Terry D. Lindstrom

The Potent BACE1 Inhibitor LY2886721 Elicits Robust Central Aβ Pharmacodynamic Responses in Mice, Dogs, and Humans

BACE1 is a key protease controlling the formation of amyloid β, a peptide hypothesized to play a significant role in the pathogenesis of Alzheimers disease (AD). Therefore, the development of potent and selective inhibitors of BACE1 has been a focus of many drug discovery efforts in academia and industry. Herein, we report the nonclinical and early clinical development of LY2886721, a BACE1 active site inhibitor that reached phase 2 clinical trials in AD. LY2886721 has high selectivity against key off-target proteases, which efficiently translates in vitro activity into robust in vivo amyloid β lowering in nonclinical animal models. Similar potent and persistent amyloid β lowering was observed in plasma and lumbar CSF when single and multiple doses of LY2886721 were administered to healthy human subjects. Collectively, these data add support for BACE1 inhibition as an effective means of amyloid lowering and as an attractive target for potential disease modification therapy in AD.

Chiral separation of neutral species by capillary electrophoresis evaluation of a theoretical model

A theoretical model has been developed for the separation of enantiomers of neutral species by employing a combination of charged and neutral cyclodextrins. A neutral compound (LY213829), an ionizable cyclodextrin (sulfobutylether-cyclodextrin), and three neutral cyclodextrins (β-cyclodextrin, trimethyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin) were chosen to test the model. The model parameters were obtained by performing two specific sets of experiments. Resolution and selectivity can be readily obtained from these model parameters. The validity of the model has been demonstrated by resolving enantiomers of LY213829 and its four isomeric sulfoxide metabolites, and the model was very successful in predicting the migration times and resolution of the LY213829 enantiomers. Baseline separation was achieved for all the analytes.

Characterization and application of a vinblastine-selected Caco-2 cell line for evaluation of P-glycoprotein

SummaryThe role of the adenosine triphosphate-binding cassette (ABC) superfamily of membrane transporters is well documented in tumor cell multidrug resistance. More recently, growing evidence of their influence on oral bioavailability, drug excretion rates, and drug-drug interaction potential at the intestinal level has stimulated much investigation. Our laboratory is interested in evaluating the apical (AP) ABC transporter P-glycoprotein (Pgp [mdr-1]) for its role in xenobiotic efflux at the intestinal level. We propagated Caco-2 cells in the presence of vinblastine (a cytotoxic, Pgp substrate) to promote transporter expression though selection. That is, the cell population expressing Pgp, or with the capacity to upregulate Pgp expression, survived and expanded in the presence of vinblastine. We have used this selected cell line (Caco-2 VinB) to develop a fluorescent-based assay to study the chemical modulators of Pgp activity. Using the Caco-2 VinB cells, we have successfully demonstrated the differential potency of previously characterized Pgp inhibitors. In addition, we conducted a morphological evaluation of the two cell lines using transmission, scanning, and confocal microscopy. Both cell strains differentiated into highly functional, polarized columnar epithelium, although the vinblastine-selected cell line had lost the phenotypic diversity observed in native Caco-2 populations. Increased Pgp expression was noted in Caco-2 VinB cells compared with the native cell line on Western blot analysis, which was localized to the AP surface using confocal microscopy and functionally demonstrated using transport assays. We believe that the Caco2 VinB cell line is a versatile tool for application in pharmaceutical drug development.

Discovery and development of a novel class of nonsteroidal aromatase inhibitors

Efforts to develop a novel class of nonsteroidal aromatase inhibitors began with the discovery that the infertility in male rats exposed to high levels of the agricultural fungicide, fenarimol (alpha-(2-chlorophenyl)-alpha-(4-chlorophenyl)-5-pyrimidine-methanol), was attributable to the inhibition of aromatase activity within the central nervous system during the critical neonatal period. Although fenarimol was not particularly potent in inhibiting rat ovarian microsome aromatase activity in vitro (50% inhibition (IC50) = 4.1 microM). Subsequent testing of a number of analogues led to the identification of LY56110 (alpha,alpha-bis(4-chlorophenyl)-5-methylpyrimidine) which exhibited an IC50 of 29 nM. LY56110 was orally active, blocking the testosterone-induced increase of uterine weight and ovarian estrogen biosynthesis in immature female rats. In rats with established DMBA-induced mammary carcinoma, complete tumor regression was observed in 80% of the animals. Development of LY56110 was, however, stopped because of its effects on hepatic microsomal enzymes and an unacceptably long half-life. Structural modifications resulted in the development of the indenopyrimidines. LY113174 (8-chloro-5-(4-chlorophenyl)-5H-indeno less than 1, 2D greater than pyrimidine) was highly effective in vitro (IC50 = 24 nM) and in vivo but was far less potent than LY56110 with respect to induction of hepatic microsomal enzymes. LY113174 exhibited an acceptable biological half-life and had no effect on cholesterol side-chain cleavage. The indenopyrimidines appear to be a novel class of nonsteroidal aromatase inhibitors which may prove useful in the treatment of estrogen-dependent diseases.

Fused bicyclic Gly-Asp β-turn mimics with potent affinity for GPIIb-IIIa. Exploration of the arginine isostere

6-[4-Amidinobenzoyl]amino]-tetralone-2-acetic acid is a potent antagonist of GPIIb-IIIa. Substitution in the meta position of the benzamidine, or replacement with a heteroaryl amidine was tolerated in this series. Use of an acyl-linked 4-alkyl piperidine as an arginine isostere also provided active compounds. Compounds from this series provided substantial systemic exposure in the rat following oral administration.

Inhibition of granulocyte cAMP-phosphodiesterase by rolipram in vivo is not sufficient to protect the canine myocardium from reperfusion injury.

Summary: The purpose of this study was to determine whether the selective type IV cAMP-phosphodiesterase inhibitor rolipram could reduce the reperfusion injury that occurs during myocardial infarction in the anesthetized dog. This question was tested in pentobarbital-anesthetized dogs subject to 90 min of regional myocardial ischemia and 5 h of reperfusion. Dogs were treated with 1 mg/kg of rolipram (i.v., 15 min before reperfusion) followed by a 1 mg/kg/h infusion over the duration of the 5 h of reperfusion. Rolipram was tested in vitro for efficacy in inhibition of isolated human neutrophil superoxide generation. Rolipram produced significant inhibition of superoxide production over the concentration range of 0.1–100 μM- rolipram when neutrophils were stimulated with a 10 -7 M concentration of the chemotactic peptide f-Met-Leu-Phe. Rolipram significantly inhibited superoxide generation from human and canine granulocytes in whole blood stimulated by zymosan. Therapeutic concentrations of rolipram in the blood of dogs were achieved during the course of the experiments with a plasma concentration of 0.761 ± 0.095 μg/ml (2.76 ± 0.34 μ- M) at the time of reperfusion, and 0.574 ± 0.098 μg/ml (2.08 ± 0.36 μ M) at the end of the reperfusion period. The relative severity of myocardial ischemia between the two treatment groups was similar as assessed with radiolabeled microsphere measurement of myocardial blood flow. Transmural myocardial blood flows were not significantly different between the two groups after coronary occlusion (control, 0.05 ± 0.01 ml/min/g, n = 6, vs. rolipram, 0.18 ± 0.07 ml/min/g, n = 6; p = 0.48). There was not a significant reduction in the accumulation of neutrophils in the myocardium as assessed by the marker enzyme myeloperoxidase (control, 2.33 ± 1.07 U/g, n = 5, vs. rolipram, 0.54 ± 0.28 U/g, n = 6; p = 0.0548). Myocardial infarct size determined postmortem was not limited by rolipram treatment (control, 31.1 ± 2.6% infarct/ area at risk, n = 6, vs. rolipram, 23.3 ± 7.8% infarct/area at risk, n = 6; p > 0.05). Even though rolipram did not result in statistically significant protection as measured by infarct size, there was a significant attenuation of the “no-reflow” phenomenon with rolipram treatment. Circumflex coronary blood flow declines that were evident in the vehicle-treated group did not occur in the rolipram treatment group. These data indicate that cAMP-phosphodiesterase inhibition with rolipram is not sufficient to provide protection against myocardial reperfusion injury.

Inhibition of microsomal biotransformation by a series of nitrogen and oxygen heterocyclic histamine h2-antagonists

A homologous series of potent, long-lasting thiazolo-pyrimidone-pyridine histamine H2-antagonists were examined for their inhibitory effects on rat hepatic ethylmorphine N-demethylation. Inhibitory potency increased in the order: 2-pyridinyl less than 3-pyridinyl less than 4-pyridinyl histamine H2-antagonist. Substitution ortho to the pyridine nitrogen decreased inhibitory potency. Hydroxylation of the pyridine heterocycle decreased inhibitory potency, whereas substituent electronic effects did not appreciably alter the inhibitory potency of these compounds. Antagonists containing oxygen heterocycles were moderately potent inhibitors compared to those containing unsubstituted pyridine as the heterocycle. A 3-(6-methylpyridine) histamine H2-antagonist was shown to be a slightly more potent inhibitor of ethinamate metabolism than cimetidine in rats. However, unlike cimetidine, it did not inhibit the plasma half-life of antipyrine in dogs at doses that were equally efficacious in inhibiting gastric acid secretion.

Disposition of the aromatase inhibitor LY56110 and associated induction and inhibition studies in rats, dogs, and monkeys.

Compound LY56110 was well absorbed but slowly excreted in the rat, dog, and monkey. Oral administration of 5 mg/kg of [14C]LY56110 (5-bis(4-chlorophenyl)methylpyrimidine) to the rat, monkey, and dog resulted in a total excretion of 68, 65, and 30% of the radioactivity within 5 days, respectively. Very low urinary excretion was observed in the rat and dog (2%), with fecal excretion being the predominant mode of elimination in all three species. The plasma radioactivity half-life was 49, 41, and greater than 100 hr in the rat, monkey, and dog, respectively. The plasma half-life of parent compound was 18 hr in the rat and 10 hr in the dog. LY56110 accounted for only 25, 12, and 1% of the plasma radioactivity area under the curve in the rat, dog, and monkey, respectively. High levels of radioactivity were observed in the target tissues of fat, adrenals, and ovaries of rats. LY56110 induced hepatic cytochromes b5 and P-450 and cytochrome c reductase in rats after 14 days of oral dosing at 10 mg/kg but not in monkeys after 10 days of oral dosing at 10 mg/kg. The compound was more potent than aminoglutethimide or cimetidine in inhibiting hepatic ethylmorphine and p-nitroanisole demethylase activity in vitro. LY56110 also inhibited ethinamate-induced sleeping time in rats in vivo. The compound induced a reverse type I binding spectrum with rat ovarian microsomes.

Stereoselective metabolism of tazofelone, an anti-inflammatory bowel disease agent, in rats and dogs and in human liver microsomes.

Incubation of (R)-tazofelone and (S)-tazofelone in rat, dog, and human liver microsomes demonstrated that the (R)-tazofelone enantiomer was more rapidly metabolized, with two diastereomeric sulfoxides as the major metabolites formed in all three species. The two diasteresomers epimerized at physiological pH, therefore total sulfoxide formation rates were measured. The formation of the total sulfoxide metabolites followed Michaelis-Menten kinetics. The K(m), Vmax, and intrinsic formation clearance (Vmax/K(m)) values were determined in rat, dog, and human liver microsomes. The intrinsic formation clearance of sulfoxide from (R)-tazofelone exceeded that of (S)-tazofelone in all three species. In vivo studies in rats and dogs dosed orally and intravenously confirmed the stereoselective metabolism of tazofelone observed in vitro. Plasma concentrations of (S)-tazofelone exceeded (R)-tazofelone in rats and dogs by a factor of 3 to 4. In rat portal plasma, both enantiomers were of approximately equal concentration after oral dosing, indicating similar absorption. The half-lives of tazofelone and total sulfoxides in rats were 3.5 and 2.8 h, respectively. In dogs, the half-lives of tazofelone and total sulfoxides were 2.2 and 5.5 h, respectively. Plasma clearance was 2.3 l/h in rats and 1.4 l/h in dogs, and the volumes of distribution were 12 and 4.5 l, respectively, in rats and dogs. Both enantiomers were highly bound to plasma proteins to a similar extent in both species.

Proof-of-concept pharmacodynamic assessment of a prototypic BACE1 inhibitor at steady-state using IV infusion dosing in the PDAPP transgenic mouse model of Alzheimer's disease

Background: BACE1 is a key protease involved in Abeta generation but has proven challenging as a drug target. Many early BACE1 inhibitors which were optimized for potency at the enzyme, failed to achieve sufficient brain penetration to demonstrate central pharmacodynamic (PD) effects in vivo. Methods: Young PDAPP transgenic mice (n 1⁄4 10/group) were surgically implanted with a central catheter and dosed IV over a period of 22 hrs with vehicle or BACE1 inhibitor at 4.3, 8.6 or 14.4 mg/ml at 60 ul/hr rate. Doses were determined by prior pharmacokinetic measurements using a single IV bolus and calculated to reach steady-state levels exceeding their cellular IC50. Plasma and brain Ab (1-X) levels were measured using human-specific total Ab (1-X) ELISAs. The proximal biomarkers of BACE1 cleavage, C99 and sAPPbeta, were also measured in brain homogenates using appropriately configured sandwich ELISAs. Compound exposure was measured in plasma and remaining brain using LC/MS. Results: Steady-state IV infusion of this small molecule BACE1 inhibitor in PDAPP transgenic mice resulted in dose-dependent decreases in Ab, C99 and sAPPbeta measured in brain. Plasma Ab levels were not measurable beyond the lowest dose due to the robust effect of treatment. The reductions in brain biomarkers were dose-dependent and a change of similar magnitude occurred in all three in both brain regions (w71-85% decrease in the high dose group). The exposures measured in the plasma and brains increased with dose and were consistent with the dose-dependent efficacy observed. There were no adverse reactions to these doses in this study. Conclusions: IV administration of a prototypic BACE1 small molecule inhibitor to steady state produces robust reduction of brain Abeta levels by a mechanism of action consistent with BACE1 inhibition in vivo. Robust BACE1 inhibition can be maintained for 22 hours without significant adverse events offering hope for targeting this protease for the treatment of AD.