Oral Bioavailability In Dogs Research Articles

Systems Biology and Peptide Engineering to Overcome Absorption Barriers for Oral Peptide Delivery: Dosage Form Optimization Case Study Preceding Clinical Translation.

Oral delivery of peptides and biological molecules promises significant benefits to patients as an alternative to daily injections, but the development of these formulations is challenging due to their low bioavailability and high pharmacokinetic variability. Our earlier work focused on the discovery of MEDI7219, a stabilized, lipidated, glucagon-like peptide 1 agonist peptide, and the selection of sodium chenodeoxycholate (Na CDC) and propyl gallate (PG) as permeation enhancer combinations. We hereby describe the development of the MEDI7219 tablet formulations and composition optimization via in vivo studies in dogs. We designed the MEDI7219 immediate-release tablets with the permeation enhancers Na CDC and PG. Immediate-release tablets were coated with an enteric coating that dissolves at pH ≥ 5.5 to target the upper duodenal region of the gastrointestinal tract and sustained-release tablets with a Carbopol bioadhesive polymer were coated with an enteric coating that dissolves at pH ≥ 7.0 to provide a longer presence at the absorption site in the gastrointestinal tract. In addition to immediate- and enteric-coated formulations, we also tested a proprietary delayed release erodible barrier layer tablet (OralogiKTM) to deliver the payload to the target site in the gastrointestinal tract. The design of tablet dosage forms based on the optimization of formulations resulted in up to 10.1% absolute oral bioavailability in dogs with variability as low as 26% for MEDI7219, paving the way for its clinical development.

Engineering of Orally Available, Ultralong-Acting Insulin Analogues: Discovery of OI338 and OI320.

Recently, the first basal oral insulin (OI338) was shown to provide similar treatment outcomes to insulin glargine in a phase 2a clinical trial. Here, we report the engineering of a novel class of basal oral insulin analogues of which OI338, 10, in this publication, was successfully tested in the phase 2a clinical trial. We found that the introduction of two insulin substitutions, A14E and B25H, was needed to provide increased stability toward proteolysis. Ultralong pharmacokinetic profiles were obtained by attaching an albumin-binding side chain derived from octadecanedioic (C18) or icosanedioic acid (C20) to the lysine in position B29. Crucial for obtaining the ultralong PK profile was also a significant reduction of insulin receptor affinity. Oral bioavailability in dogs indicated that C18-based analogues were superior to C20-based analogues. These studies led to the identification of the two clinical candidates OI338 and OI320 (10 and 24, respectively).

Albendazole solid dispersions prepared using PEG6000 and Poloxamer188: formulation, characterization and in vivo evaluation

This study aimed to optimize the preparation process of albendazole (ABZ) solid dispersion (SD) and enhance its dissolution rate and oral bioavailability in dogs. The ABZ-SD formulations were prepared by a fusion method with ABZ and polyethylene glycol 6000 (PEG 6000), poloxamer 188 (P 188) polymers at various weight ratios or the combination of PEG 6000&P 188. The characterizations of the optimal formulations were performed by scanning electron microscopy (SEM), powder X-ray diffraction (PXRD), Fourier transform infrared spectroscopy (FTIR), in vitro dissolution test and molecular docking. The in vivo pharmacokinetic study was conducted in beagle dogs. As a result, ABZ solid dispersion based on PEG 6000&P 188 (1:2) was successfully prepared. The ABZ-SD formulation could significantly improve the apparent solubility and dissolution rate of ABZ compared with commercial tablets. Furthermore, the water solubility of ABZ-SD was improved mainly based on hydrogen bond association. Besides, at an oral dosage of 15 mg/kg ABZ, the SDs had higher Cmax values and areas under the curve (AUCs) compared to those of commercial ABZ tablets. Preparation of ABZ-loaded SDs by PEG 6000&P 188 is a promising strategy to improve the oral bioavailability of ABZ.

Discovery of a 4-Azetidinyl-1-thiazoyl-cyclohexane CCR2 Antagonist as a Development Candidate.

We have discovered a novel series of 4-azetidinyl-1-aryl-cyclohexanes as CCR2 antagonists. Divergent SAR studies on hCCR2 and hERG activities led to the discovery of compound 8d, which displayed good hCCR2 binding affinity (IC50, 37 nM) and potent functional antagonism (chemotaxis IC50, 30 nM). It presented an IC50 of >50 μM in inhibition of the hERG channel and had no effect on the QTc interval up to 10 mg/kg (i.v.) in anesthetized guinea pig and dog CV studies. It also displayed high selectivity over other chemokine receptors and GPCRs, and amendable oral bioavailability in dogs and primates. In a thioglycollate-induced inflammation model in hCCR2KI mice, it had ED50 of 3 mg/kg on inhibition of the influx of leukocytes, monocytes/macrophages, and T-lymphocytes.

Mechanism for Effective Lymphoid Cell and Tissue Loading Following Oral Administration of Nucleotide Prodrug GS-7340

GS-7340 is a prodrug of tenofovir (TFV) that more efficiently delivers TFV into lymphoid cells and tissues than the clinically used prodrug TFV disoproxil fumarate, resulting in higher antiviral potency at greatly reduced doses and lower systemic TFV exposure. First-pass extraction by the intestine and liver represents substantial barriers to the oral delivery of prodrugs designed for rapid intracellular hydrolysis. In order to understand how GS-7340 reduces first-pass clearance to be an effective oral prodrug, its permeability and stability were characterized in vitro and detailed pharmacokinetic studies were completed in dogs. GS-7340 showed concentration-dependent permeability through monolayers of caco-2 cells and dose-dependent oral bioavailability in dogs, increasing from 1.7% at 2 mg/kg to 24.7% at 20 mg/kg, suggesting saturable intestinal efflux transport. Taking into account a 65% hepatic extraction measured in portal vein cannulated dogs, high dose GS-7340 is nearly completely absorbed. Consistent with the proposed role of intestinal efflux transport, coadministration of low dose GS-7340 with a transport inhibitor substantially increased GS-7340 exposure. The result of effective oral absorption and efficient lymphoid cell loading was reflected in the high and persistent levels of the pharmacologically active metabolite, TFV diphosphate, in peripheral blood mononuclear cells following oral administration to dogs. In conclusion, GS-7340 reaches the systemic circulation to effectively load target cells by saturating intestinal efflux transporters, facilitated by its high solubility, and by maintaining sufficient stability in intestinal and hepatic tissue.

5-Benzothiazole substituted pyrimidine derivatives as HCV replication (replicase) inhibitors

Based on a previously identified HCV replication (replicase) inhibitor 1, SAR efforts were conducted around the pyrimidine core to improve the potency and pharmacokinetic profile of the inhibitors. A benzothiazole moiety was found to be the optimal substituent at the pyrimidine 5-position. Due to potential reactivity concern, the 4-chloro residue was replaced by a methyl group with some loss in potency and enhanced rat in vivo profile. Extensive investigations at the C-2 position resulted in identification of compound 16 that demonstrated very good replicon potency, selectivity and rodent plasma/target organ concentration. Inhibitor 16 also demonstrated good plasma levels and oral bioavailability in dogs, while monkey exposure was rather low. Chemistry optimization towards a practical route to install the benzothiazole moiety resulted in an efficient direct C–H arylation protocol.

Solid Dispersion of an HIV Protease Inhibitor

LB71350 is an HIV protease inhibitor with poor aqueous solubility and extensive first pass effect. The purpose of the present study was to test the feasibility of solid dosage form of LB71350 with improved bioavailability utilizing solid dispersion. Three different compositions with varying ratio of (LB71350: Gelucire 44/14: Tween 20) were studied. Capsule filling of these solid dispersion compositions was tested using a semi-automatic capsule filling system. Oral bioavailability in dog was tested. Chemical and physical stability at 4, 25 and 40˚C was monitored by HPLC assay, dissolution test, powder XRD and microscopy. The capsule filling system yielded uniform products of drug loading up to 10%. Oral bioavailability in dog was improved compared to the aqueous suspension of crystalline LB71350. Capsules were chemically stable for up to 6 months at 40˚C. However, there were temperature and composition dependent physical changes. Decrease in dissolution rates after storage at 40˚C was due to the polymorphic change. In conclusion, manufacturing process, bioavailability, and physico-chemical stability have been considered to propose a solid dispersion capsule formulation for the HIV protease inhibitor with poor physico-chemical properties. A new less soluble crystalline form identified during the physical stability test warrants further study.

Use of An Intravenous Microdose of 14C-labeled drug and Accelerator Mass Spectrometry to measure Absolute Oral Bioavailability in Dogs; Cross-comparison of Assay Methods by Accelerator Mass Spectrometry and Liquid Chromatography-Tandem Mass Spectrometry

A technique utilizing simultaneous intravenous microdosing of (14)C-labeled drug with oral dosing of non-labeled drug for measurement of absolute bioavailability was evaluated using R-142086 in male dogs. Plasma concentrations of R-142086 were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and those of (14)C-R-142086 were measured by accelerator mass spectrometry (AMS). The absence of metabolites in the plasma and urine was confirmed by a single radioactive peak of the parent compound in the chromatogram after intravenous microdosing of (14)C-R-142086 (1.5 microg/kg). Although plasma concentrations of R-142086 determined by LC-MS/MS were approximately 20% higher than those of (14)C-R-142086 as determined by AMS, there was excellent correlation (r=0.994) between both concentrations after intravenous dosing of (14)C-R-142086 (0.3 mg/kg). The oral bioavailability of R-142086 at 1 mg/kg obtained by simultaneous intravenous microdosing of (14)C-R-142086 was 16.1%, this being slightly higher than the value (12.5%) obtained by separate intravenous dosing of R-142086 (0.3 mg/kg). In conclusion, on utilizing simultaneous intravenous microdosing of (14)C-labeled drug in conjunction with AMS analysis, absolute bioavailability could be approximately measured in dogs, but without total accuracy. Bioavailability in humans may possibly be approximately measured at an earlier stage and at a lower cost.

Enhancement of oral bioavailability of an HIV-attachment inhibitor by nanosizing and amorphous formulation approaches

BMS-488043 is an HIV-attachment inhibitor that exhibited suboptimal oral bioavailability upon using conventional dosage forms prepared utilizing micronized crystalline drug substance. BMS-488043 is classified as a Biopharmaceutics Classification System (BCS) Class-II compound with a poor aqueous solubility of 0.04 mg/mL and an acceptable permeability of 178 nm/s in the Caco2 cell-line model. Two strategies were evaluated to potentially enhance the oral bioavailability of BMS-488043. The first strategy targeted particle size reduction through nanosizing the crystalline drug substance. The second strategy aimed at altering the drug's physical form by producing an amorphous drug. Both strategies provided an enhancement in oral bioavailability in dogs as compared to a conventional formulation containing the micronized crystalline drug substance. BMS-488043 oral bioavailability enhancement was ∼5- and 9-folds for nanosizing and amorphous formulation approaches, respectively. The stability of the amorphous coprecipitated drug prepared at different compositions of BMS-488043/polyvinylpyrrolidone (PVP) was evaluated upon exposure to stressed stability conditions of temperature and humidity. The drastic effect of exposure to humidity on conversion of the amorphous drug to crystalline form was observed. Additionally, the dissolution behavior of coprecipitated drug was evaluated under discriminatory conditions of different pH values to optimize the BMS-488043/PVP composition and produce a stabilized, amorphous BMS-488043/PVP (40/60, w/w) spray-dried intermediate (SDI), which was formulated into an oral dosage form for further development and evaluation.

The discovery of highly potent CGRP receptor antagonists

Rational modification of a previously identified spirohydantoin lead structure has identified a series of potent spiroazaoxindole CGRP receptor antagonists. The azaoxindole was found to be a general replacement for the hydantoin that consistently improved in vitro potency. The combination of the indanylspiroazaoxindole and optimized benzimidazolinones led to highly potent antagonists (e.g., 25, CGRP K i = 40 pM). The closely related compound 27 demonstrated good oral bioavailability in dog and rhesus.

Phase 1, pharmacokinetic (PK) and pharmacodynamic (PD) study of oral alvespimycin (A; KOS-1022; 17-DMAG): Two different schedules in patients with advanced malignancies

14059 Background: A is ∼3–5 fold more potent compared to 17-AAG (the first Hsp90 inhibitor to enter clinical testing), based on in vitro cytotoxicity or the MTD in toxicology studies; it is water-soluble; and oral bioavailability in dogs is estimated at 40%. Toxicity in the dog included kidney, intestinal and liver findings. This study was conducted to determine the toxicity, MTD, recommended phase 2 dose (RP2D), PK and PD of A in pts with solid tumors. Methods: Escalating doses of A were given PO on 2 different schedules: every other day or daily for 4 out of 6 weeks. An initial IV dose was given to calculate absolute bioavailability. PK was evaluated after the IV dose, Day 1 and 21 of oral dosing. PBLs were collected to investigate changes in intracellular signaling proteins by immunoblot (Days 1 and 21 at 1, 3, 24 and 48 hours post-dose). Results: 28 pts were enrolled: 24 on the QOD schedule at doses of 5 (n=4), 10 (n=4), 20 (n=8), 30 (n=5) and 40 mg (n=3); 4 pts received 10mg on the QD schedule. 50% were male, median age/ECOG PS 55 and 0; median prior regimen 3. DLT has not yet been observed. Common drug-related toxicities (n=23): fatigue 43%, nausea 24%, anorexia 19%, proteinuria 19%, and peripheral edema 14%. Of these, fatigue and peripheral edema appear to be possibly dose-related. Drug-related Grade 3–4 toxicity (one patient each) included anemia, neutropenia, peripheral edema, hypokalemia, pain in extremity and hypoxia. For pts with full PK data (n=14), bioavailability equaled 51% and 49% on Day 1 and 21, and was not dose-dependent. Mean Day 21 AUCinf for the 5 to 30 mg/m2 levels equaled 91, 166, 542 and 1889 ng*h/mL. One pt with 3-fold increase in AUCinf comparing Day 1 and 21 dose had been started on dronabinol, a CYP2C9 inhibitor. One pt with fibrosarcoma (4 prior regimens) had necrotic changes in the tumor in the axilla with improved symptoms (active at 5+ months). Additional pts with SD include hemangioendothelioma (7 months), melanoma (6+ months), and renal cell (5 months). Induction in Hsp70 at the 30 mg dose level was seen pre-dose on Day 21 with maximal induction at 24 hours post-dose. Conclusions: Dose escalation continues on both schedules in order to define a RP2D. Toxicity is acceptable. Early signs of activity have been observed. [Table: see text]

Disposition of abouthiouzine: a novel antihyperthyroid drug

Abouthiouzine is a novel antithyroid agent with a profile of fewer reported adverse effects than other currently used drugs. The purpose of this current work was to explore, for the first time, the disposition of abouthiouzine following intravenous and oral administration using an animal model; also, to study its plasma protein binding properties. Abouthiouzine (2 mg/kg intravenously) was administered to healthy male Vole rabbits and Beagle dogs. A dose of 20 mg/kg of the drug was also given orally to another group of Beagle dogs. Abouthiouzine plasma concentrations were measured using an HPLC method, and its pharmacokinetic parameters were determined by non-compartmental analysis. Abouthiouzine plasma protein binding was determined using an ultrafiltration technique. The drug was quickly eliminated from the rabbit and dog systemic circulations with terminal half-lives (T(1/2 lambda)) of 0.7 h and 1.9 h, respectively. The calculated T(1/2 lambda) following the oral administration in dogs was 1.8 h. Total abouthiouzine clearance (CL) in rabbits was 7.84+/-0.87 ml/min/kg, and 4.03+/-0.83 ml/min/kg in dogs. The apparent volume of distribution at steady state (V(ss)) in rabbits and dogs was 360.09+/-63.41 ml/kg and 481.10+/-62.64 ml/kg, respectively. The absolute oral bioavailability in dogs was approximately 16%, which may indicate poor absorption characteristics of the pure drug and/or an extensive first past effect. Protein binding studies have demonstrated that abouthiouzine has moderate-to-high binding properties ( approximately 63%-86%). Further studies are needed to evaluate the route of elimination of abouthiouzine in these animal models including any metabolite formation and the role of enterohepatic recycling in this process.

Discovery of potent, selective 4-fluoroproline-based thrombin inhibitors with improved metabolic stability

Previous reports from our laboratories described potent tripeptide thrombin inhibitors which incorporate heterocycle-substituted chlorophenyl groups in the P1 position. Using these as lead compounds for further optimization, we identified sites of metabolism and designed analogs with 4-fluoroproline in P2 and cyclopropane-containing side chains in P3 as an approach to reducing metabolism and improving their oral pharmacokinetic performance. The large (300-fold) difference in potency between analogs containing (4 R)- and (4 S)-4-fluoroproline was rationalized by analyzing inhibitor–enzyme interactions in crystal structures of related compounds and by molecular modeling which indicated that the more potent (4 R)-4-fluoroproline isomer stabilizes a proline ring conformation that is preferred for binding to the enzyme. An optimal compound from this work, 41, exhibits high potency in a coagulation assay in human plasma (2×APTT = 190 nM), excellent selectivity versus the digestive enzyme trypsin ( K i = 3300 nM), and excellent oral bioavailability in dogs with moderate clearance ( F = 100%, CL = 12 mL/min/kg).

The effects of inhibiting cytochrome P450 3A, p‐glycoprotein, and gastric acid secretion on the oral bioavailability of methadone in dogs

Methadone is an opioid, which has a high oral bioavailability (>70%) and a long elimination half-life (>20 h) in human beings. The purpose of this study was to evaluate the effects of ketoconazole [a CYP3A and p-glycoprotein (p-gp) inhibitor] and omeprazole (an H+,K(+)-ATPase proton-pump inhibitor) on oral methadone bioavailability in dogs. Six healthy dogs were used in a crossover design. Methadone was administered i.v. (1 mg/kg), orally (2 mg/kg), again orally following oral ketoconazole (10 mg/kg q12 h for two doses), and following omeprazole (1 mg/kg p.o. q12 h for five doses). Plasma concentrations of methadone were analyzed by high-pressure liquid chromatography or fluorescence polarization immunoassay. The mean +/- SD for the elimination half-life, volume of distribution, and clearance were 1.75 +/- 0.25 h, 3.46 +/- 1.09 L/kg, and 25.14 +/- 9.79 mL/min.kg, respectively following i.v. administration. Methadone was not detected in any sample following oral administration alone or following oral administration with omeprazole. Following administration with ketoconazole, detectable concentrations of methadone were present in one dog with a 29% bioavailability. MDR-1 genotyping, encoding p-gp, was normal in all dogs. In contrast to its pharmacokinetics humans, methadone has a short elimination half-life, rapid clearance, and low oral bioavailability in dogs and the extent of absorption is not affected by inhibition of CYP3A, p-gp, and gastric acid secretion.

Synthesis of 1 H-pyridin-2-one derivatives as potent and selective farnesyltransferase inhibitors

Two novel series of potent and selective FTase inhibitors have been synthesized using structure-based design. Medicinal chemistry efforts led to the discovery of compound 4e with potent cellular activity and good oral bioavailability in dog. A synthetic route toward novel heterocycles 1,5-dimethyl-6-oxo-4-aryl-1,6-dihydro-pyridine-2-carbonitrile was established. The structure of compound 5c was confirmed by X-ray crystallography.

Design, synthesis, and biological evaluation of monopyrrolinone-based HIV-1 protease inhibitors.

The design, synthesis, and biological evaluation of a series of HIV-1 protease inhibitors [(-)-6, (-)-7, (-)-23, (+)-24] based upon the 3,5,5-trisubstituted pyrrolin-4-one scaffold is described. Use of a monopyrrolinone scaffold leads to inhibitors with improved cellular transport properties relative to the earlier inhibitors based on bispyrrolinones and their peptide counterparts. The most potent inhibitor (-)-7 displayed 13% oral bioavailability in dogs. X-ray structure analysis of the monopyrrolinone compounds cocrystallized with the wild-type HIV-1 protease provided valuable information on the interactions between the inhibitors and the HIV-1 enzyme. In each case, the inhibitors assumed similar orientations for the P2'-P1 substituents, along with an unexpected hydrogen bond of the pyrrolinone NH with Asp225. Interactions with the S2 pocket, however, were not optimal, as illustrated by the inclusion of a water molecule in two of the three inhibitor-enzyme complexes. Efforts to increase affinity by displacing the water molecule with second and third generation inhibitors did not prove successful. Lack of success with this venture is a testament to the difficulty of accurately predicting the many variables that influence and build binding affinity. Comparison of the inhibitor positions in three complexes with that of Indinavir revealed displacements of the protease backbones in the enzyme flap region, accompanied by variations in hydrogen bonding to accommodate the monopyrrolinone ring. The binding orientation of the pyrrolinone-based inhibitors may explain their sustained efficacy against mutant strains of the HIV-1 protease enzyme as compared to Indinavir.

Substituted oxazole benzenesulfonamides as potent human β 3 adrenergic receptor agonists

As a part of our investigation into the development of orally bioavailable β 3 adrenergic receptor agonists, we have identified a series of substituted oxazole derivatives that are potent β 3 agonists with excellent selectivity against other β receptors. Several of these compounds showed excellent oral bioavailability in dogs. One example, cyclopentylethyloxazole 5f is a potent β 3 agonist (EC 50=14 nM, 84% activation) with 340-fold and 160-fold selectivity over β 1 and β 2 receptors, respectively, and has 38% oral bioavailability in dogs.

Synthesis and SAR of benzyl and phenoxymethylene oxadiazole benzenesulfonamides as selective β 3 adrenergic receptor agonist antiobesity agents

Benzyl and phenoxymethylene substituted oxadiazoles are potent and orally bioavailable β 3 adrenergic receptor (AR) agonists. The 4-trifluormethoxy substituted 5-benzyl oxadiazole 5f has an EC 50 of 8 nM in the β 3 AR agonist assay with 100-fold selectivity over β 1 and β 2 AR binding inhibition activity. Its oral bioavailability in dogs is 30 ± 4%, with a half-life of 3.8 ± 0.4 h. In the anesthetized rhesus, 5f evoked a dose-dependent glycerolemia (ED 50Gly=0.15 mg/kg). Under these conditions a heart rate increase of 15% was observed at a dose level of 10 mg/kg.

Discovery of an orally bioavailable alkyl oxadiazole β 3 adrenergic receptor agonist

5- n-Pentyl oxadiazole substituted benzenesulfonamide 8 is a potent and selective β 3 adrenergic receptor agonist (β 3 EC 50=23 nM, β 1 IC 50=3000 nM, β 2 IC 50=3000 nM). The compound has high oral bioavailability in dogs (62%) and rats (36%) and is among the most orally bioavailable β 3 adrenergic receptor agonists reported to date.

New highly potent dipeptidic growth hormone secretagogues with low molecular weight

Based on NN703, low molecular weight growth hormone secretagouges (GHSs) with a reduced number of hydrogen binding sites were designed by removal of the C-terminal amide group. The compounds were highly potent in combination with high efficacy in a rat pituitary cell assay, being characterized with EC 50 values down to 0.8 nM. Selected compounds were tested in in vivo animal models. The oral bioavailability in dogs was 16–44%. Also, the ED 50 values of the compounds were determined both in dog and swine.