetrahydroisoxazolo?4,5-cpyridin-3-ol (1, THPO) shows some selectivity for glial (IC(50) = 268 microM) versus neuronal (IC(50) = 530 microM) GABA uptake, exo-THPO (8) was shown to be more potent as an inhibitor of glial (IC(50) = 200 microM) rather than neuronal (IC(50) = 900 microM) GABA uptake. This selectivity was more pronounced for 9, which showed IC(50) values of 40 and 500 microM as an inhibitor of glial and neuronal GABA uptake, respectively. These effects of 8 and 9 proved to be enantioselective, (R)-(-)-8 and (R)-(+)-9 being the active inhibitors of both uptake systems. The selectivity of 9 as a glial GABA uptake inhibitor was largely lost by replacing the N-methyl group of 9 by an ethyl group, compound 10 being an almost equipotent inhibitor of glial (IC(50) = 280 microM) and neuronal (IC(50) = 400 microM) GABA uptake. The remaining target compounds, 11-17, were very weak or inactive as inhibitors of both uptake systems. Compounds 9-13 and 15 were shown to be essentially inactive against isoniazide-induced convulsions in mice after subcutaneous administration. The isomeric pivaloyloxymethyl derivatives of 9, compounds 43 and 44, were synthesized and tested as potential prodrugs in the isoniazide animal model. Both 43 (ED(50) = 150 micromol/kg) and 44 (ED(50) = 220 micromol/kg) showed anticonvulsant effects, and this effect of 43 was shown to reside in the (R)-(+)-enantiomer, 45 (ED(50) = 44 micromol/kg). Compound 9 also showed anticonvulsant activity when administered intracerebroventricularly (ED(50) = 59 nmol).     

Metabolism of Testosterone During in Vitro Transport Across CACO-2 Cell Monolayers: Evidence for β-Hydroxysteroid Dehydrogenase Activity in Differentiated CACO-2 Cells

Testosterone has previously been used as a model compound for the determination of unstirred water layer thickness in the CACO-2 transport model. We have found, however, that testosterone is metabolized during in vitro transport across the CACO-2 cell monolayers. This suggests that testosterone is not an ideal model substance. Testosterone is metabolized to androstenedione, indicating the formation of 17--hydroxysteroid dehydrogenase by differentiated CACO-2 cells. No reverse metabolism is observed, thus androstenedione is considered superior to testosterone for determination of unstirred water layer thickness in the CACO-2 system. Permeability coefficients for testosterone and androstenedione obtained under identical transport conditions were 66 (±7) * 10 ^–6 (n = 26) and 84 (±7) * 10^–6 (n = 9) cm/sec, respectively. The unstirred water layer thicknesses at different agitation rates are determined for the CACO-2 transport model used in our laboratory utilizing androstenedione as a model compound. The system is capable of controlling the water layer thickness from about 200 to 1000 µm.     

Assessment of the combined approach of N-alkylation and salt formation to enhance aqueous solubility of tertiary amines using bupivacaine as a model drug.

Quaternary prodrug types of poorly water-soluble tertiary amines have been shown to exhibit significantly enhanced solubilities as compared to the parent amine. In the present study the combined effect of N-alkylation and salt formation to enhance aqueous solubility of tertiary amines have been investigated using bupivacaine as a model compound. X-ray structure analyses of selected salts were included to investigate the potential existence of correlations between salt solubility and crystal packing modes. Alkyl groups were methyl, ethyl, propyl, and butyl and the derivatives were isolated as their iodide salts. Chloride, mesylate, formate, acetate, glycolate, and tosylate salts were obtained by anion exchange of the N-methyl-bupivacaine derivative. N-Alkylation and salt formation afforded quaternary ammonium salts possessing pH-independent aqueous solubilities far exceeding that of the parent tertiary amine (up to a factor of 3200 at pH 8). A moderate reduction in solubility with increasing length of the alkyl chain was observed for the iodide salts of the N-alkylated bupivacaine derivatives. In case of the N-methyl-bupivacaine derivative variation of the counterion had a significant impact on the solubility with the iodide salt being 200 times less soluble than the chloride salt. X-ray analysis revealed that both the alkyl substituent and the anionic counterion influenced salt packing modes, however, in an unpredictable manner making establishment of quantitative correlations between crystal packing and solubility difficult even for a series of closely related derivatives.     

A dual echo approach to removing motion artefacts in fMRI time series

In fMRI, subject motion can severely affect data quality. This is a particular problem when movement is correlated with the experimental paradigm as this potentially causes artefactual activation. A method is presented that uses linear regression, to utilise the time course of an image acquired at very short echo time (TE) as a voxel‐wise regressor for a second image in the same echo train, that is acquired with high BOLD sensitivity. The value of this approach is demonstrated using task‐locked motion combined with visual stimulation. Results obtained at both 1.5 and 3 T show improvements in functional activation maps for individual subjects. The method is straightforward to implement, does not require extra scan time and can easily be embedded in a multi‐echo acquisition framework. Copyright © 2009 John Wiley & Sons, Ltd.     

Estimating meat withdrawal times in pigs exposed to melamine contaminated feed using a physiologically based pharmacokinetic model

Recently melamine was found to have contaminated the feed of multiple food production species leading to concern over the ability to establish an appropriate withdrawal interval and protect the safety of the food supply. To establish an appropriate withdrawal interval, a physiologically based pharmacokinetic (PBPK) model for melamine was developed for rats and extrapolated to pigs. The rat model underpredicted plasma concentrations, but better predicted tissue residues. Correlation values for plasma, kidney, and liver were 0.59, 0.76, and 0.73, respectively. The pig model underpredicted early plasma time points but had greater accuracy at later time points which is relevant to withdrawal times. Correlation (R²) between predicted and observed plasma values was 0.89 with a negative intercept of −0.76. The pig model estimated a withdrawal interval (based on kidney tissue residues) of 19.2 and 20.9 h for single oral exposures of 3.0 and 5.12 mg/kg of melamine, respectively. Chronic oral dosing (3.0 and 5.12 mg/kg twice daily for 7 days) yielded withdrawal intervals of 20 and 21.3 h, respectively. PBPK models, such as this one, provide evidence of the usefulness in species extrapolation over a range of dosing scenarios and can be used to protect the food supply after accidental exposure in the face of little in the target species.     

Bioavailability of progesterone from rectal dosage forms in rabbits

The systemic availability of progesterone in two rectal dosage forms was investigated in rabbits. The progesterone plasma concentration was determined as total radioactivity (progesterone and its metabolites) after a single dose of 5 mg/kg [³H]-labelled progesterone in an adeps solidus suppository (1) and in a propylene glycol enema (2) given in a randomized cross-over fashion. The equivalent dose was given intravenously (3) to the same rabbits.The maximum plasma concentration (C_max) after (1) was 0.82 ± 0.43 μg/ml and significantly lower than after (2), which was 3.81 ± 1.08 μg/ml (mean ± S.E.; P < 0.05). The time to reach the maximum plasma concentration (T_max) was for (1) 1.75 ± 0.25 h and for (2) 0.56 ± 0.32 h (mean ± S.E.; P < 0.05). The mean plasma concentration vs time curve after (3) indicates that a muhicompartment system is involved in the disposition of progesterone. The plasma half-life (t_{$\text{1}\text{2}$}) estimated from 0–6 h was 5.10 ± 1.12 (mean ± S.E.).The systemic availability (F%) of (1) from 0–6 h (AUC_0–6h) was 14 ± 8% and significantly lower than that of (2), which was 44 ± 10% (mean ± S.E.; P < 0.05).The results indicate a delayed and possibly lower absorption of progesterone from the suppositories as compared to the enema.     

Pharmacokinetics of felodipine in chronic hemodialysis patients

Five chronic hemodialysis patients (1 woman and 4 men, aged, 46-68 yr) were given an oral dose of 10 mg felodipine followed by 0.057 mg [3H] felodipine IV. After 5 hours, a hemodialysis treatment lasting 4 hours was performed. Blood and dialysate flows were 200 mL/min and 500 mL/min, respectively. Capillary dialyzers with 1.3 m2 cellulose acetate membrane were used. The pharmacokinetic characteristics and reduction in diastolic BP were similar to those in hypertensive patients with normal renal function and in uremic patients who were not treated with dialysis. There was no measurable removal of felodipine by hemodialysis. Dialyzer clearance of radioactive metabolites was about 10 mL/min, and only 8.9% of the dose was eliminated by the treatment. The half-life of radioactive metabolites was 10 days (6-14 days) in three patients dialyzed thrice weekly. Since the metabolites are biologically inactive, no adjustment of dose is required in hemodialysis patients.     

Analysis of diltiazem in Lipoderm transdermal gel using reversed-phase high-performance liquid chromatography applied to homogenization and stability studies

A simple and novel method for the extraction and quantification of diltiazem hydrochloride was developed and applied to homogenization and stability studies. The method used solid phase extraction coupled with reversed-phase high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection. Validation showed inter-day recoveries ranging from 84.00 to 96.52% with relative standard deviations ranging from 12.01 to 15.94%. Intra-day recoveries ranged from 67.95 to 106.1% with relative standard deviations less than 5%. The method showed excellent linearity from 50 to 250 mg/ml in undiluted gel (R2 = 0.996). The homogenization study showed good homogenization using both 50 and 100 depression techniques. Diltiazem was stable at a concentration of 246 mg/ml for 30 days and at a concentration of 99.6 mg/ml for 60 days no matter the storage conditions explored in this study.