Pharmacokinetics and metabolism studies on the glucagon-like peptide-1 (GLP-1)-derived metabolite GLP-1(9-36)amide in male Beagle dogs.

Glucagon-like peptide-1 (GLP-1)(7-36)amide is a 30-amino acid peptide hormone that is secreted from intestinal enteroendocrine L-cells in response to nutrients. GLP-1(7-36)amide possesses potent insulinotropic actions in the augmentation of glucose-dependent insulin secretion. GLP-1(7-36)amide is rapidly metabolized by dipeptidyl peptidase-IV to yield GLP-1(9-36)amide as the principal metabolite. Contrary to the earlier notion that peptide cleavage products of native GLP-1(7-36)amide [including GLP-1(9-36)amide] are pharmacologically inactive, recent studies have demonstrated cardioprotective and insulinomimetic effects with GLP-1(9-36)amide in mice, dogs and humans. In the present work, in vitro metabolism and pharmacokinetic properties of GLP-1(9-36)amide have been characterized in dogs, since this preclinical species has been used as an animal model to demonstrate the in vivo vasodilatory and cardioprotective effects of GLP-1(9-36)amide. A liquid chromatography tandem mass spectrometry assay was developed for the quantitation of the intact peptide in hepatocyte incubations as opposed to a previously reported enzyme-linked immunosorbent assay. Although GLP-1(9-36)amide was resistant to proteolytic cleavage in dog plasma and bovine serum albumin (t1/2>240 min), the peptide was rapidly metabolized in dog hepatocytes with a t1/2 of 110 min. Metabolite identification studies in dog hepatocytes revealed a variety of N-terminus cleavage products, most of which, have also been observed in human and mouse hepatocytes. Proteolysis at the C-terminus was not observed in GLP-1(9-36)amide. Following the administration of a single intravenous bolus dose (20 µg/kg) to male Beagle dogs, GLP-1(9-36)amide exhibited a mean plasma clearance of 15 ml/min/kg and a low steady state distribution volume of 0.05 l/kg, which translated into a short elimination half life of 0.05 h. Following subcutaneous administration of GLP-1(9-36)amide at 50 µg/kg, systemic exposure of GLP-1(9-36)amide as ascertained from maximal plasma concentrations and area under the plasma concentration-time curve from zero to infinity was 44 ng/ml and 32 ng h/ml, respectively. The subcutaneous bioavailability of GLP-1(9-36)amide in dogs was 57%. Our findings raise the possibility that the cardioprotective effects of GLP-1(9-36)amide in the conscious dog model of pacing-induced heart failure might be due, at least in part, to the actions of additional downstream metabolites, which are obtained from proteolytic cleavage of the peptide backbone in the parent compound in the liver.

Pharmacological inhibition to examine the role of DGAT1 in dietary lipid absorption in rodents and humans.

Alterations in fat metabolism, in particular elevated plasma concentrations of free fatty acids and triglycerides (TG), have been implicated in the pathogenesis of Type 2 diabetes, obesity, and cardiovascular disease. Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1), a member of the large family of membrane-bound O-acyltransferases, catalyzes the final step in triacylglycerol formation. In the intestine, DGAT1 is one of the acyltransferases responsible for the reesterficiation of dietary TG. Following a single dose of a selective pharmacological inhibitor of DGAT1, PF-04620110, a dose-dependent inhibition of TG and vitamin A absorption postprandially was demonstrated in rodents and human subjects. In C57/BL6J mice, acute DGAT1 inhibition alters the temporal and spatial pattern of dietary lipid absorption. To understand the impact of DGAT1 inhibition on enterocyte lipid metabolism, lipomic profiling was performed in rat intestine and plasma as well as human plasma. DGAT1 inhibition causes an enrichment of polyunsaturated fatty acids within the TG class of lipids. This pharmacological intervention gives us insight as to the role of DGAT1 in human dietary lipid absorption.

In vitro metabolism of the glucagon-like peptide-1 (GLP-1)-derived metabolites GLP-1(9-36)amide and GLP-1(28-36)amide in mouse and human hepatocytes.

Previous studies have revealed that the glucoincretin hormone glucagon-like peptide-1 (GLP-1)(7-36)amide is metabolized by dipeptidyl peptidase-IV (DPP-IV) and neutral endopeptidase 24.11 (NEP) to yield GLP-1(9-36)amide and GLP-1(28-36)amide, respectively, as the principal metabolites. Contrary to the previous notion that GLP-1(7-36)amide metabolites are pharmacologically inactive, recent studies have demonstrated cardioprotective and insulinomimetic effects with both GLP-1(9-36)amide and GLP-1(28-36)amide in animals and humans. In the present work, we examined the metabolic stability of the two GLP-1(7-36)amide metabolites in cryopreserved hepatocytes, which have been used to demonstrate the in vitro insulin-like effects of GLP-1(9-36)amide and GLP-1(28-36)amide on gluconeogenesis. To examine the metabolic stability of the GLP-1(7-36)amide metabolites, a liquid chromatography-tandem mass spectrometry assay was developed for the quantitation of the intact peptides in hepatocyte incubations. GLP-1(9-36)amide and GLP-1(28-36)amide were rapidly metabolized in mouse [GLP-1(9-36)amide: t(1/2) = 52 minutes; GLP-1(28-36)amide: t(1/2) = 13 minutes] and human hepatocytes [GLP-1(9-36)amide: t(1/2) = 180 minutes; GLP-1(28-36)amide: t(1/2) = 24 minutes), yielding a variety of N-terminal cleavage products that were characterized using mass spectrometry. Metabolism at the C terminus was not observed for either peptides. The DPP-IV and NEP inhibitors diprotin A and phosphoramidon, respectively, did not induce resistance in the two peptides toward proteolytic cleavage. Overall, our in vitro findings raise the intriguing possibility that the insulinomimetic effects of GLP-1(9-36)amide and GLP-1(28-36)amide on gluconeogenesis and oxidative stress might be due, at least in part, to the actions of additional downstream metabolites, which are obtained from the enzymatic cleavage of the peptide backbone in the parent compounds.

In vitro-in vivo correlation for low-clearance compounds using hepatocyte relay method.

In vitro-in vivo correlation (IVIVC) of intrinsic clearance in preclinical species of rat and dog was established using the hepatocyte relay method to support high-confidence prediction of human pharmacokinetics for low-clearance compounds. Good IVIVC of intrinsic clearance was observed for most of the compounds, with predicted values within 2-fold of the observed values. The exceptions involved transporter-mediated uptake clearance or metabolizing enzymes with extensive extrahepatic contribution. This is the first assay available to address low clearance challenges in preclinical species for IVIVC in drug discovery. It extends the utility of the hepatocyte relay method in addressing low clearance issues.

Demonstration of the innate electrophilicity of 4-(3-(benzyloxy)phenyl)-2-(ethylsulfinyl)-6-(trifluoromethyl)pyrimidine (BETP), a small-molecule positive allosteric modulator of the glucagon-like peptide-1 receptor.

4-(3-(Benzyloxy)phenyl)-2-(ethylsulfinyl)-6-(trifluoromethyl)pyrimidine (BETP) represents a novel small-molecule activator of the glucagon-like peptide-1 receptor (GLP-1R), and exhibits glucose-dependent insulin secretion in rats following i.v. (but not oral) administration. To explore the quantitative pharmacology associated with GLP-1R agonism in preclinical species, the in vivo pharmacokinetics of BETP were examined in rats after i.v. and oral dosing. Failure to detect BETP in circulation after oral administration of a 10-mg/kg dose in rats was consistent with the lack of an insulinotropic effect of orally administered BETP in this species. Likewise, systemic concentrations of BETP in the rat upon i.v. administration (1 mg/kg) were minimal (and sporadic). In vitro incubations in bovine serum albumin, plasma, and liver microsomes from rodents and humans indicated a facile degradation of BETP. Failure to detect metabolites in plasma and liver microsomal incubations in the absence of NADP was suggestive of a covalent interaction between BETP and a protein amino acid residue(s) in these matrices. Incubations of BETP with glutathione (GSH) in buffer revealed a rapid nucleophilic displacement of the ethylsulfoxide functionality by GSH to yield adduct M1, which indicated that BETP was intrinsically electrophilic. The structure of M1 was unambiguously identified by comparison of its chromatographic and mass spectral properties with an authentic standard. The GSH conjugate of BETP was also characterized in NADPH- and GSH-supplemented liver microsomes and in plasma samples from the pharmacokinetic studies. Unlike BETP, M1 was inactive as an allosteric modulator of the GLP-1R.

Immune-mediated agranulocytosis caused by the cocaine adulterant levamisole: a case for reactive metabolite(s) involvement.

The United States Public Health Service Administration is alerting medical professionals that a substantial percentage of cocaine imported into the United States is adulterated with levamisole, a veterinary pharmaceutical that can cause blood cell disorders such as severe neutropenia and agranulocytosis. Levamisole was previously approved in combination with fluorouracil for the treatment of colon cancer; however, the drug was withdrawn from the U.S. market in 2000 because of the frequent occurrence of agranulocytosis. The detection of autoantibodies such as antithrombin (lupus anticoagulant) and an increased risk of agranulocytosis in patients carrying the human leukocyte antigen B27 genotype suggest that toxicity is immune-mediated. In this perspective, we provide an historical account of the levamisole/cocaine story as it first surfaced in 2008, including a succinct review of levamisole pharmacology, pharmacokinetics, and preclinical/clinical evidence for levamisole-induced agranulocytosis. Based on the available information on levamisole metabolism in humans, we propose that reactive metabolite formation is the rate-limiting step in the etiology of agranulocytosis associated with levamisole, in a manner similar to other drugs (e.g., propylthiouracil, methimazole, captopril, etc.) associated with blood dyscrasias. Finally, considering the toxicity associated with levamisole, we propose that the 2,3,5,6-tetrahydroimidazo[2,1-b]thiazole scaffold found in levamisole be categorized as a new structural alert, which is to be avoided in drug design.

A novel relay method for determining low-clearance values.

A novel relay method has been developed using cryopreserved human hepatocytes to measure intrinsic clearance of low-clearance compounds. The relay method involved transferring the supernatant from hepatocyte incubations to freshly thawed hepatocytes at the end of the 4-h incubation to prolong the exposure time to active enzymes in hepatocytes. An accumulative incubation time of 20 h or longer in hepatoctyes can be achieved using the method. The relay method was validated using seven commercial drugs (diazepam, disopyramide, theophylline, timolol, tolbutamide, S-warfarin, and zolmitriptan) that were metabolized by various cytochrome P450s with low human in vivo intrinsic clearance at approximately 2 to 15 ml · min⁻¹ · kg⁻¹. The results showed that the relay method produced excellent predictions of human in vivo clearance. The difference between in vitro and in vivo intrinsic clearance was within 2-fold for most compounds, which is similar to the standard prediction accuracy for moderate to high clearance compounds using hepatocytes. The relay method is a straightforward, relatively low cost, and easy-to-use new tool to address the challenges of low clearance in drug discovery and development.

Intrinsic electrophilicity of a 4-substituted-5-cyano-6-(2-methylpyridin-3-yloxy)pyrimidine derivative: structural characterization of glutathione conjugates in vitro.

Isopropyl 9-anti-[5-cyano-6-(2-methyl-pyridin-3-yloxy)-pyrimidin-4-yloxy]-3-oxa-7-aza-bicyclo[3.3.1]nonane-7-carboxylate (1) represents a prototypic compound from a lead chemical series of G protein-coupled receptor 119 agonists, intended for treatment of type 2 diabetes. When compound 1 was incubated with NADPH-supplemented human liver microsomes in the presence of glutathione, two thioether conjugates M4-1 and M5-1 were observed. Omission of NADPH from the microsomal incubations prevented the formation of M5-1 but not M4-1. The formation of M4-1 was also discerned in incubations of 1 and glutathione with human liver cytosol, partially purified glutathione transferase, and in phosphate buffer at pH 7.4. M4-1 was isolated, and its structure ascertained from LC-MS/MS and NMR analysis. The mass spectral and NMR data suggested that M4-1 was obtained from a nucleophilic displacement of the 6-(2-methylpyridin-3-yloxy) group in 1 by glutathione. In addition, mass spectral studies revealed that M5-1 was derived from an analogous displacement reaction on a monohydroxylated metabolite of 1; the regiochemistry of hydroxylation was established to be on the isopropyl group. Of great interest were the findings that replacement of the 5-cyano group in 1 with a 5-methyl group resulted in 2, which was practically inert toward reaction with glutathione. This observation suggests that the electron-withdrawing potential of the C5 cyano group serves to increase the electrophilicity of the C6 carbon (via stabilization of the transition state) and favors reaction with the nucleophilic thiol. The mechanistic insights gained from these studies should assist medicinal chemistry efforts toward the design of analogs that retain primary pharmacology but are latent toward reaction with biological nucleophiles, thus mitigating the potential for toxicological outcome due to adduction with glutathione or proteins.

Utility of linear and nonlinear models for retention prediction in liquid chromatography.

Linear solvation strength model in reversed-phase liquid chromatography assumes linear relationship between ln k and Φ. In this work we show that this assumption is true only in narrow range of mobile phase strength. The ln k versus Φ relationship could be more accurately described by three-parametric non-linear model in a wide range of eluent strength. We investigated the consequences of non-linearity on retention prediction accuracy and analyte retention behavior in reversed-phase chromatography. When the ln k versus Φ is measured in narrow range of mobile phase strength (ΔΦ ~ 0.1-0.2) both linear and nonlinear models provide comparable retention prediction results. We propose that the linear trend of ln k versus Φ relationship is obtained in the range flanking the elution factor ke (value of retention factor at the column end). We calculated and plotted changes of retention factor of analytes along the column. The visualization illustrates the ranges of retention factor values participating in separation during gradient. For typical gradient slopes employed in liquid chromatography practice and small molecules the elution factor ke value is between 2 and 8. As a simplified generalization for typical gradient slopes we propose using linear ln k versus Φ trend in the k range between 1 and 30. The spreadsheet was utilized to compare the retention prediction accuracy of linear and non-linear retention models. When fitting ln k versus Φ trend in k range 1-30 the simple linear model is in good agreement with nonlinear model with retention time prediction error 0.3-4.7% (for gradient slope 0.013-0.260).

Chromatographic performance of microfluidic liquid chromatography devices: Experimental evaluation of straight versus serpentine packed channels.

We prepared a series of planar titanium microfluidic (µLC) columns, each 100 mm long, with 0.15, 0.3 and 0.5 mm i.d.'s. The microfluidic columns were packed with 1.8 µm C18 sorbent and tested under isocratic and gradient conditions. The efficiency and peak capacity of these devices were monitored using a micro LC instrument with minimal extra column dispersion. Columns with serpentine channels were shown to perform worse than those with straight channels. The loss of efficiency and peak capacity was more prominent for wider i.d. columns, presumably due to on-column band broadening imparted by the so-called "race-track" effect. The loss of chromatographic performance was partially mitigated by tapering the turns (reduction in i.d. through the curved region). While good performance was obtained for 0.15 mm i.d. devices even without turn tapering, the performance of 0.3 mm i.d. columns could be brought on par with capillary LC devices by tapering down to 2/3 of the nominal channel width in the turn regions. The loss of performance was not fully compensated for in 0.5 mm devices even when tapering was employed; 30% loss in efficiency and 10% loss in peak capacity was observed. The experimental data for various devices were compared using the expected theoretical relationship between peak capacity Pc and efficiency N; (Pc-1) = N0.5 × const. While straight µLC columns showed the expected behavior, the devices with serpentine channels did not adhere to the plot. The results suggest that the loss of efficiency due to the turns is more pronounced than the corresponding loss of peak capacity.

Impact of instrument and column parameters on high-throughput liquid chromatography performance.

The speed and separation performance of high-throughput liquid chromatography (HT LC) was investigated. We evaluated the contributions of various experimental parameters to the total analysis cycle time, including column length (column void time), gradient delay, flow rate, auto-sampler (A/S) speed, and software related delays. The best case injection-to-injection cycle time of 22s was achieved using 12s gradient time and 2.1×20mm columns packed with 1.7µm C18 particles. The total 22s analytical duty cycle consisted of 2.5s column void time, 1.8s gradient delay, 12s gradient time, and approximately 5.7s for software setup delay time that served as column re-equilibration time. The achieved peak capacity for an alkylphenone sample was approximately 35, giving a peak production rate of 95 peaks per minute. We estimated the impact of LC system dispersion on peak capacity and peak production rate (peak capacity per unit of time). For HT LC scenarios (5-50mm columns and 4.8-120s long gradients) we observed that even a minor amount of system dispersion (2µL2) reduces the achievable peak capacity and peak productivity rate significantly. HT LC-MS analysis using 2.1×5mm guard column with duty cycle of 22s was successfully demonstrated.

Experimental evaluation of chromatographic performance of capillary and microfluidic columns with linear or curved channels.

We prepared 0.3 or 0.15mm i.d. columns from both fused silica capillaries and planar titanium wafers with machined grooves. Both types of devices were packed with sub-two micron C18 sorbent. Chromatographic efficiency and peak capacity were tested using LC instruments with low extra column dispersion (300nL2 or 30nL2 for 0.3 or 0.15mm i.d. columns, respectively). Micro column testing in gradient mode was less affected by extra column (pre-column) dispersion. To exploit this feature we developed a method for estimation of column efficiency from gradient analysis using the theoretical relationship (Pc-1)=N0.5×const. The validity of this relationship was experimentally verified using 2.1mm i.d. and 0.3mm i.d. columns. The (Pc-1) versus N relationship was experimentally determined with straight columns, which in turn was employed for the estimation of microfluidic column efficiency. Microfluidic devices with serpentine channels exhibited lower isocratic efficiency than straight capillary columns, but the loss of peak capacity was less significant. The loss chromatographic efficiency due to zone dispersion in serpentine microfluidic channels was more apparent for 0.3 than 0.15mm i.d. devices. Gradient performance of 0.15×100mm microfluidic columns was comparable to state-of-the-art 2.1×100mm columns packed with the same sorbent.

Repetitive injection method: a tool for investigation of injection zone formation and its compression in microfluidic liquid chromatography.

Sample introduction in microfluidic liquid chromatography often generates wide zones rather than peaks, especially when a large sample volume (relative to column volume) is injected. Formation of wide injection zones can be further amplified when the sample is dissolved in a strong eluent. In some cases sample breakthrough may occur, especially when the injection is performed into short trapping columns. To investigate the band formation and subsequent zone focusing under gradient elution in situations such as these, we developed the Repetitive Injection Method (RIM), based on the temporally resolved introduction of two discrete peaks to a column, mimicking both the leading and trailing edges of a larger, singly injected sample zone. Using titanium microfluidic 0.32 mm I.D. columns, the results of RIM experiments were practically identical to injection of a correspondingly larger single zone volume. It was also experimentally shown that zone width (spacing between two injected peaks) decreases during gradient elution. We utilized RIM experiments to investigate wide sample zones created by strong sample solvent, and subsequent gradient zone focusing for a series of compounds. This experimental work was compared with computationally simulated chromatograms. The success of sample focusing during injection and gradient elution depends not only on an analyte's absolute retention, but also on how rapidly the analyte's retention changes during the mobile phase gradient.

Wide injection zone compression in gradient reversed-phase liquid chromatography.

Chromatographic zone broadening is a common issue in microfluidic chromatography, where the sample volume introduced on column often exceeds the column void volume. To better understand the propagation of wide chromatographic zones on a separation device, a series of MS Excel spreadsheets were developed to simulate the process. To computationally simplify these simulations, we investigated the effects of injection related zone broadening and its gradient related zone compression by tracking only the movements of zone boundaries on column. The effects of sample volume, sample solvent, gradient slope, and column length on zone broadening were evaluated and compared to experiments performed on 0.32mm I.D. microfluidic columns. The repetitive injection method (RIM) was implemented to generate experimental chromatograms where large sample volume scenarios can be emulated by injecting two discrete small injection plugs spaced in time. A good match between predicted and experimental RIM chromatograms was observed. We discuss the performance of selected retention models on the accuracy of predictions and use the developed spreadsheets for illustration of gradient zone focusing for both small molecules and peptides.

Solvent selectivity and strength in reversed-phase liquid chromatography separation of peptides.

A set of tryptic peptides was analyzed in reversed-phase liquid chromatography using gradient elution with acetonitrile, methanol, or isopropanol. We used these retention data as training sets to develop retention prediction models of peptides for the three organic eluents used. The coefficients of determination, R(2), between predicted and observed data were approximately 0.95 for all systems. Retention coefficient values of twenty amino acids calculated from a model were utilized to investigate differences in separation selectivity between acetonitrile, methanol, or isopropanol eluents. The experimentally observed difference in separation selectivity appears to be a complex interplay of multiple amino acids, each contributing to a different degree to overall peptide retention. While retention contribution of hydrophilic amino acids was higher in methanol than acetonitrile, peptides containing aromatic amino acids (tyrosine, phenylalanine, tryptophan) exhibit relatively lower retention in methanol compared to acetonitrile. The differences between acetonitrile and isopropanol eluents were less pronounced. We also compared the relative elution strength of the three organic eluents for peptides. The relationship between the elution strength of two solvents is not linear, rather it was best fitted by a cubic polynomial function. Three solvents can be arranged in the order of increasing elution power as methanol