Effect of age and gender on the pharmacokinetics of R667, a novel agent for the treatment of emphysema, in healthy volunteers.

The purpose of the present study was to assess the impact of age and gender on the pharmacokinetics (PK) of R667. Thirty six healthy male and female volunteers (12 m 18-45 years; 12 m and 12 f > or = 65 years) received a single 1 mg oral dose of R667. Serial blood samples were collected for determination of plasma R667 and metabolite concentrations. The PK parameters of R667 were similar between elderly males and young males (Cmax = 9.8 vs 9.8 ng/mL; AUC(0-last) = 50.9 vs 47.3 ng x h/mL, respectively). Exposure of R667 increased in elderly females compared to elderly males (Cmax = 13.1 vs 9.8 ng/mL; AUC(0-last) = 60.8 vs 47.3 ng x h/mL, respectively). When the CL/F was corrected for BSA and V/F corrected for weight these differences were no longer evident. In conclusion these exposure differences are not considered clinically relevant, and no dose adjustment of R667 is required based on gender or age.

Bioanalytical method development and validation for a large peptide HIV fusion inhibitor (Enfuvirtide, T-20) and its metabolite in human plasma using LC-MS/MS.

A method for measuring a human immunodeficiency virus (HIV) cell membrane fusion inhibitor (T-20/Ro 29-9800) and its metabolite (M-20/Ro 50-6343) in human plasma by liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed. The relatively large peptide analytes and their corresponding deuterated (d(10)) peptides used as internal standard were isolated from plasma by protein precipitation with two volumes of acetonitrile to plasma. A large pore size reversed-phase C(18) column was employed to elute the peptides. A triple quadrupole mass spectrometer with electrospray interface operating in positive ion and multiple reaction monitoring modes with transitions m/z 1124-->1343 for both T-20 and M-20 was utilized for peak detection. The advantages of the method were a simple sample preparation, specific and sensitive MS/MS detection, and a wide dynamic range of 10-2000 ng/ml for T-20. The method was validated and used for analyzing samples from clinical studies to provide pharmacokinetic profiles of the HIV fusion inhibitor peptide drug and its metabolite.

Antitumor efficacy of AG3340 associated with maintenance of minimum effective plasma concentrations and not total daily dose, exposure or peak plasma concentrations.

Oral administration of AG3340, a novel metalloprotease (MMP) inhibitor, suppresses the growth of human colon adenocarcinoma (COLO-320DM) tumors in vivo (Proc Am Assoc Cancer Res 39: 2059, 1998). In this report, we tested the hypothesis that the growth inhibition of these tumors is associated with maintaining minimum effective plasma concentrations of AG3340. Nude mice were given a total oral daily dose of 25 or 200 mg/kg; 6.25 mg/kg was given four times per day (QID) (25 mg/kg/day), and 100 mg/kg was given in two daily doses (BID) (200 mg/kg/day). Peak plasma concentrations (Cmax) of 83 +/- 43 (mean +/- SD) and 1998 +/- 642 ng/ml were detected 30 min after a single dose with 6.25 mg/kg and 100 mg/kg AG3340, respectively. AUC(0-24 h) values estimated from dosing with 25 and 200 mg/kg/day AG3340 were 672 and 10882 ng*h/ml, respectively. Importantly, both regimen inhibited tumor growth equivalently (74 to 82%). Efficacy was also compared at a total daily dose of 25 mg/kg by giving AG3340: QID (6.25 mg/kg per dose), BID (12.5 mg/kg per dose), and once daily (25 mg/kg per dose). The Cmax of these regimens was 83 +/- 43, 287 +/- 175 and 462 +/- 495 ng/ml, respectively. AG3340 did not inhibit tumor growth with the latter two regimens. The efficacy of 6.25 mg/kg QID (25 mg/kg/day) was superior to the efficacy of 25 mg/kg BID (50 mg/kg/day), substantiating the independence of efficacy from the total daily dose and Cmax. Expectedly, peak to trough fluctuations were significantly smaller with the QID regimen than with BID and QD dosing. After 24 h, the trough was greater than 1 ng/ml with QID dosing but was less than 1 ng/ml after QD and BID dosing. These results suggest that the antitumor efficacy of AG3340 was associated with maintaining minimum effective plasma concentrations of AG3340 and demonstrate that the antitumor efficacy of AG3340 was independent of the total daily dose, peak plasma concentration, and drug exposure in this tumor model.

A proposed mechanism for p-aminoclonidine allergenicity based on its relative oxidative lability.

p-Aminoclonidine (apraclonidine) is a selective alpha 2 adrenergic agonist used to reduce intraocular pressure in the treatment of glaucoma. Use of apraclonidine is frequently associated with severe local allergic effects which warrant discontinuation of the drug in affected patients. We have assessed the oxidative lability of apraclonidine relative to a panel of adrenergic agonists and/or known allergens; amodiaquine, epinephrine, clonidine, and brimonidine. These compounds were compared by their electrochemical potentials as well as their oxidative lability in the presence of several oxidative enzyme systems (i.e., horseradish peroxidase, lactoperoxidase, myeloperoxidase, and diamine oxidase). The half-lives for enzymatic oxidation of these compounds were found to parallel the electrochemical oxidation potentials in the order: amodiaquine approximately epinephrine < apraclonidine << clonidine approximately brimonidine. The production of a reactive electrophilic intermediate of apraclonidine was demonstrated through the formation of two glutathione apraclonidine adducts from the horseradish peroxidase/H2O2-mediated oxidation of apraclonidine in the presence of glutathione. A mechanism for apraclonidine allergenicity in vivo is proposed wherein apraclonidine is bioactivated through oxidation to the bis-iminoquinone followed by protein conjugation to form an apraclonidine-protein hapten that elicits the immune response.

Identification of trimoprostil metabolites excreted in rat bile formed by oxidation and taurine conjugation.

14C-Trimoprostil was administered iv and orally to male rats. Radioactivity was excreted mainly via the feces after both routes of drug administration. Bile duct-cannulated rats excreted an average of 76% of an iv dose in the bile within 6 hr after dosing. Four biliary metabolites were isolated by HPLC and identified by proton NMR spectroscopy and mass spectrometry. These metabolites were taurine conjugates of: trimoprostil (approximately 11% of dose), 5,6-dihydro-2,3-dinor trimoprostil (approximately 5% of dose), 3,4-dehydro trimoprostil (less than 5% of dose) and 5,6-dihydro-2,3,4,5-tetranor trimoprostil (less than 5% of dose).

Metabolites of an antipsychotic chiral pyrroloisoquinoline and their effect on prolactin secretion in the rat.

Racemic-2, 6-dimethyl-3-ethyl- 4,4a,5,6,7,8,8a, 9-octahydro-4a,8a-trans-1H-pyrrolo[2, 3-g]isoquinoline-4-one hydrochloride (rac-l HCl) appeared to be equipotent to haloperidol in increasing serum prolactin levels in rats although only about one-twentieth as potent as haloperidol in reversing dopamine-inhibited prolactin release by rat anterior pituitary cells in vitro. The metabolism of 14C-labeled rac-l HCl was studied in rats and the activity of metabolites was evaluated in the in vitro prolactin release assay. Four metabolites, two C11-monohydroxy diastereomers and one C10-and one C12-monohydroxy metabolite, plus parent drug were isolated from the urine of rats administered [14C]rac-l HCl. These were shown to be optically active, indicating that the racemic drug was stereoselectively metabolized. All of the identified metabolites plus those in extracts of urine and feces proved inactive in the in vitro prolactin release assay. In addition, the brains of three rats removed 1 hr after single 3.6 mg/kg oral doses of [14C]rac-l HCl contained essentially only unchanged drug. We conclude that the potency of rac-l HCl in raising serum prolactin levels is not due to the formation of active metabolite(s) in vivo.

Metabolism of an anxiolytic imidazobenzodiazepine by the dog.

14C-labeled 8-chloro-6-(2-chlorophenyl)-4H-imidazo [1,5a][1,4]-benzodiazepine-3-carboxamide hydrochloride, 1 X HCl, was administered iv to two dogs. 14C-1 X HCl was extensively metabolized; in one dog, only 2% of the administered radioactivity was excreted as unchanged 1. The 4-hydroxy derivative of 1, compound 2, accounted for an additional 5% of the dose. Three urinary metabolites were identified by enzyme hydrolysis and NMR spectroscopy as conjugates of the phenolic 3'-, 4'-, and 5'-hydroxy derivatives of 1. In both dogs, the 4'-hydroxy derivative was the major identified metabolite (21 and 15% of the dose). Oral administration of 100 mg/kg/day of unlabeled 1 to dogs for 11 weeks resulted in the excretion of four urinary metabolites, the conjugated 3'-hydroxy and 4'-hydroxy derivatives of both 1 and 2. This prominent excretion of conjugated phenolic metabolites does not fit the usual pattern of 1,4-benzodiazepine metabolism in the dog.

Biotransformation of [14C]midazolam in the rat in vitro and in vivo.

1. Aerobic incubation of the maleate salt of midazolam, 8-chloro-6-(2-fluorophenyl)-1-methyl-4H-imidazo[1,5-a][1,4]benzodiazepine, with rat liver 9000 g supernatant fluid fortified with an NADPH-generating system yielded three metabolites. These were identified with an 1-hydroxymethyl derivative formed by oxidation of the methyl substituent on the imidazo moiety, the derivative hydroxylated at C-4 of the diazepine ring, and the corresponding dihydroxy derivative. 2. Pretreatment of rats with phenobarbital stimulated the oxidative metabolism in vitro of midazolam by 9000 g liver supernatant. 3. Rats given 2.5 mg/kg of [14C]midazolam maleate i.v. excreted 81% of the administered radioactivity in the faeces and 10% in the urine in 24 h. The highest levels of 14C were in the liver and the lowest in the brain during the first day. 4. Four phenolic derivatives of midazolam were identified in rat bile. These were the 4'-hydroxy and the 4'-hydroxy-1-hydroxymethyl derivatives of midazolam and two benzophenones. Evidence that the benzophenones were artifacts derived from the 4-hydroxy derivatives of the two biliary benzodiazepine metabolites is presented. These biliary metabolites were excreted as glucuronide and/or sulphate conjugates. 5. The Propensity of the rat to excrete phenolic metabolites of midazolam in bile is in agreement with the metabolism of several other benzodiazepines in this species.

The metabolism of 14C-methoxsalen by the dog.

Single doses of 14C-methoxsalen (5 mg/kg) were administered iv to three dogs. Almost as much administered radioactivity was excreted in the feces as in the urine, suggesting that biliary secretion of metabolites was an important route of excretion. 14C-Methoxsalen disappeared rapidly from plasma, although plasma levels of radioactivity persisted for 5 weeks after drug administration. Evidence was obtained which suggested that the persistent plasma radioactivity was due to a metabolite bound to plasma protein. Four urinary metabolites were isolated. Three of the metabolites resulted from opening of the furan ring; these are 7-hydroxy-8-methoxy-2-oxo-2H-1-benzopyran-6-acetic acid (A), alpha,7-dihydroxy-8-methoxy-2-oxo-2H-1-benzopyran-6-acetic acid (B), and an unknown conjugate of A at the 7-hydroxy position. The fourth metabolite, formed by opening of the pyrone ring, is an unknown conjugate of (Z)-3-(6-hydroxy-7-methoxybenzofuran-5-yl)-2-propenoic acid.

Biotransformation of prochiral 2-phenyl-1,3-di(4-pyridyl)-2-propanol to a chiral N-oxide metabolite.

The prochiral compound, 2-phenyl-1,3-di(4-pyridyl)-2-propanol (PPP) labeled with 3H in the phenyl ring, was administered to rats, dogs, and a human subject. Paper chromatography of the urine indicated that a major metabolite common to all three species was excreted. This metabolite was isolated from the urine of chronically dosed dogs and was identified by mass, nuclear magnetic resonance (NMR), and infrared spectrometry as the N-oxide, 2-phenyl-1-(4-pyridyl)-3-(4-pyridyl-1-oxide)-2-propanol. In addition, polarimetry indicated that this metabolite was levorotatory. Examination of the enantiomeric purity of a crystallized sample of the metabolite by NMR spectroscopy of resolvable diastereomeric salts formed with lasalocid revealed the presence of only the levorotatory enantiomer. Accordingly, this metabolic N-oxide formation in the dog was at least stereoselective, and perhaps stereospecific. The N-oxidation of PPP was also demonstrated in vitro with 9000 g supernatant fraction of rat liver fortified with an NADPH generating system, and this reaction was inducible by phenobarbital, indicating that it is mediated by the cytochrome P-450 mixed-function oxidase system. This study, in addition to providing another example of the pyridyl N-oxidation pathway, illustrates the necessity of considering the stereochemical aspects of the metabolism of prochiral drugs.

A comparative diuretic and tissue distribution study of bumetanide and furosemide in the dog.

Intravenous dose-response data obtained from renal clearance studies in anesthetized dogs indicated that bumetanide was approximately 30-fold more potent than furosemide in enhancing sodium excretion. After the administration of 0.01 mg/kg of bumetanide or 1.0 mg/kg of furosemide, the relationship between i.v. diuretic activity and tissue distribution was evaluated. In dog renal clearance experiments, bumetanide and furosemide significantly enhanced urine flow, sodium and potassium excretion. Inulin clearance as an estimate of glomerular filtration rate was not altered by either drug, but sodium reabsorption was decreased with bumetanide (13%) and furosemide (12%). At these diuretic doses, both compounds were bound to dog plasma protein to about the same extent (86-91%), although total plasma levels were 100-fold higher for furosemide. Within 1/2 hour after the i.v. administration of 14C-bumetanide or 14C-furosemide, 86 to 99% of the 14C in urine, plasma, kidney, and liver appeared as unchanged drug. One minute after maximal diuresis bumetanide was found to have a higher affinity (3-fold) for kidney compared to furosemide. These data offer a possible explanation for the i.v. diuretic potency difference between these two compounds. Furthermore, the lack of significant difference in plasma protein binding and the absence of urinary metabolites of either drug suggest that other factors may also contribute to the marked differences in diuretic activity between bumetanide and furosemide.

Identification of the urinary metabolites of 14C-bumetanide in the rat and their excretion by rats and dogs.

Unchanged bumetanide, 3-(n-butylamino)-4-phenoxy-5-sulfamoylbenzoic acid, and five metabolites were excreted in the urine of rats given 50 mg of 14C-labeled drug per kg intravenously. The metabolites, which were identified by mass spectrometry and/or nuclear magnetic resonance spectroscopy, arose by metabolic alteration of the n-butyl sidechain. In metabolites I-V, the butylamino group was converted to -NHCH2CH2CH2COOH, -NHCH2CH2CH2CH2OH,, -NHCH2CH2CHOHCH3, -NHCH2CH2CHOHCH2OH and -NH2, respectively. The total urinary and fecal excretion of labeled drug and metabolites after iv and oral administration of 14C-bumetanide was estimated in dogs given 0.5 mg/kg and in rats given 5 mg/kg. In the dog, the primary excretion product was unchanged drug, although evidence was obtained that the acyl glucuronide of bumetanide was secreted in dog bile. The major metabolite excreted by the rat was I, and negligible quantities of intact drug were excreted in the urine after oral or iv administration. Optical activity was found for the two metabolites that contained a chiral center (III and IV), indicating that they were formed by a stereoselective hydroxylation.