Discovery of VU6007496: Challenges in the Development of an M1 Positive Allosteric Modulator Backup Candidate

Herein we report progress toward a backup clinical candidate to the M1 positive allosteric modulator (PAM) VU319/ACP-319. Scaffold-hopping from the pyrrolo[2,3-b]pyridine-based M1 PAM VU6007477 to isomeric pyrrolo[3,2-b]pyridine and thieno[3,2-b]pyridine congeners identified several backup contenders. Ultimately, VU6007496, a pyrrolo[3,2-b]pyridine, advanced into late stage profiling, only to be plagued with unanticipated, species-specific metabolism and active/toxic metabolites which were identified in our phenotypic seizure liability in vivo screen, preventing further development. However, VU6007496 proved to be a highly selective and CNS penetrant M1 PAM, with minimal agonism, that displayed excellent multispecies IV/PO pharmacokinetics (PK), CNS penetration, no induction of long-term depression (or cholinergic toxicity) and robust efficacy in novel object recognition (minimum effective dose = 3 mg/kg p.o.). Thus, VU6007496 can serve as another valuable in vivo tool compound in rats and nonhuman primates, but not mouse, to study selective M1 activation.


Procedures for Biological Experiments
Calcium mobilization assays: All functional cell-based assays were performed in stable Chinese Hamster Ovary (CHO) cell lines constitutively expressing human M 5 or human M 1 receptors.For full muscarinic selectivity, CHO cells expressing human M 3 , human M 2 plus G qi5 or human M 4 plus G qi5 were used.Cells were plated at 15,000 cells per 20 L per well in black 384-well, TC-treated, clear-bottomed plates (Greiner) in Ham's F12 medium supplemented with 10% FBS and 20 mM HEPES.Cells were incubated overnight at 37 °C under 5% CO 2 .The following day, the medium was removed and replaced with 1.2 µM Fluo-4 AM (Invitrogen) in assay buffer (Hank's Balanced Salt Solution supplemented with 20 mM HEPES and 2.5 mM Probenecid, pH 7.4) and the cells were incubated for 50 minutes at 37 °C under 5% CO 2 .Dye was then removed and replaced with 20 µL of fresh assay buffer.Test compounds at a 10 mM concentration in DMSO were serially diluted in DMSO (either 1:3 or 1:5 dilution) to create a 10-point concentration series.The DMSO solutions were then diluted in assay buffer resulting in compound solutions at 2-times the final assay concentration with the highest assay concentration of 30 µM.The compound plate, cell plate, and plates containing EC 20 and EC 80 acetylcholine concentrations were placed in a Hamamatsu FDSS 6000 or 7000 kinetic imaging plate reader equipped to measure Ex 480 /Em 540 fluorescence.Data were collected at 1 frame per second.After 2 seconds of collecting baseline fluorescence, 20 L of the compound solutions were added to the cell plate.This was followed by the addition of an EC 20 concentration of acetylcholine at 142 seconds.At 267 seconds, an EC 80 concentration of acetylcholine was added along with a maximally effective acetylcholine concentration in wells not containing a compound to allow data normalization.The fluorescence signal was collected for a total of 300 seconds.Compound concentration response curves (CRCs) were collected in triplicate across three separate plates.Data were imported and analyzed in Dotmatics Informatics software.
by normalizing all data in the individual kinetic traces to the initial fluorescence read.The magnitude of each agonist addition was then determined and normalized to the average maximum response.This percent maximum response was plotted against log [compound] and fit to a four parameter logistical equation to determine log(IC 50 ).The IC 50 determined using the EC 80 of acetylcholine is the value reported.Compound CRC curves that did not plateau below 10% ACh max were assigned a low potency value of >10 µM.

Drug Metabolism Methods:
In vitro The unbound fraction (f u ) was calculated following the equation below, and mean values for each species were calculated from 3 replicates.

Plasma protein binding and
A similar approach was used to determine the degree of brain homogenate binding, which employed the same methodology and procedure with the following modifications: 1) a final compound concentration of 1 μM was used, 2) naïve rat brains were homogenized in DPBS (1:3 composition of brain: DPBS, w/w) using a Mini-Bead Beater™ machine in order to obtain brain homogenate, which was then treated in the same manner as the plasma samples in the previously described plasma protein binding assay.Fraction unbound for both plasma and brain samples was determined using Equation 4.
Equation 4 Determination of fraction unbound in plasma.
The diluted fraction unbound (f u 2) in brain was calculated in the same manner by using brain homogenate rather than plasma.Undiluted fraction unbound for the brain was calculated using Equation 5 Equation 5 Determination of fraction unbound in brain.F u 2 represents the diluted fraction unbound.

Intrinsic clearance:
Human or rat hepatic microsomes (0.5 mg/mL) and 1 µM test compound were incubated in 100 mM potassium phosphate pH 7.4 buffer with 3 mM MgCl 2 at 37 °C with constant shaking.After a 5 min preincubation, the reaction was initiated by the addition of NADPH (1 mM).At selected time intervals (0, 3, 7, 15, 25, and 45 min), aliquots were taken and subsequently placed into a 96-well plate containing cold acetonitrile with internal standard (50 ng/mL carbamazepine).Plates were then centrifuged at 3000 rcf (4 °C) for 10 min, and the supernatant was transferred to a separate 96-well plate and diluted 1:1 with water for LC/MS/MS analysis.The in vitro half-life ( 1/2 , min, Eq. 1), intrinsic clearance (CL int , mL/min/kg, Eq. 2), and subsequent predicted hepatic clearance (CL hep , mL/min/kg, Eq. 3) was determined employing the following equations: where k represents the slope from linear regression analysis of the natural log percent remaining

In vivo DMPK experimental:
Determination of brain to plasma ratio:

Animal care and use
All animal study procedures were approved by the Institutional Animal Care and Use Committee and were conducted in accordance with the National Institutes of Health regulations of animal care covered in Principles of Laboratory Animal Care (National Institutes of Health).All rats were fasted overnight prior to testing.

In-life phase
For determination of the brain over plasma ratio (K p ), compounds were formulated in 8% ethanol, 32% PEG400 and 60% DMSO (v/v/v) and administered as a single 0. mL of 70:30 IPA:water in a mini bead beater for 3 min, and centrifuged at 3,500 g for 5 min.5 uL of the supernatant was diluted in 15 uL of blank plasma for quantification of the analytes.Plasma samples were centrifuged at 3,500 g for 5 min.A standard curve was generated by diluting the analytes DMSO stocks with blank plasma to obtain a final concentration of 10,000 ng/ml followed by a serial dilution down to 0.5 ng/ml.Quality controls were generated by a serial dilution of the 5,000 ng/ml standard curve solution in blank plasma to obtain 3 concentrations of 500, 50, and 5 ng/ml.20 uL of brain diluted in plasma, plasma, blank plasma, standard curve and QC samples were loaded in a V-bottom 96-well plate.120 uL of acetonitrile containing 0.05 uM carbamazepine (internal standard) was added to each well and the plate was centrifuged at 3,500 g for 5 min.60 uL of the supernatant of each well (protein free) was transferred to a new 96-well plate containing 60 uL of water.The plates were sealed for analysis by LC-MS/MS.
Plasma and brain tissue samples originating from in vivo studies were analyzed by electrospray ionization using an AB Sciex Q-TRAP 5500 (Foster City, CA) that was coupled to a Shimadzu LC-20AD pump (Columbia, MD) and a Leap Technologies CTC PAL auto-sampler (Carrboro, NC).Analytes were separated by gradient elution using a C18 column (3 x 50 mm, 3 mm; Fortis Technologies Ltd, Cheshire, UK) that was thermostated at 40 °C.HPLC mobile phase A was 0.1% formic acid in water (pH unadjusted); mobile phase B was 0.1% formic acid in acetonitrile (pH unadjusted).A 10% B gradient was held for 0.2 min and was linearly increased to 90% B over 0.8 min, with an isocratic hold for 0.5 min, before transitioning to 10% B over 0.05 min.The column was re-equilibrated (1 min) before the next sample injection.The total run time was 2.55 min, and the HPLC flow rate was 0.5 ml/min.The source temperature was set at 500 °C, and mass spectral analyses were performed using a Turbo-Ion spray source in positive ionization mode (5.0-kV spray voltage) and using multiple-reaction monitoring of transitions specific for the analytes.All data were analyzed using AB Sciex Analyst 1.5.1 software.
Brain plasma concentration ratio (K p ) was calculated by dividing brain concentration by plasma concentration for each animal.Unbound brain to unbound plasma concentration ratio (K p,uu ) is calculated using the following formula: K p,uu = (Brain ng/g x brain fu)/(plasma ng/ml x plasma fu).

Pharmacokinetic profiles in rats following oral single escalating doses
Single escalating oral dosing in Sprague-Dawley rats was performed at Frontage Laboratories according to their non-GLP Standard Operating Procedure and IACUC protocols.In short, compounds were formulated in 10% Tween 80 in water and dosed at 10 mg/kg.At different times, arterial blood was collected from a femoral artery catheter, and compound concentration was determined in plasma by LC-MS/MS following their non-GLP protocol.PK parameters were determined using Phoenix WinNonlin software (version 6.3).

In-vitro determination of blood-brain barrier penetration potential
Blood-brain barrier penetration was determined using MDR1-MDCK cell monolayers by Absorption Systems, following their protocol.In short, compounds were incubated at 5 mM final concentration on one side of the cell monolayer for 2 hours.Compounds concentration on either side of the monolayer was determined by LC-MS/MS and apparent permeability and efflux ratio were determined as described in Wang, Q. et al. 1

Behavioral Manifestations of Seizure Activity
To evaluate induction of behavioral manifestation of seizure activity, C57Bl/6 mice received administration of vehicle or 100 mg/kg M 1 PAM.Compounds were formulated in 30% Captisol (11) or 10% Tween 80 (2-4 and 12) (pH 7.0) at a concentration of 10 mg/mL and injected i.p. (n = 3).Animals were monitored continuously and scored for behavioral manifestations of seizure activity at 5, 10, 15, and 30 min, and 1 and 3 h.Behavioral manifestations of seizures were scored using a modified Racine scoring system. 2 Briefly, a score of 0 represents no behavior alterations; score 1, immobility, mouth and facial movements, or facial clonus; score 2, head nodding, tail extension; score 3, forelimb clonus, repetitive movements; score 4, rearing and tonic clonic seizure; and score 5, continuous rearing and falling, severe generalized tonic clonic seizure.

Novel Object Recognition Task
Rats were habituated for 10 min for 2 consecutive days in an empty novel object recognition (NOR) arena consisting of dark-colored plexiglass box (40 × 64 × 33 cm 3 ).On day 3, rats were administered vehicle (0.5% natrasol/0.015%Tween80) or M 1 PAM (0.1-3 mg/kg, p.o.., 10 mL/kg, n = 15-18) and returned to their home cage for 30 min.Rats were then placed in the NOR arena containing two identical objects for 10 min.Following the exposure period, rats were placed back into their home cages for 24 h.The rats were then returned to the arena in which one of the previously exposed (familiar) objects was replaced by a novel object and were video recorded for 5 min while they explored the two objects.Time spent exploring each object was scored by an observer blinded to the experimental conditions and the recognition index was calculated as [(time spent exploring novel object) -(time spent exploring familiar object)]/total time exploring objects.
Layer II/III was visualized using an Olympus BX50WI upright microscope (Olympus, Lake Success, NY) microscope according to landmarks illustrated in the Allen mouse brain atlas (Lein et al, 2007) and the recording electrode was laterally placed approximately 200µM away from layer 2/3 into layer V so that the recording and stimulating electrodes were parallel to each other.
Inputoutput curves were generated to determine the stimulus intensity that produced approximately 70% of the maximum fEPSP slope before each experiment, which was then used as the baseline stimulation.Similarly, the recording electrode for ofEPSP was placed in layer V and an inputoutput curve was generated to produce approximately 70% of the maximal ofEPSP slope.Data were digitized using a Multiclamp 700B, using a sampling rate of 20,000kHz and were filtered at 0.5kHz, with a Digidata 1322A, pClamp 9.2 and Clampex 10.6.2 software (Molecular Devices) running on a Dell PC (Round Rock, TX).Offline data analysis to calculate fEPSP slope or ofEPSP slope was performed using Clampfit 10.2 (Molecular Devices).

Table S2 .
Effects of VU6007496 on Ion Channel Current (Charles River Study 200609.WVN)