Identification and pharmacological profile of SPP1, a potent, functionally selective and brain penetrant agonist at muscarinic M1 receptors.

BACKGROUND AND PURPOSE: We aimed to identify and develop novel, selective muscarinic M1 receptor agonists as potential therapeutic agents for the symptomatic treatment of Alzheimer's disease. EXPERIMENTAL APPROACH: We developed and utilized a novel M1 receptor occupancy assay to drive a structure activity relationship in a relevant brain region while simultaneously tracking drug levels in plasma and brain to optimize for central penetration. Functional activity was tracked in relevant native in vitro assays allowing translational (rat-human) benchmarking of structure-activity relationship molecules to clinical comparators. KEY RESULTS: Using this paradigm, we identified a series of M1 receptor selective molecules displaying desirable in vitro and in vivo properties and optimized key features, such as central penetration while maintaining selectivity and a partial agonist profile. From these compounds, we selected spiropiperidine 1 (SPP1). In vitro, SPP1 is a potent, partial agonist of cortical and hippocampal M1 receptors with activity conserved across species. SPP1 displays high functional selectivity for M1 receptors over native M2 and M3 receptor anti-targets and over a panel of other targets. Assessment of central target engagement by receptor occupancy reveals SPP1 significantly and dose-dependently occupies rodent cortical M1 receptors. CONCLUSIONS AND IMPLICATIONS: We report the discovery of SPP1, a novel, functionally selective, brain penetrant partial orthosteric agonist at M1 receptors, identified by a novel receptor occupancy assay. SPP1 is amenable to in vitro and in vivo study and provides a valuable research tool to further probe the role of M1 receptors in physiology and disease.

Population Pharmacokinetic/ Pharmacodynamic Modelling of Auditory-Evoked Event-Related Potentials with Lorazepam.

Event-related potentials (ERPs) are commonly used in Neuroscience research, particularly the P3 waveform because it is associated with cognitive brain functions and is easily elicited by auditory or sensory inputs. ERPs are affected by drugs such as lorazepam, which increase the latency and decrease the amplitude of the P3 wave. In this study, auditory-evoked ERPs were generated in 13 older healthy volunteers using an oddball tone paradigm, after administration of single 0.5 and 2 mg doses of lorazepam. Population pharmacokinetics (PK)/pharmacodynamics (PD) models were developed using nonlinear mixed-effects methods in order to assess the effect of lorazepam on the latency and amplitude of the P3 waveforms. The PK/PD models showed that doses of 0.3 mg of lorazepam achieved approximately half of the maximum effect on the latency of the P3 waveform. For P3 amplitude, half the maximum effect was achieved with a dose of 1.2 mg of lorazepam. The PK/PD models also predicted an efficacious dose range of lorazepam, which was close to the recommended therapeutic range. The use of longitudinal P3 latency data allowed better predictions of the lorazepam efficacious dose range than P3 amplitude or aggregate exposure-response data, suggesting that latency could be a more sensitive parameter for drugs with similar mechanisms of action as lorazepam and that time course rather than single time-point ERP data should be collected. Overall, the results suggest that P3 ERP waveforms could be used as potential non-specific biomarkers for functional target engagement for drugs with brain activity, and PK/PD models can aid trial design and choice of doses for development of new drugs with ERP activity.

An assessment of the central disposition of intranasally administered insulin lispro in the cerebrospinal fluid of healthy volunteers and beagle dogs.

Intranasally administered regular insulin and insulin aspart have shown cognitive benefit for patients with Alzheimer's disease (AD). To support development of intranasally administered insulin analogs for AD, the central disposition of intranasal insulin lispro in the cerebrospinal fluid (CSF) of healthy volunteers was investigated. Healthy volunteers (N = 8) received two sequential doses of intranasal insulin lispro (48 or 80 IU followed by 160 IU) by Aero Pump in an open-label, single-period study with serial CSF and serum sampling over 5 hours after each dose. CSF insulin lispro was also measured in beagle dogs (N = 6/dose group) that received either 24 IU/kg (equivalent local nasal (IU/cm2) dose to the human 160 IU dose) or 192 IU/kg intranasally, using the same device. Insulin lispro was measured in the CSF and serum using a validated enzyme-linked immunosorbent assay method, and pharmacokinetic parameters were calculated by standard noncompartmental methods. Intranasal administration of insulin lispro was well tolerated. Insulin lispro concentrations in the CSF of humans at all dose levels were below the limit of quantification. Serum insulin lispro concentrations were quantifiable only up to 1-2 hours in the majority of subjects. In contrast to insulin lispro in the CSF of humans, insulin lispro was detectable in the CSF at both dose levels in dogs, and serum concentrations of insulin lispro were generally higher in dogs than in healthy volunteers. The absence of insulin lispro in CSF from healthy volunteers and the lack of robust exposure-response analyses will hinder the development of intranasally administered insulin lispro for AD.

Brain Exposure of Two Selective Dual CDK4 and CDK6 Inhibitors and the Antitumor Activity of CDK4 and CDK6 Inhibition in Combination with Temozolomide in an Intracranial Glioblastoma Xenograft.

Effective treatments for primary brain tumors and brain metastases represent a major unmet medical need. Targeting the CDK4/CDK6-cyclin D1-Rb-p16/ink4a pathway using a potent CDK4 and CDK6 kinase inhibitor has potential for treating primary central nervous system tumors such as glioblastoma and some peripheral tumors with high incidence of brain metastases. We compared central nervous system exposures of two orally bioavailable CDK4 and CDK6 inhibitors: abemaciclib, which is currently in advanced clinical development, and palbociclib (IBRANCE; Pfizer), which was recently approved by the U.S. Food and Drug Administration. Abemaciclib antitumor activity was assessed in subcutaneous and orthotopic glioma models alone and in combination with standard of care temozolomide (TMZ). Both inhibitors were substrates for xenobiotic efflux transporters P-glycoprotein and breast cancer resistant protein expressed at the blood-brain barrier. Brain Kp,uu values were less than 0.2 after an equimolar intravenous dose indicative of active efflux but were approximately 10-fold greater for abemaciclib than palbociclib. Kp,uu increased 2.8- and 21-fold, respectively, when similarly dosed in P-gp-deficient mice. Abemaciclib had brain area under the curve (0-24 hours) Kp,uu values of 0.03 in mice and 0.11 in rats after a 30 mg/kg p.o. dose. Orally dosed abemaciclib significantly increased survival in a rat orthotopic U87MG xenograft model compared with vehicle-treated animals, and efficacy coincided with a dose-dependent increase in unbound plasma and brain exposures in excess of the CDK4 and CDK6 Ki values. Abemaciclib increased survival time of intracranial U87MG tumor-bearing rats similar to TMZ, and the combination of abemaciclib and TMZ was additive or greater than additive. These data show that abemaciclib crosses the blood-brain barrier and confirm that both CDK4 and CDK6 inhibitors reach unbound brain levels in rodents that are expected to produce enzyme inhibition; however, abemaciclib brain levels are reached more efficiently at presumably lower doses than palbociclib and are potentially on target for a longer period of time.

Preclinical characterization of the CDK4/6 inhibitor LY2835219: in-vivo cell cycle-dependent/independent anti-tumor activities alone/in combination with gemcitabine.

The G1 restriction point is critical for regulating the cell cycle and is controlled by the Rb pathway (CDK4/6-cyclin D1-Rb-p16/ink4a). This pathway is important because of its inactivation in a majority of human tumors. Transition through the restriction point requires phosphorylation of retinoblastoma protein (Rb) by CDK4/6, which are highly validated cancer drug targets. We present the identification and characterization of a potent CDK4/6 inhibitor, LY2835219. LY2835219 inhibits CDK4 and CDK6 with low nanomolar potency, inhibits Rb phosphorylation resulting in a G1 arrest and inhibition of proliferation, and its activity is specific for Rb-proficient cells. In vivo target inhibition studies show LY2835219 is a potent inhibitor of Rb phosphorylation, induces a complete cell cycle arrest and suppresses expression of several Rb-E2F-regulated proteins 24 hours after a single dose. Oral administration of LY2835219 inhibits tumor growth in human tumor xenografts representing different histologies in tumor-bearing mice. LY2835219 is effective and well tolerated when administered up to 56 days in immunodeficient mice without significant loss of body weight or tumor outgrowth. In calu-6 xenografts, LY2835219 in combination with gemcitabine enhanced in vivo antitumor activity without a G1 cell cycle arrest, but was associated with a reduction of ribonucleotide reductase expression. These results suggest LY2835219 may be used alone or in combination with standard-of-care cytotoxic therapy. In summary, we have identified a potent, orally active small-molecule inhibitor of CDK4/6 that is active in xenograft tumors. LY2835219 is currently in clinical development.

Semi-mechanistic pharmacokinetic/pharmacodynamic modeling of the antitumor activity of LY2835219, a new cyclin-dependent kinase 4/6 inhibitor, in mice bearing human tumor xenografts.

PURPOSE: Selective inhibition of cyclin-dependent kinases 4 and 6 (CDK4/6) represents a promising therapeutic strategy. However, despite documented evidence of clinical activity, limited information is available on the optimal dosing strategy of CDK4/6 inhibitors. Here, we present an integrated semi-mechanistic pharmacokinetic/pharmacodynamic model to characterize the quantitative pharmacology of LY2835219, a CDK4/6 inhibitor, in xenograft tumors. EXPERIMENTAL DESIGN: LY2835219 plasma concentrations were connected to CDK4/6 inhibition and cell-cycle arrest in colo-205 human colorectal xenografts by incorporating the biomarkers, phospho-(ser780)-Rb, topoisomerase II α, and phosphohistone H3, into a precursor-dependent transit compartment model. This biomarker model was then connected to tumor growth inhibition (TGI) by: (i) relating the rate of tumor growth to mitotic cell density, and (ii) incorporating a concentration-dependent mixed cytostatic/cytotoxic effect driving quiescence and cell death at high doses. Model validation was evaluated by predicting LY2835219-mediated antitumor effect in A375 human melanoma xenografts. RESULTS: The model successfully described LY2835219-mediated CDK4/6 inhibition, cell-cycle arrest, and TGI in colo-205, and was validated in A375. The model also demonstrated that a chronic dosing strategy achieving minimum steady-state trough plasma concentrations of 200 ng/mL is required to maintain durable cell-cycle arrest. Quiescence and cell death can be induced by further increasing LY2835219 plasma concentrations. CONCLUSIONS: Our model provides mechanistic insight into the quantitative pharmacology of LY2835219 and supports the therapeutic dose and chronic dosing strategy currently adopted in clinical studies.

A novel CDK9 inhibitor shows potent antitumor efficacy in preclinical hematologic tumor models.

DNA-dependent RNA polymerase II (RNAP II) largest subunit RPB1 C-terminal domain (CTD) kinases, including CDK9, are serine/threonine kinases known to regulate transcriptional initiation and elongation by phosphorylating Ser 2, 5, and 7 residues on CTD. Given the reported dysregulation of these kinases in some cancers, we asked whether inhibiting CDK9 may induce stress response and preferentially kill tumor cells. Herein, we describe a potent CDK9 inhibitor, LY2857785, that significantly reduces RNAP II CTD phosphorylation and dramatically decreases MCL1 protein levels to result in apoptosis in a variety of leukemia and solid tumor cell lines. This molecule inhibits the growth of a broad panel of cancer cell lines, and is particularly efficacious in leukemia cells, including orthotopic leukemia preclinical models as well as in ex vivo acute myeloid leukemia and chronic lymphocytic leukemia patient tumor samples. Thus, inhibition of CDK9 may represent an interesting approach as a cancer therapeutic target, especially in hematologic malignancies.

Cryopreservation of avian spermatozoa.

This chapter describes a simple system for cryopreservation of avian spermatozoa as pellets, formed by dropping volumes of sperm suspension directly into liquid nitrogen with dimethylacetamide as cryoprotectant. The method originates from the group at the Research Institute of Farm Animal Breeding and Genetics at St. Petersburg-Pushkin and is described here for chicken spermatozoa, but has also been adapted successfully for other species, such as Houbara bustards and pheasants.

Discovery of non-covalent dipeptidyl peptidase IV inhibitors which induce a conformational change in the active site.

A series of non-covalent inhibitors of the serine protease dipeptidyl peptidase IV (DPP-IV) were found to adopt a U-shaped binding conformation in X-ray co-crystallization studies. Remarkably, Tyr547 undergoes a 70 degrees side-chain rotation to accommodate the inhibitor and allows access to a previously unexposed area of the protein backbone for hydrogen bonding.

Species specificity in avian sperm:perivitelline interaction.

The interaction of chicken spermatozoa with the inner perivitelline layer from different avian species in vitro during a 5 min co-incubation was measured as the number of points of hydrolysis produced per unit area of inner perivitelline layer. The average degree of interaction, as a proportion of that between chicken spermatozoa and their homologous inner perivitelline layer, was: equal to or greater than 100% within Galliformes (chicken, turkey, quail, pheasant, peafowl and guineafowl); 44% within Anseriformes (goose, duck); and less than 30% in Passeriformes (Zebra Finch) and Columbiformes (collared-dove). The homologue of the putative chicken sperm-binding proteins, chicken ZP1 and ZP3, were identified by Western blotting with anti-chicken ZP1/ZP3 antibody in the perivitelline layers of all species. The functional cross-reactivity between chicken spermatozoa and heterologous inner perivitelline layer appeared to be linked to known phylogenetic distance between the species, although it was not related to the relative affinity of the different ZP3 homologues for anti-chicken ZP3. This work demonstrates that sperm interaction with the egg investment does not represent such a stringent species-specific barrier in birds as it does in mammals and marine invertebrates. This may be a factor in the frequency of hybrid production in birds.

Excess nuclear DNA in spermatozoa of guinea fowl.

The proportion of spermatozoa with elongated nuclei in ejaculates from a strain of guinea fowl was estimated, subjectively, to range from approximately 1 to 6%. It was confirmed by image analysis that in an ejaculate from one male, the distribution of nuclear lengths was bimodal, with a distinct population comprising 10% of spermatozoa having a mean nuclear length that was 52% greater than that of the remaining 90%. Furthermore, the mean DNA content of the 'large-nuclei' population was 1.85 times (not significantly different from twice) that of the main sperm population. The proportion of large-nuclei spermatozoa that was motile was less than that of normal sperm (31% versus 59%) and the velocity of motile spermatozoa was also less (24 microm/s versus 72 microm/s). The poor motility of the large-nuclei spermatozoa in vitro was reflected in their limited performance in vivo, since only 1.1% were found associated with the egg outer perivitelline layer. This is the first report to quantify the occurrence of, presumed, polyploid spermatozoa in a domestic bird. The incidence of such spermatozoa in commercial guinea fowl and other domestic poultry and the genesis and effects on fertility of such spermatozoa may be significant.