Discovery, Optimization, and Biological Characterization of 2,3,6-Trisubstituted Pyridine-Containing M4 Positive Allosteric Modulators.

Herein we describe the discovery and optimization of a new series of 2,3-disubstituted and 2,3,6-trisubstituted muscarinic acetylcholine receptor 4 (M4 ) positive allosteric modulators (PAMs). Iterative libraries enabled rapid exploration of one-dimensional structure-activity relationships (SAR) and identification of potency-enhancing heterocycle and N-alkyl pyrazole substituents. Further optimization led to identification of the potent, receptor-subtype-selective, brain-penetrant tool compound 24 (7-[3-[1-[(1-fluorocyclopentyl)methyl]pyrazol-4-yl]-6-methyl-2-pyridyl]-3-methoxycinnoline). It is efficacious in preclinical assays that are predictive of antipsychotic effects, producing dose-dependent reversal of amphetamine-induced hyperlocomotion in rats and mice, but not in M4 knockout mice. Cholinergic-related adverse effects observed in rats treated with 24 at unbound plasma concentrations more than 3-fold higher than an efficacious dose in the hyperlocomotion assay were fewer and less severe than those observed in rats treated with the nonselective M4 agonist xanomeline, suggesting a receptor-subtype-selective PAM has the potential for an improved safety profile.

Microbial biotransformation - an important tool for the study of drug metabolism.

Metabolite identification is an integral part of both preclinical and clinical drug discovery and development. Synthesis of drug metabolites is often required to support definitive identification, preclinical safety studies and clinical trials. Here we describe the use of microbial biotransformation as a tool to produce drug metabolites, complementing traditional chemical synthesis and other biosynthetic methods such as hepatocytes, liver microsomes and recombinant human drug metabolizing enzymes. A workflow is discussed whereby microbial strains are initially screened for their ability to form the putative metabolites of interest, followed by a scale-up to afford quantities sufficient to perform definitive identification and further studies. Examples of the microbial synthesis of several difficult-to-synthesize hydroxylated metabolites and three difficult-to-synthesize glucuronidated metabolites are described, and the use of microbial biotransformation in drug discovery and development is discussed.

Preclinical to Human Translational Pharmacology of the Novel M1 Positive Allosteric Modulator MK-7622.

The current standard of care for treating Alzheimer's disease is acetylcholinesterase inhibitors, which nonselectively increase cholinergic signaling by indirectly enhancing activity of nicotinic and muscarinic receptors. These drugs improve cognitive function in patients, but also produce unwanted side effects that limit their efficacy. In an effort to selectively improve cognition and avoid the cholinergic side effects associated with the standard of care, various efforts have been aimed at developing selective M1 muscarinic receptor activators. In this work, we describe the preclinical and clinical pharmacodynamic effects of the M1 muscarinic receptor-positive allosteric modulator, MK-7622. MK-7622 attenuated the cognitive-impairing effects of the muscarinic receptor antagonist scopolamine and altered quantitative electroencephalography (qEEG) in both rhesus macaque and human. For both scopolamine reversal and qEEG, the effective exposures were similar between species. However, across species the minimum effective exposures to attenuate the scopolamine impairment were lower than for qEEG. Additionally, there were differences in the spectral power changes produced by MK-7622 in rhesus versus human. In sum, these results are the first to demonstrate translation of preclinical cognition and target modulation to clinical effects in humans for a selective M1 muscarinic receptor-positive allosteric modulator.

Discovery and optimization of 2-pyridinone aminal integrase strand transfer inhibitors for the treatment of HIV.

HIV integrase strand transfer inhibitors (InSTIs) represent an important class of antiviral therapeutics with proven efficacy and excellent tolerability for the treatment of HIV infections. In 2007, Raltegravir became the first marketed strand transfer inhibitor pioneering the way to a first-line therapy for treatment-naïve patients. Challenges with this class of therapeutics remain, including frequency of the dosing regimen and the genetic barrier to resistance. To address these issues, research towards next-generation integrase inhibitors has focused on imparting potency against RAL-resistent mutants and improving pharmacokinetic profiles. Herein, we detail medicinal chemistry efforts on a novel class of 2-pyridinone aminal InSTIs, inpsired by MK-0536, which led to the discovery of important lead molecules for our program. Systematic optimization carried out at the amide and aminal positions on the periphery of the core provided the necessary balance of antiviral activity and physiochemical properties. These efforts led to a novel aminal lead compound with the desired virological profile and preclinical pharmacokinetic profile to support a once-daily human dose prediction.

Discovery of 2-Pyridinone Aminals: A Prodrug Strategy to Advance a Second Generation of HIV-1 Integrase Strand Transfer Inhibitors.

The search for new molecular constructs that resemble the critical two-metal binding pharmacophore required for HIV integrase strand transfer inhibition represents a vibrant area of research within drug discovery. Here we present the discovery of a new class of HIV integrase strand transfer inhibitors based on the 2-pyridinone core of MK-0536. These efforts led to the identification of two lead compounds with excellent antiviral activity and preclinical pharmacokinetic profiles to support a once-daily human dose prediction. Dose escalating PK studies in dog revealed significant issues with limited oral absorption and required an innovative prodrug strategy to enhance the high-dose plasma exposures of the parent molecules.

Understanding and reducing the experimental variability of in vitro plasma protein binding measurements.

The experimental measurement of plasma protein binding is a useful in vitro Absorption Distribution Metabolism and Excretion(ADME) assay currently conducted in both screening and definitive early development candidate modes. The fraction unbound is utilized to calculate important pharmacokinetic (PK) parameters such as unbound clearance and unbound volume of distribution in animals that can be used to make human PK and dose predictions and estimate clinically relevant drug-drug interaction potential. Although these types of assays have been executed for decades, a rigorous statistical analysis of sources of variability has not been conducted because of the tedious nature of the manual experiment. Automated conduct of the incubations using a 96-well equilibrium dialysis device as well as high-throughput liquid chromatography-mass spectrometry quantitation has now made this level of rigor accessible and useful. Sources of variability were assessed including well position, day-to-day, and site-to-site reproducibility. Optimal pH conditions were determined using a design of experiments method interrogating buffer strength, CO2 % and device preparation conditions. Variability was minimized by implementing an in-well control that is concurrently analyzed with new chemical entity analytes. Data acceptance criteria have been set for both the in-well control and the range of analyte variability, with a sliding scale tied to analyte-binding characteristics. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:3302-3309, 2014.

Atmospheric Pressure Chemical Ionization Tandem Mass Spectrometry of Carotenoids.

Carotenoids are natural pigments synthesized by plants and photosynthetic microorganisms, some of which, like ß-carotene, are precursors of vitamin A, and others such as lutein and lycopene might function in the prevention of age-related macular degeneration and prostate cancer, respectively. Mass spectrometry provides high sensitivity and selectivity for the identification and quantitative analysis of carotenoids in biological samples, and previous studies have described how atmospheric pressure chemical ionization (APCI) offers distinct advantages over electrospray and fast atom bombardment for the analysis of specific carotenoids. Since APCI product ion tandem mass spectra have been reported for only a few carotenoids, a detailed investigation of twelve carotenes and xanthophylls was carried out using both positive ion and negative ion APCI tandem mass spectrometry with collision-induced dissociation. Using protonated molecules as precursor ions in positive ion mode and radical anions in negative ion mode, characteristic fragment ions were identified that may be used to distinguish between carotenoids.

Antioxidant effects of lycopene in African American men with prostate cancer or benign prostate hyperplasia: a randomized, controlled trial.

Consumption of tomato products is associated with a decreased risk of developing prostate cancer, and lycopene, the red carotenoid in the tomato, is a potent antioxidant that might contribute to this chemoprevention activity. A double-blind, randomized, placebo-controlled trial of 105 African American men veterans, recommended for prostate biopsy to detect cancer, was carried out to investigate whether oral administration of lycopene increases lycopene levels in blood and prostate tissue and lowers markers of oxidative stress. Urology patients were randomly assigned to receive 30 mg/d of lycopene as a tomato oleoresin or placebo for 21 days prior to prostate biopsy for possible diagnosis of prostate cancer. A total of 47 men had a diagnosis of prostate cancer, and 58 men had a diagnosis of benign prostate hyperplasia. Diet, smoking, and drinking habits were assessed. For the men receiving lycopene, the mean lycopene concentration increased from 0.74 ± 0.39 to 1.43 ± 0.61 µmol/L in plasma (P < 0.0001) and from 0.45 ± 0.53 to 0.59 ± 0.47 pmol/mg in prostate tissue (P = 0.005). No significant changes in the DNA oxidation product 8-oxo-deoxyguanosine and the lipid peroxidation product malondialdehyde were observed in prostate tissue and plasma, respectively, as a result of lycopene administration.

Multitargeted therapy of cancer by lycopene.

Lycopene (psi,psi-carotene) is the most abundant carotenoid in tomatoes and is the red pigment of not only tomatoes but also rosehips, watermelon, papaya, pink grapefruit, and guava. Unlike beta-carotene, lycopene lacks a beta-ionone ring and therefore has no pro-vitamin A activity. However, the 11 conjugated and two non-conjugated double bonds in lycopene make it highly reactive towards oxygen and free radicals, and this anti-oxidant activity probably contributes to its efficacy as a chemoprevention agent. The reactivity of lycopene also explains why it isomerizes rapidly in blood and tissues from the biosynthetic all-trans form to a mixture of cis-isomers. Prospective and retrospective epidemiological studies indicating an inverse relationship between lycopene intake and prostate cancer risk have been supported by in vitro and in vivo experiments showing that oral lycopene is bioavailable, accumulates in prostate tissue and is localized to the nucleus of prostate epithelial cells. In addition to antioxidant activity, in vitro experiments indicate other mechanisms of chemoprevention by lycopene including induction of apoptosis and antiproliferation in cancer cells, anti-metastatic activity, and the upregulation of the antioxidant response element leading to the synthesis of cytoprotective enzymes. Lycopene is a substrate for carotene-9',10'-monooxygenase (CMO2) and can be converted to apo-10'-carotenal. Although Phase I and II studies have been published that establish the safety of lycopene supplementation, carefully designed and adequately powered clinical studies of lycopene are still needed to confirm its efficacy as a chemoprevention agent.

Systematic investigation of lycopene effects in LNCaP cells by use of novel large-scale proteomic analysis software.

Lycopene, the red pigment of tomatoes, is a carotenoid with potent antioxidant properties. Although lycopene might function as a prostate cancer chemoprevention agent, little is known about its effects at the cellular level. To define general changes induced by treatment of cells with lycopene, and to gain insights into the possible chemoprevention properties of lycopene, we investigated changes in protein expression after lycopene treatment in human LNCaP cells. The high throughput proteomics data were then visualized and analyzed by novel biological protein pathway modeling software. Differentially expressed proteins were identified, and the data were analyzed by protein pathway simulation software without need for specialized programming by importing pathway models from a number of sources or creating their own. One notable outcome was the identification of a group of upregulated proteins involved in detoxification of reactive oxygen species. This finding suggests that a possible mechanism of lycopene chemoprevention is the stimulation of detoxification enzymes associated with the antioxidant response element. Novel biological pathway modeling software enhances analysis of large proteomics data. When applied to the analysis of proteins differentially expressed in prostate cancer cells upon treatment with lycopene, the upregulation of detoxification enzymes was identified.

Absorption and subcellular localization of lycopene in human prostate cancer cells.

Lycopene, the red pigment of the tomato, is under investigation for the chemoprevention of prostate cancer. Because dietary lycopene has been reported to concentrate in the human prostate, its uptake and subcellular localization were investigated in the controlled environment of cell culture using the human prostate cancer cell lines LNCaP, PC-3, and DU145. After 24 hours of incubation with 1.48 micromol/L lycopene, LNCaP cells accumulated 126.6 pmol lycopene/million cells, which was 2.5 times higher than PC-3 cells and 4.5 times higher than DU145 cells. Among these cell lines, only LNCaP cells express prostate-specific antigen and fully functional androgen receptor. Levels of prostate-specific antigen secreted into the incubation medium by LNCaP cells were reduced 55% as a result of lycopene treatment at 1.48 micromol/L. The binding of lycopene to the ligand-binding domain of the human androgen receptor was carried out, but lycopene was not found to be a ligand for this receptor. Next, subcellular fractionation of LNCaP cells exposed to lycopene was carried out using centrifugation and followed by liquid chromatography-tandem mass spectrometry quantitative analysis to determine the specific cellular locations of lycopene. The majority of lycopene (55%) was localized to the nuclear membranes, followed by 26% in nuclear matrix, and then 19% in microsomes. No lycopene was detected in the cytosol. These data suggest that the rapid uptake of lycopene by LNCaP cells might be facilitated by a receptor or binding protein and that lycopene is stored selectively in the nucleus of LNCaP cells.

Quantitative analysis of lycopene isomers in human plasma using high-performance liquid chromatography-tandem mass spectrometry.

An analytical method for the determination of the concentrations of total lycopene and its cis and all-trans isomers in human plasma has been developed using high-performance liquid chromatography-tandem mass spectrometry (LC-MS-MS). This method was based on the observation that, during negative ion atmospheric pressure chemical ionization with collision-induced dissociation, a unique fragment of m/z 467 was formed from the molecular ion of m/z 536 by elimination of a terminal isoprene group. The use of multiple reaction monitoring facilitated the selective detection of lycopene isomers and an internal standard without interference from the isobaric carotenoids a-carotene and beta-carotene, which are also abundant in human plasma. Measurement of total lycopene was carried out using a C18 high-performance liquid chromatography (HPLC) column and an isocratic mobile phase consisting of acetonitrile/methyl tert-butyl ether (95:5) so that all lycopene isomers eluted as a single chromatographic peak. all-trans-Lycopene was separated from its various cis isomers by using a C30 carotenoid column and a gradient solvent system from methanol to methyl tert-butyl ether. The effects of sample preparation and handling parameters on the stability of lycopene were evaluated such as the stability of lycopene in the HPLC autosampler and the effect of saponification upon lycopene isomerization. For example, the half-life of all-trans-lycopene in the HPLC mobile phase in the autosampler at 4 degrees C was determined to be approximately 16 h. Also, saponification of plasma samples was determined to cause lycopene degradation and isomerization so that lycopene recovery was reduced. The accuracy and interassay precision of this LC-MS-MS assay for lycopene showed a standard deviation of less than 10% over the range of 5-500 pmol injected on-column. The limit of detection was 11.2 fmol injected on-column, and the limit of quantitation was 22.8 fmol.