Phosphatase and tensin homologue deleted on chromosome ten (PTEN) as a molecular target in lung epithelial wound repair.

BACKGROUND AND PURPOSE: Epithelial injury contributes to lung pathogenesis. Our work and that of others have identified the phosphoinositide-3 kinase (PI3K)/Akt pathway as a vital component of survival in lung epithelia. Therefore, we hypothesized that pharmacological inhibition of PTEN, a major suppressor of this pathway, would enhance wound closure and restore lung epithelial monolayer integrity following injury. EXPERIMENTAL APPROACH: We evaluated the ability of two bisperoxovanadium derivatives, bpV(phen) and bpV(pic), in differentiated primary human airway epithelia and BEAS2B cultures for their ability to inhibit PTEN, activate the PI3K/Akt pathway and restore epithelial monolayer integrity following mechanical injury. KEY RESULTS: BpV(phen) and bpV(pic) induced Akt phosphorylation in primary and BEAS2B cells in a dose and time dependent manner. Minimal toxicity was observed as measured by lactate dehydrogenase (LDH) release. To verify that Akt phosphorylation is specifically induced by PTEN inhibition, the PTEN positive cell line, DU145, and two PTEN negative cell lines, LNCaP and PC3, were examined. PTEN positive cells demonstrated a dose responsive increase in Akt phosphorylation whereas PTEN negative cells showed no response indicating that bpV(phen) directly suppresses PTEN without affecting auxiliary pathways. Next, we observed that exposure to either compound resulted in accelerated wound closure following mechanical injury. Similar effects were observed after transfection with a dominant negative isoform of PTEN and PTEN specific siRNA. CONCLUSIONS AND IMPLICATIONS: From these studies, we conclude that PTEN is a valid target for future studies directed at restoring epithelial barrier function after lung injury.

Discovery of antiandrogen activity of nonsteroidal scaffolds of marketed drugs.

Finding good drug leads de novo from large chemical libraries, real or virtual, is not an easy task. High-throughput screening is often plagued by low hit rates and many leads that are toxic or exhibit poor bioavailability. Exploiting the secondary activity of marketed drugs, on the other hand, may help in generating drug leads that can be optimized for the observed side-effect target, while maintaining acceptable bioavailability and toxicity profiles. Here, we describe an efficient computational methodology to discover leads to a protein target from safe marketed drugs. We applied an in silico "drug repurposing" procedure for identification of nonsteroidal antagonists against the human androgen receptor (AR), using multiple predicted models of an antagonist-bound receptor. The library of marketed oral drugs was then docked into the best-performing models, and the 11 selected compounds with the highest docking score were tested in vitro for AR binding and antagonism of dihydrotestosterone-induced AR transactivation. The phenothiazine derivatives acetophenazine, fluphenazine, and periciazine, used clinically as antipsychotic drugs, were identified as weak AR antagonists. This in vitro biological activity correlated well with endocrine side effects observed in individuals taking these medications. Further computational optimization of phenothiazines, combined with in vitro screening, led to the identification of a nonsteroidal antiandrogen with improved AR antagonism and marked reduction in affinity for dopaminergic and serotonergic receptors that are the primary target of phenothiazine antipsychotics.

Pharmacokinetics of S-3-(4-acetylamino-phenoxy)-2-hydroxy-2-methyl-N-(4-nitro- 3-trifluoromethyl-phenyl)-propionamide in rats, a non-steroidal selective androgen receptor modulator.

1: S-3-(4-acetylamino-phenoxy)-2-hydroxy-2-methyl-N-(4-nitro-3-trifluoromethyl-phenyl)-propionamide (also known as S-4) is a non-steroidal selective androgen receptor modulator demonstrating tissue-selective androgenic and anabolic effects. The purpose of the present study was to examine the systemic pharmacokinetics, elimination and oral bioavailability of S-4 in rats. 2: Thirty-five male Sprague-Dawley rats weighing approximately 250 g were randomly assigned to one of seven treatment groups. Intravenous doses of 0.5, 1, 10, and 30 mg kg(-1) were given via a jugular catheter. Oral doses of 1, 10 and 30 mg kg(-1) were administered via gavage. Plasma concentrations were determined using a validated high-performance liquid chromatography or by a high-performance liquid chromatography/mass spectrometry method. 3: Clearances ranged between 1.0 and 2.1 ml min(-1) kg(-1) and varied with dose. The volume of distribution was approximately 0.448 l kg(-1) in all treatment groups. Oral bioavailability was also dose dependent, with the lower doses showing complete oral bioavailability. The half-life of S-4 over the dose range tested was between 2.6 and 5.3 h. 4: It was demonstrated that S-4 is rapidly absorbed, slowly cleared, and has a moderate volume of distribution in rats. The pharmacokinetics and oral bioavailability of S-4 indicate that it is an excellent candidate for clinical development.

Mass spectrometric characterization of the human androgen receptor ligand-binding domain expressed in Escherichia coli.

The ligand-binding domain (LBD) of the human androgen receptor (hAR LBD), encompassing amino acids (AAs) 647-919, was expressed in Escherichia coli with an N-terminal polyhistidine tag (His(10)-hAR LBD) from a pET-16b vector. The overexpressed protein was initially insoluble in inclusion bodies, and was subsequently solubilized in 8 M guanidine hydrochloride (GdnHCl). The solubilized His(10)-hAR LBD was purified to apparent homogeneity by metal ion affinity chromatography in the presence of 6 M GdnHCl. The isolated protein migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular mass of 33-34 kDa, as expected from the plasmid construct. Immunoblot analysis with C-terminal antibodies raised against a peptide corresponding to the last 19 AAs (AAs 901-919) of hAR revealed that the purified protein contained an immunoreactive epitope present within the AR and was of the appropriate size. Further characterization, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS), showed a single protein species of average mass 34 580 Da, confirming the size and purity of the purified His(10)-hAR LBD. Detailed tryptic peptide mapping analysis, using MALDI/TOF-MS, identified a total of eight peptides with a 30% coverage of the LBD, including the last tryptic peptide in the hAR sequence. These data confirm that the purified protein was the intact hAR LBD. AA sequencing of these tryptic peptides, using an HPLC-coupled electrospray ionization ion trap mass spectrometer (LC/ESI-ITMS and MS/MS), unambiguously confirmed that the peptides were from the hAR LBD. The purified His(10)-hAR LBD in 6 M GdnHCl could be renatured as determined by ligand-binding activity, with a similar equilibrium dissociation constant (K(d)) for [(3)H]-mibolerone and a similar steroid specificity to the AR isolated from rat ventral prostate.

Recombinant expression and purification of human androgen receptor in a baculovirus system.

A full-length human androgen receptor (hAR) cDNA was used to produce recombinant baculovirus. Spodoptera frugiperda (Sf9) cells infected with this virus expressed protein with an N-terminal hexahistidine tag (His(6)-hAR) in soluble and insoluble forms. The soluble cytosolic His(6)-hAR demonstrated similar association and dissociation half-times for mibolerone, similar binding affinity for mibolerone, and similar steroid specificity as bona fide AR. Under native conditions, the soluble cytosolic His(6)-hAR was purified to apparent homogeneity in the presence of dihydrotestosterone, using metal ion affinity chromatography. The insoluble pellet fraction was solubilized with strong denaturant 6 M guanidine HCl, and His(6)-hAR was purified from it in the presence of 6 M guanidine HCl. Both the solubilized crude pellet fraction and the solubilized/purified His(6)-hAR could be renatured to bind mibolerone. The baculovirus system will therefore provide an efficient means for producing hAR for ligand-binding assays, as well as purifying hAR for detailed molecular analyses.

Identification of a novel phosphorylation site in human androgen receptor by mass spectrometry.

An N-terminal hexahistidine-tagged full-length human androgen receptor protein (His(6)-hAR) was overexpressed and purified to apparent homogeneity in the presence of dihydrotestosterone (DHT) in our previous studies. In-gel trypsin digestion of the purified DHT-bound His(6)-hAR, and tryptic peptide mapping using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS), detected a total of 17 peptides (21% coverage of hAR) with 9 peptides originating from the ligand-binding domain (LBD, 31% coverage of LBD). Amino acid sequencing analysis of the tryptic peptides from a separate in-gel digestion of the His(6)-hAR, using HPLC-coupled electrospray ionization ion trap mass spectrometry (LC/ESI-ITMS and MS/MS), unambiguously confirmed 21 peptides with 19% coverage of the hAR, of which 11 peptides originated from the LBD (35% coverage of LBD). These 21 peptides included 11 out of the 17 peptides detected by MALDI/TOF-MS. In addition, a novel serine phosphorylation site (Ser(308)) within the N-terminal transactivation domain of hAR was identified.

Homology modeling using multiple molecular dynamics simulations and docking studies of the human androgen receptor ligand binding domain bound to testosterone and nonsteroidal ligands.

To facilitate the rational design of novel and more potent androgen receptor ligands, three-dimensional models for the human androgen receptor ligand binding domain bound to testosterone have been developed. These models of the androgen receptor were based on the crystal structure of the highly homologous human progesterone receptor ligand binding domain. The homology modeled androgen receptor was refined using unrestrained multiple molecular dynamics simulations in explicit solvent. Key H-bonding partners with the 17-hydroxy group and 3-keto group of testosterone are Asn705 and Thr877, and Gln711 and Arg752, respectively. These models show the presence of a unique unoccupied cavity within the androgen receptor binding pocket which may be valuable in the development of novel selective androgen receptor ligands. A qualitative analysis of amino acid mutations within the hAR binding pocket that affect ligand binding are consistent with these androgen receptor models. In addition to testosterone, the binding modes of several hydroxyflutamide-like nonsteroidal ligands for the androgen receptor are investigated using flexible docking with FlexX followed by refinement of the initial complexes with molecular dynamics simulations. These docking studies indicate that Asn705 is an important determinant in binding hydroxyflutamide and its derivatives by participating in H-bond interactions with the alpha-hydroxy moiety of these ligands. In addition, the nitro functionality mimics the 3-keto group of the natural ligand testosterone and is involved in H-bonding interactions with Gln711 and Arg752. From these docking studies, we suggest a mechanism for the enantioselective binding of chiral hydroxyflutamide derivatives and expand upon the previously reported structure-activity relationship for hydroxyflutamide and its derivatives.

Time-variant increase in methylprednisolone clearance in patients with acute respiratory distress syndrome: a population pharmacokinetic study.

Methylprednisolone (MP) disposition was evaluated in 20 individuals who participated in an ongoing randomized, double-blind, placebo-controlled study designed to evaluate the efficacy of MP in the treatment of acute respiratory distress syndrome (ARDS). MP (1 mg/kg) was given as a loading infusion over 30 minutes followed by a 1 mg/kg/day continuous i.v. infusion. Patients were switched to oral MP upon restoration of oral intake. MP plasma concentrations (n = 110) were determined using a specific HPLC method. Population pharmacokinetic analysis was performed using nonlinear mixed-effects models, implemented in NONMEM, version V. MP plasma concentration data were described by a one-compartment open model with a time-dependent, non-linear increase in the clearance (CL) of MP during the course of therapy. Initial clearance of MP (CLo) in ARDS patients at the start of therapy increased to a maximal value (CLmax) after approximately 7 days. The estimate of CLmax was similar to the CL of MP in healthy individuals reported previously. Population mean estimates (+/- SE) of parameters in the model were as follows: CLo = 13.2 +/- 2.4 L/h, CLmax = 25.0 +/- 3.6 L/h, time of half-maximal increase in CL (T50) = 41.1 +/- 8.2 h, gamma (Hill coefficient) = 3.8 +/- 0.6, and volume of distribution (Vd) = 137 +/- 30.2 L. Disease progression indices and patient demographics were evaluated as covariates, and no significant correlation was found. Means (+/- SD) of plasma protein binding differed between healthy individuals (72% +/- 4%) and ARDS patients (46% +/- 11%) (p < 0.001). The pharmacokinetics of MP in ARDS patients has not been described previously.

Predictive ability of level A in vitro-in vivo correlation for ringcap controlled-release acetaminophen tablets.

PURPOSE: The goal of this study was to establish and validate an in vitro-in vivo correlation (IVIVC) for two sustained-release formulations (i.e., a matrix tablet and a RingCap banded matrix tablet) containing 750 mg of acetaminophen. METHODS: The in vitro dissolution and in vivo disposition of these formulations were examined by using a USP type III dissolution apparatus and a single-dose, three-way, crossover study that included an immediate-release acetaminophen dosage form, respectively. An IVIVC was established by using the mean fraction dissolved (FD) and mean fraction absorbed (FA) and used to simulate the plasma concentration-time profile of acetaminophen after administration of the matrix tablet (i.e., internal validation) and RingCap banded matrix tablet (i.e., external validation). RESULTS: A statistically significant relationship (r2 = 0.997, P < 0.001) existed between the FD and FA for matrix tablets and was best described by the equation (FA) = 0.984 x (FD) + 0.0133. The percent predictions errors in CMAX and AUCL were <10% when predicting the plasma concentration-time profiles for the two formulations, validating the internal and external predictability of the IVIVC. CONCLUSIONS: The data (i) show that in vitro dissolution data are a good predictor of in vivo fraction absorbed for acetaminophen, (ii) support the general use of in vitro dissolution data for readily soluble and readily absorbed drugs, (iii) suggest that acetaminophen may serve as a model drug for evaluating novel sustained-release delivery systems, and (iv) provide a tangible example of the limitations of current methods for predicting and validating IVIVC.

Cytochrome P-450 2C9 sensitizes human prostate tumor cells to cyclophosphamide via a bystander effect.

The goal of the present study was to examine the ability of cytochrome P450-2C9 (CYP2C9) to activate cyclophosphamide (CPA) and elicit tumor cell death. A CYP2C9-deficient human lymphoblastoid cell line (AHH-1 cells) and a derivative cell line (H2C9 cells) stably transfected with a cDNA encoding CYP2C9 were used. The catalytic activity present in cell lines was examined by measuring the conversion of diclofenac, a CYP2C9-specific substrate, to its 4'-hydroxy metabolite by high-pressure liquid chromatography. Initial rate plots were constructed and the maximal rate of formation (V(max)) and the Michaelis-Menten constant (K(m)) for diclofenac metabolism were determined. Cytotoxicity was studied by exposing the cells to 0.01 to 4 mM CPA in the presence or absence of sulfaphenazole, a CYP2C9-specific inhibitor. Cell survival was quantitated by determination of the level of tritiated thymidine incorporation. H2C9 cells quickly metabolized diclofenac, indicating the presence of high levels of CYP2C9. Kinetic experiments demonstrated a V(max) and K(m) of 0.62+/-0.012 pmol/min/10(6) cells and 6.16+/-0.62 microM, respectively, for diclofenac metabolism. Diclofenac 4'-hydroxylase activity was undetectable in AHH-1 cells. H2C9 cells were more sensitive to the cytotoxic effects of CPA (50% inhibitory concentration [IC(50)], 0.80+/-0.03 mM) than AHH-1 cells (IC(50), 4.07+/-0.35 mM). The cytotoxicity (IC(50), 1.99+/-0.14 mM) of CPA to H2C9 cells was blocked by sulfaphenazole, demonstrating that the chemosensitivity of these cells is a consequence of intracellular prodrug activation. H2C9 cells mediated a bystander killing effect for CYP2C9-negative PPC-1 cells, reducing the IC(50) of CPA from about 14 to 3.62+/-0.73 mM in PPC-1 cells when they were cocultured with H2C9 cells. These results suggest that the enzyme-prodrug system of CYP2C9 and CPA may be an effective combination for gene-directed enzyme prodrug therapy. Ongoing studies are examining the utility of this system for use in prostate cancer cells.

Mdr1 limits CYP3A metabolism in vivo.

We determined whether the drug efflux protein P-glycoprotein (Pgp) could influence the extent of CYP3A-mediated metabolism of erythromycin, a widely used model substrate for CYP3A. We compared CYP3A metabolism of erythromycin (a Pgp substrate) using the erythromycin breath test in mice proficient and deficient of mdr1 drug transporters. We first injected mdr1(+/+) mice with [(14)C]N-methyl erythromycin and measured the rate of appearance of (14)CO(2) in the breath as a measure of hepatic CYP3A activity. Animals treated with CYP3A inducers or inhibitor showed accelerated or diminished (14)CO(2) in the breath, respectively. The erythromycin breath test was next administered to mdr1a(-/-) and mdr1a/1b(+/+) and (-/-) mice. These animals had equivalent levels of immunoreactive CYP3A and CYP3A activity as measured by erythromycin N-demethylase activity in liver microsomes. Nevertheless, the rate of (14)CO(2) appearance in the breath showed no relationship with these measurements of CYP3A, but changed proportionally to expression of mdr1. The average breath test (14)CO(2) area under the curves were 1.9- and 1.5-fold greater in mdr1a/1b(-/-) and mdr1a(-/-) mice, respectively, compared with (+/+) mice, and CER(max) was 2-fold greater in mdr1a/1b(-/-) compared with (+/+) mice. We conclude that Pgp, by limiting intracellular substrate availability can be an important determinant of CYP3A metabolism of numerous medications that are substrates for CYP3A and Pgp.

Creation of polarized cells coexpressing CYP3A4, NADPH cytochrome P450 reductase and MDR1/P-glycoprotein.

PURPOSE: To develop model polarized cell systems expressing cytochrome P4503A4. NADPH P450 reductase, and P-glycoprotein (Pgp). METHODS: LLC-PK1 and derivative L-MDR1 cells stably expressing Pgp, the product of the multidrug resistance gene (MDR1), were transfected stably using either a mammalian neomycin selectable expression vector (CYP3A4-Neo) or an episomal vector based on Epstein-Barr virus (CYP3A4-Hygro). These CYP3A4 expressing cells were compared with LLC-PK1, L-MDR1, or Caco-2 cells transduced with Adenovirus-3A4 vector (Ad3A4) with or without simultaneous Adenovirus-P450 Reductase (AdRed) transduction. Cells were characterized for expression of CYP3A4 protein and CYP3A4 mediated metabolism towards midazolam and testosterone. Analysis of membrane integrity and drug transport assays were performed to determine whether infection with recombinant Ad3A4 +/- AdRed affected Pgp function. RESULTS: The rank order of optimal CYP3A4 expression and activities in LLC-PKI and L-MDR1 cells from highest to lowest was cells cotransduced with Ad3A4 plus AdRed >> Ad3A4 >>> CYP3A4-Hygro > CYP3A4-Neo. Similarly, coexpression of Ad3A4 plus AdRed led to enhanced CYP3A4 mediated metabolism in Caco-2 cells over cells with Ad3A4 alone. Incubation of transwell cultured cells expressing Ad3A4/AdRed with midazolam led to readily detectable metabolite in the medium. In microsomes from Caco-2 and LLC-PK1 cells, each co-transduced with Ad3A4/AdRed, Vmax values for testosterone 6beta-hydroxylase activity ranged from 414 to 1350 pmoles/min/mg, respectively. For either Caco-2 or LLC-MDR1 cells, TEER values and the rate of apical to basal and basal to apical transport of vinblastine or digoxin were similar in cells with and without Ad3A4/Red transduction. CONCLUSIONS: Polarized cellular systems coexpressing Ad3A4, AdRed, and the MDR1/Pgp transporter were developed and characterized. The results document the utility of these polarized model systems for simultaneous drug transport/drug metabolism studies. Since the experimental approach can be adapted to study the interplay of multiple enzyme/ transporting systems, it may find significant application as a screening tool for the pharmaceutical industry and as a more basic research tool to study the kinetics of intestinal drug bioavailability.

Chiral nonsteroidal affinity ligands for the androgen receptor. 1. Bicalutamide analogues bearing electrophilic groups in the B aromatic ring.

A series of chiral analogues of bicalutamide bearing electrophilic groups (isothiocyanate, N-chloroacetyl, and N-bromoacetyl) on aromatic ring B of the parent molecule were synthesized. These compounds were designed as affinity ligands for the androgen receptor (AR). We prepared the (R)- and (S)-optical isomers of these compounds as pure enantiomers. The AR binding affinities of these compounds were measured in a competitive binding assay with the radiolabeled high-affinity AR ligand, [(3)H]mibolerone. In accordance with our previous results for the enantiomers of bicalutamide, we found that all (R)-isomers demonstrated much higher binding affinity to the AR as compared to their corresponding (S)-isomers. The para-substituted affinity ligands in ring B bound the AR with higher affinities than the corresponding meta-substituted analogues. Oxidation of thioester affinity ligands to their sulfonyl analogues for the para-substituted compounds decreased AR binding affinities and similar modification increased binding affinities for corresponding meta-analogues. The least potent para-substituted sulfonyl compounds had higher AR binding affinities than the most potent meta-substituted sulfonyl compounds. Overall, the para-substituted unoxidized molecules demonstrated the highest AR binding affinity. Subsequent research using AR exchange assays and Scatchard analyses showed that the isothiocyanate affinity ligands (R)-7, (R)-9, and (R)-10 reported herein are the first specific chemoaffinity ligands for the AR.

Affinity labeling of the androgen receptor with nonsteroidal chemoaffinity ligands.

We synthesized a series of potential chemoaffinity ligands for the androgen receptor (AR) by means of structural modifications of bicalutamide, a known nonsteroidal antiandrogen used in the treatment of hormone-dependent prostate cancer. We determined AR binding affinities of these ligands, identified chemoaffinity ligands by exchange assays, and confirmed irreversible binding to the AR by Scatchard analyses. AR binding affinity was determined in a competitive binding assay with a radiolabeled high-affinity AR ligand, [3H]mibolerone ([3H]MIB). For exchange assays, AR were incubated with an excess of each ligand, and then adsorbed onto hydroxyapatite (HAP). HAP-bound AR then were incubated with [3H]MIB to determine the remaining exchangeable specific binding sites. To determine the concentration of binding sites (Bmax), using Scatchard analysis, AR were incubated with a fixed concentration of ligand and increasing [3H]MIB concentrations. The ligands showed a wide range of AR binding affinities. In the exchange assays, three isothiocyanate derivatives of R-bicalutamide, the p-isothiocyanate (R-4), the p-thio-isothiocyanate (R-6), and the m-isothiocyanate (R-3), reduced exchangeable specific binding of [3H]MIB by 85, 84, and 50%, respectively. The S-isomer of p-thio-isothiocyanate (S-6), which showed 700-fold lower AR binding affinity than R-6, did not reduce exchangeable specific binding of [3H]MIB. In Scatchard analyses, the isothiocyanate derivatives R-3, R-4, and R-6 showed significant and progressive reduction in Bmax at increasing concentrations. The results indicate that initial specific reversible AR binding was required for subsequent covalent labeling, and that R-3, R-4, and R-6 bound the AR specifically and irreversibly. These isothiocyanate derivatives of R-bicalutamide are the first specific chemoaffinity ligands for the AR, and will provide valuable tools for the molecular characterization of the ligand binding domain of the AR.

Use of intravenous valproate in three pediatric patients with nonconvulsive or convulsive status epilepticus.

OBJECTIVE: To report the pharmacokinetics of intravenous valproate (VPA) in children with generalized convulsive status epilepticus (GCSE) or nonconvulsive status epilepticus (NCSE). To provide loading and maintenance dosing for patients with hepatic induction secondary to concurrent anticonvulsants. CASE SUMMARY: Two patients (10 y, 34 mo) with GCSE refractory to benzodiazepines, phenobarbital, phenytoin, and pentobarbital received intravenous VPA. Apparent volume of distribution (Vd) following a 20 mg/kg loading dose was 0.29 L/kg. Maintenance infusions of 4-6 mg/kg/h produced steady-state total concentrations of 66 mg/L and 92.4 mg/L (unbound concentration 44.6 mg/L). Clearance ranged from 63-66 mL/h/kg. An eight-year-old with NCSE received intravenous VPA (13.4 mg/kg load followed by 9 mg/kg every 8 h). Total and unbound steady-state VPA concentrations were 32.9 mg/L and 21.2 mg/L, respectively. Elimination half-life was eight hours. DISCUSSION: We constructed a pharmacokinetic simulation using VPA parameters from children receiving mono- or polyanticonvulsants. Our Vd and elimination half-life rates were comparable with published pediatric values. Patients on hepatic inducers had clearance rates 2.5 times those of children receiving oral anticonvulsant polytherapy. Unbound fractions (48.3% and 66%) were significantly higher than normal. CONCLUSIONS: A 20 mg/kg loading dose should produce a concentration after the bolus dose of approximately 75 mg/L. Initial infusion should consider hepatic induction (noninduced = 1 mg/kg/h, polyanticonvulsant therapy = 2 mg/kg/h, and high-dose pentobarbital = 4 mg/kg/h). Adjustments should be based on response and serum concentrations.

Pharmacokinetics of aminolevulinic acid after oral and intravenous administration in dogs.

The purpose of these studies was to examine the pharmacokinetics, oral bioavailability, and systemic side effects of aminolevulinic acid (ALA) in beagle dogs after oral and i.v. administration. Oral and i.v. doses of ALA (128 mg of ALA hydrochloride, equivalent to 100 mg of ALA) were administered to four animals using a crossover design. Animals were allowed a 2-week washout period between doses. Plasma ALA concentrations were determined using precolumn fluorescent derivatization and reversed-phase HPLC. Plasma concentrations after i.v. administration declined rapidly with a terminal half-life of 19.5 +/-2.5 min (mean +/- S.D.). Total body clearance and volume of distribution at steady state averaged 6.79+/-1.77 ml/min/kg and 259+/- 128 ml/kg, respectively. Peak plasma concentrations of ALA after oral administration ranged from 1.27 to 9.42 microgram/ml. Oral bioavailability in these animals averaged 41.2+/-14.8% (range, 23.5-58.5%). These studies demonstrate that oral administration may provide a convenient and efficient route of delivery of ALA for photodynamic therapy in patients.

Pharmacokinetics of chlorpheniramine, phenytoin, glipizide and nifedipine in an individual homozygous for the CYP2C9*3 allele.

Genetic polymorphisms in the cytochrome P450 (CYP) family are widely known to contribute to interindividual differences in the pharmacokinetics of many drugs. Several alleles for the CYP2C9 gene have been reported. Individuals homozygous for the Leu359 variant (CYP2C9*3) have been shown to have significantly lower drug clearances compared with Ile359 (CYP2C9*1) homozygous individuals. A male Caucasian who participated in six bioavailability studies in our laboratory over a period of several years showed extremely low clearance of two drugs: phenytoin and glipizide (both substrates of CYP2C9), but not for nifedipine (a CYP3A4 substrate) and chlorpheniramine (a CYP2D6 substrate). His oral clearance of phenytoin was 21% of the mean of the other 11 individuals participating in the study, and his oral clearance of glipizide, a second generation sulfonylurea structurally similar to tolbutamide, was only 188% of the mean of the other 10 individuals. However, his oral clearance of nifedipine and chlorpheniramine did not differ from individuals in other studies performed at our laboratories. An additional blood sample was obtained from this individual to determine if he possessed any of the known CYP2C9 or CYP2C19 allelic variants that would account for his poor clearance of the CYP2C9 substrates (phenytoin and glipizide) compared with the CYP3A4 (nifedipine) and CYP2D6 (chlorpheniramine) substrates. The results of the genotype testing showed that this individual was homozygous for the CYP2C9*3 allele and did not possess any of the known defective CYP2C19 alleles. This study establishes that the Leu359 mutation is responsible for the phenytoin and glipizide/tolbutamide poor metabolizer phenotype.

Pharmacokinetics of aminolevulinic acid after intravesical administration to dogs.

PURPOSE: To examine the stability and systemic absorption of aminolevulinic acid (ALA) in dogs during intravesical administration. METHODS: Nine dogs received an intravesical dose of ALA either with no prior treatment, after receiving ammonium chloride for urinary acidification, or after receiving sodium bicarbonate for urinary alkalinization. Urine and blood samples collected during and after administration were monitored for ALA using an HPLC assay developed in our laboratories. Concentrations of pyrazine 2,5-dipropionic acid, the major ALA degradation product, and radiolabeled inulin, a nonabsorbable marker for urine volume, were also determined. RESULTS: Less than 0.6% of intravesical ALA doses was absorbed into plasma. Urine concentrations decreased to 37% of the initial concentration during the 2 hour instillation. Decreases in urinary ALA and radiolabeled inulin concentrations were significantly correlated, indicating that urine dilution accounted for over 80% of observed decreases in urinary ALA. ALA conversion to pyrazine 2,5-dipropionic acid was negligible. CONCLUSIONS: These studies demonstrate that ALA is stable and poorly absorbed into the systemic circulation during intravesical instillation. Future studies utilizing intravesical ALA for photodiagnosis of bladder cancer should include measures to restrict fluid intake as a means to limit dilution and maximize ALA concentrations during instillation.

Discovery of nonsteroidal androgens.

Nonsteroidal androgens have not been reported. During studies to identify affinity ligands for the androgen receptor in our laboratory, we synthesized several electrophilic nonsteroidal ligands for the androgen receptor and examined their receptor binding affinity and ability to stimulate receptor-mediated transcriptional activation. We found that three of these ligands (1) bound the androgen receptor with affinity similar to that of dihydrotestosterone (the endogenous ligand) and (2) mimicked the effects of dihydrotestosterone on receptor-mediated transcriptional activation (i.e., they were receptor agonists). These studies demonstrate that nonsteroidal ligands can be structurally modified to produce agonist activity. These ligands thus represent the first members of a novel class of androgens with potential therapeutic applications in male fertility and hormone replacement therapy.

Medetomidine analogs as alpha 2-adrenergic ligands. 3. Synthesis and biological evaluation of a new series of medetomidine analogs and their potential binding interactions with alpha 2-adrenoceptors involving a "methyl pocket".

The synthesis and the biological evaluation of a new series of medetomidine analogs are reported. The substitution pattern at the phenyl ring of the tetralin analogs had a distinct influence on the alpha 2-adrenoceptor binding affinity. 4-Methylindan analog 6 was the most potent alpha 2-adrenoceptor binding ligand among these 4-substituted imidazoles, and its alpha 2-adrenoceptor selectivity was greater than the 5-methyl tetralin analog 4c. Ligand-pharmacophore and receptor modeling were combined to rationalize alpha 2-adrenoceptor binding data of the imidazole analogs in terms of ligand-receptor interactions. The structure-activity relationships that were apparent from this and previous studies were qualitatively rationalized by the binding site models of the alpha 2-adrenoceptor. The benzylic methyl group of medetomidine or the naphthyl analog 2a was superimposable with the alpha-methyl group of (-)-alpha-methylnorepinephrine and fit into the proposed "methyl pocket" of the alpha 2-adrenoceptor defined by the residues Leu110, Leu169, Phe391, and Thr395.