Intra-Articular Hyaluronic Acid and Chondroitin Sulfate: Pharmacokinetic Investigation in Osteoarthritic Rat Models.

BACKGROUND: Viscosupplementation of synovial fluid with intra-articular (IA) injections of hyaluronic acid (HA) is widely used for symptomatic treatment of osteoarthritis (OA). Herein we present HCS, a new combination of chemicals, associating HA and chondroitin sulfate (CS), both members of the glycosaminoglycan (GAGs) family, which are major components of the joint. HA provides viscosity to the synovial fluid and CS provides elasticity to the cartilage. Reduced levels of HA and CS are observed in OA joints and are associated with progressive cartilage damage and loss. OBJECTIVE: The objective of the study was to evaluate the pharmacokinetic (PK) properties of both HA and CS after IA administration in a validated OA animal model. METHODS: Motion impairment measurements and histological examinations were used to validate the ability of an IA injection of mono-iodoacetate (MIA) in the knee of rats to induce OA symptoms. Then, the PK properties of HA and CS after IA administration were characterized and each active ingredient was independently profiled: HA was labeled with tritium (3H-HA) and CS was labeled with carbon 14. (14C-CS) The final radio-labeled solution reproduced the cold HCS formulation. RESULTS: Four male Sprague-Dawley rats received a 1 mg MIA injection on day 1, then motor impairment was monitored from day 4 to day 18. Chondrocyte necrosis, loss of GAGs and other cartilage damage were observed. Twelve other rats received a MIA IA injection on day 1 then a radio-labeled HCS IA injection (50 µL) on day 8. Plasma and knee cartilage were collected postadministration and the terminal half-life was similar in both matrices (about 5 days), for both 3H-HA and 14C-CS. CONCLUSIONS: Despite differences in their molecular size, HA and CS showed PK behavior similarly characterized by prolonged residence inside the joint and slow release in plasma, favoring long-term beneficial effects. (Curr Ther Res Clin Exp. 2020; 92:XXX-XXX).

Characterization of the novel positive allosteric modulator of the metabotropic glutamate receptor 4 ADX88178 in rodent models of neuropsychiatric disorders.

There is growing evidence that activation of metabotropic glutamate receptor 4 (mGlu4) leads to anxiolytic- and antipsychotic-like efficacy in rodent models, yet its relevance to depression-like reactivity remains unclear. Here, we present the pharmacological evaluation of ADX88178 [5-methyl-N-(4-methylpyrimidin-2-yl)-4-(1H-pyrazol-4-yl)thiazol-2-amine], a novel potent, selective, and brain-penetrant positive allosteric modulator of the mGlu4 receptor in rodent models of anxiety, obsessive compulsive disorder (OCD), fear, depression, and psychosis. ADX88178 dose-dependently reduced the number of buried marbles in the marble burying test and increased open-arm exploration in the elevated plus maze (EPM) test, indicative of anxiolytic-like efficacy. Target specificity of the effect in the EPM test was confirmed using male and female mGlu4 receptor knockout mice. In mice, ADX88178 reduced the likelihood of conditioned freezing in the acquisition phase of the fear conditioning test, yet had no carryover effect in the expression phase. Also, ADX88178 dose-dependently reduced duration of immobility in the forced swim test, indicative of antidepressant-like efficacy. ADX88178 reduced DOI (2,5-dimethoxy-4-iodoamphetamine)-mediated head twitches (albeit with no dose-dependency), and MK-801 [(5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine]-induced locomotor hyperactivity in mice, but was inactive in the conditioned avoidance response test in rats. The compound showed good specificity as it had no effect on locomotor activity in mice and rats at efficacious doses. Thus, allosteric activation of mGlu4 receptors can be a promising new therapeutic approach for treatment of anxiety, OCD, fear-related disorders, and psychosis.

Scaling approaches for the prediction of human clearance of LNA-i-mir-221: A retrospective validation.

LNA-i-miR-221 is a novel microRNA(miRNA)-221 inhibitor designed for the treatment of human malignancies. It has recently undergone phase 1 clinical trial (P1CT) and early pharmacokinetics (PKs) data in cancer patients are now available. We previously used multiple allometric interspecies scaling methods to draw inferences about LNA-i-miR-221 PKs in humans and estimated the patient dose based on the safe and pharmacodynamic (PD) active dose observed in mice, therefore providing a framework for the definition of safe starting and escalation doses for the P1CT. The preliminary data collected during the P1CT showed that the LNA-i-miR-221 anticipated doses, according to our human PK estimation approach, were indeed well tolerated and effective. PD data demonstrated concentration-dependent downregulation of miR-221 and upregulation of its CDKN1B/p27 and PTEN canonical targets as well as stable disease in 8 (50.0%) patients and partial response in 1 (6.3%) colorectal cancer case. Here, we detail the experimentally evaluated PK parameters of LNA-i-miR-221 in human, using both a non-compartmental and a population PKs approach. The population approach was adequately described by a three-compartments model with first-order elimination. The recorded age, sex and body weight of patients were evaluated as potential covariates. The estimated typical population parameter values were clearance (CL = 200 mL/h/kg), central volume of distribution (V1 = 45 mL/kg), peripheral volume of distribution (V2 = 200 mL/kg, volume of the second peripheral compartment V3 = 930 mL/h/kg) and inter-compartmental clearance (Q2 = 480 mL/h/kg and Q3 = 68 mL/h/kg). Age was found to be a predictor of Q3, with a statistically significant correlation. This work aimed also at retrospectively comparing the measured plasmatic clearance values with those predicted by different allometric scaling approaches. Our comparative analysis showed that the most accurate prediction was achieved by applying the single species allometric scaling approach and that the use of more than one species in allometric scaling to predict therapeutic oligonucleotides PKs would not necessarily generate the best prediction. Finally, our predictive approach was found accurate not only in predicting the main PK parameters in human but suggesting the range of effective and safe dose to be applied in the next clinic phase 2.

N-(2-alkylaminoethyl)-4-(1,2,4-oxadiazol-5-yl)piperazine-1-carboxamides as highly potent smoothened antagonists.

Smoothened (Smo) antagonists are emerging as new therapies for the treatment of neoplasias with aberrantly reactivated hedgehog (Hh) signaling pathway. A novel series of 4-[3-(quinolin-2-yl)-1,2,4-oxadiazol-5-yl]piperazinyl ureas as smoothened antagonists was recently described, herein the series has been further optimized through the incorporation of a basic amine into the urea. This development resulted in identification of some exceptionally potent smoothened antagonists with low serum shifts, however, reductive ring opening on the 1,2,4-oxadiazole in rats limits the applicability of these compounds in in vivo studies.

Identification of MK-5710 ((8aS)-8a-methyl-1,3-dioxo-2-[(1S,2R)-2-phenylcyclo- propyl]-N-(1-phenyl-1H-pyrazol-5-yl)hexahydro-imidazo[1,5-a]pyrazine-7(1H)-carboxamide), a potent smoothened antagonist for use in Hedgehog pathway dependent malignancies, part 2.

The Hedgehog (Hh-) signaling pathway is a key developmental pathway which gets reactivated in many human tumors, and smoothened (Smo) antagonists are emerging as novel agents for the treatment of malignancies dependent on the Hh-pathway, with the most advanced compounds demonstrating encouraging results in initial clinical trials. A novel series of potent bicyclic hydantoin Smo antagonists was reported in the preceding article, these have been resolved, and optimized to identify potent homochiral derivatives with clean off-target profiles and good pharmacokinetic properties in preclinical species. While showing in vivo efficacy in mouse allograft models, unsubstituted bicyclic tetrahydroimidazo[1,5-a]pyrazine-1,3(2H,5H)-diones were shown to epimerize in plasma. Alkylation of the C-8 position blocks this epimerization, resulting in the identification of MK-5710 (47) which was selected for further development.

Allometric Scaling Approaches for Predicting Human Pharmacokinetic of a Locked Nucleic Acid Oligonucleotide Targeting Cancer-Associated miR-221.

: LNA-i-miR-221 is a novel phosphorothioate backbone 13-mer locked nucleic acid oligonucleotide-targeting microRNA-221 designed for the treatment of human malignancies. To understand the pharmacokinetic properties of this new agent, including unbound/total clearance, we investigated the LNA-i-miR-221 protein binding in three different species, including rat (Sprague-Dawley), monkey (Cynomolgus), and human. To this end, we generated a suitable ultrafiltration method to study the binding of LNA-i-miR-221 to plasma proteins. We identified that the fraction of LNA-i-miR-221 (at concentration of 1 and 10 µM) bound to rat, monkey, and human plasma proteins was high and ranged from 98.2 to 99.05%. This high protein binding of LNA-i-miR-221 to plasma proteins in all the species tested translates into a pharmacokinetic advantage by preventing rapid renal clearance. The integration of these results into multiple allometric interspecies scaling methods was then used to draw inferences about LNA-i-miR-221 pharmacokinetics in humans, thereby providing a framework for definition of safe starting and escalation doses and moving towards a first human clinical trial of LNA-i-miR-221.

Design and synthesis of bicyclic pyrimidinones as potent and orally bioavailable HIV-1 integrase inhibitors.

HIV integrase is one of the three enzymes encoded by HIV genome and is essential for viral replication, but integrase inhibitors as marketed drugs have just very recently started to emerge. In this study, we show the evolution from the N-methylpyrimidinone structure to bicyclic pyrimidinones. Introduction of a suitably substituted amino moiety modulated the physical-chemical properties of the molecules and conferred nanomolar activity in the inhibition of spread of HIV-1 infection in cell culture. An extensive SAR study led to sulfamide (R)- 22b, which inhibited the strand transfer with an IC50 of 7 nM and HIV infection in MT4 cells with a CIC95 of 44 nM, and ketoamide (S)- 28c that inhibited strand transfer with an IC50 of 12 nM and the HIV infection in MT4 cells with a CIC95 of 13 nM and exhibited a good pharmacokinetic profile when dosed orally to preclinical species.

High-throughput microsomal stability assay for screening new chemical entities in drug discovery.

In this work, the authors present a novel, robotic, automated protocol for assessing a metabolic stability protocol assembled on a Hamilton platform and a new strategy for pooling samples (cassette analysis). To increase the high throughput of the liquid chromatography (LC) step, fast chromatography and automated liquid chromatography tandem mass spectrometry (LC/MS/MS) analytical methods were also developed, and a rapid data analysis system was generated that converts peak areas obtained by LC/MS/MS in intrinsic clearance values. All of the steps of the microsomal stability assay were carefully studied and optimized. Standard errors and confidence intervals of the measured clearances were also automatically generated in the process to allow an immediate evaluation of the significance of observed values. Methods based on pooling analysis of 2 and 4 different analytes were compared with a standard method without pooling. A simple statistical treatment was used to show their equivalence. The different protocols developed were analyzed in terms of the best compromise between accuracy and high-throughput capabilities.

Optimization of a series of potent and selective ketone histone deacetylase inhibitors.

Histone deacetylase (HDAC) inhibitors offer a promising strategy for cancer therapy and the first generation HDAC inhibitors are currently in the clinic. Herein we describe the optimization of a series of ketone small molecule HDAC inhibitors leading to potent and selective class I HDAC inhibitors with good dog PK.

Direct comparison of UDP-glucuronosyltransferase and cytochrome P450 activities in human liver microsomes, plated and suspended primary human hepatocytes from five liver donors.

UDP-glucuronosyltransferases (UGTs) and cytochrome P450s (CYPs) are the major enzymes involved in hepatic metabolism of drugs. Hepatic drug metabolism is commonly investigated using human liver microsomes (HLM) or primary human hepatocytes (PHH). We describe the development of a sensitive assay to phenotype activities of six major hepatic UGT isoforms (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9 and UGT2B7) in intact PHH by analysis of glucuronidation of selective probe substrates. The non-selective, general substrate 7-hydroxycoumarin was included for comparison. For each liver donor preparation (five donors) UGT activities in cryopreserved suspended and plated PHH were compared to HLM prepared from the same donors. Standard CYP reaction phenotyping of seven major isoforms was performed in parallel. For all donors, CYP- and UGT-isoforms activity profiles were comparable in PHH and HLM, indicating that reaction phenotyping with selective probe substrates in intact cells primarily reflects respective CYP or UGT activity. System-dependent effects on UGT and CYP isoform activity were still found. While UGT activity of UGT1A1 was equivalent in plated and suspended PHH, UGT1A3, UGT1A6 and UGT2B7 activity was higher in suspended PHH and UGT1A9 and UGT1A4 activity was higher in plated PHH. The well-known decrease in activity of most CYP isoforms in plated compared to suspended PHH was confirmed. Importantly, we found a significant loss in CYP2C19 and CYP2B6 in HLM, activity being lower than in intact cells. Taken together, these findings implicate that, dependent on the UGT or CYP isoforms involved in the metabolism of a given compound, the outcome of metabolic assays is strongly dependent on the choice of the in vitro system. The currently described UGT- and CYP- activity profiling method can be used as a standard assay in intact cells and can especially aid in reaction phenotyping of in vitro systems for which a limited number of cells are available.

Extrahepatic metabolism may complicate the IVIVC in rats.

As the liver is generally considered the organ most involved in metabolic transformations, metabolism in other organs is often overlooked and in vitro screening systems largely adopted in drug discovery are generally based on liver tissue fractions. First pharmacokinetics of new chemical entities (NCEs) are initially based on preclinical species; rat is used in the majority of the cases to assess early in vitro-in vivo correlation (IVIVC). It is important, in this perspective, to address as early as possible the relevant differences between rat and human and the limits using pharmacokinetic studies in this species as a model for the human PK. In this paper the author reports at least three clear examples in drug discovery where the use of hepatic in-vitro systems resulted in a very poor IVIVC due to relevant extrahepatic metabolism in rats.

Discovery of a potent class I selective ketone histone deacetylase inhibitor with antitumor activity in vivo and optimized pharmacokinetic properties.

The optimization of a potent, class I selective ketone HDAC inhibitor is shown. It possesses optimized pharmacokinetic properties in preclinical species, has a clean off-target profile, and is negative in a microbial mutagenicity (Ames) test. In a mouse xenograft model it shows efficacy comparable to that of vorinostat at a 10-fold reduced dose.

Discovery of 2-{4-[(3S)-piperidin-3-yl]phenyl}-2H-indazole-7-carboxamide (MK-4827): a novel oral poly(ADP-ribose)polymerase (PARP) inhibitor efficacious in BRCA-1 and -2 mutant tumors.

We disclose the development of a novel series of 2-phenyl-2H-indazole-7-carboxamides as poly(ADP-ribose)polymerase (PARP) 1 and 2 inhibitors. This series was optimized to improve enzyme and cellular activity, and the resulting PARP inhibitors display antiproliferation activities against BRCA-1 and BRCA-2 deficient cancer cells, with high selectivity over BRCA proficient cells. Extrahepatic oxidation by CYP450 1A1 and 1A2 was identified as a metabolic concern, and strategies to improve pharmacokinetic properties are reported. These efforts culminated in the identification of 2-{4-[(3S)-piperidin-3-yl]phenyl}-2H-indazole-7-carboxamide 56 (MK-4827), which displays good pharmacokinetic properties and is currently in phase I clinical trials. This compound displays excellent PARP 1 and 2 inhibition with IC(50) = 3.8 and 2.1 nM, respectively, and in a whole cell assay, it inhibited PARP activity with EC(50) = 4 nM and inhibited proliferation of cancer cells with mutant BRCA-1 and BRCA-2 with CC(50) in the 10-100 nM range. Compound 56 was well tolerated in vivo and demonstrated efficacy as a single agent in a xenograft model of BRCA-1 deficient cancer.

High-throughput radiometric CYP2C19 inhibition assay using tritiated (S)-mephenytoin.

A rapid and sensitive radiometric assay for assessing the potential of drugs to inhibit cytochrome P450 (P450) 2C19 in human liver microsomes is described. The new assay, which does not require high-performance liquid chromatography (HPLC) separation or mass spectrometric detection, is based on the release of tritium as tritiated water that occurs upon CYP2C19-mediated 4'-hydroxylation of (S)-mephenytoin labeled with tritium in the 4' position. Because this reaction is subject to an NIH shift, tritium was also introduced into the 3'- and 5'-positions of the tracer to enhance formation of a tritiated water product. Tritiated water was separated from the substrate using 96-well solid-phase extraction plates. The reaction is NADPH-dependent and sensitive to CYP2C19 inhibitors. IC(50) values for 15 diverse drugs differed less than 2.5-fold from those determined by quantification of the unlabeled 4'-hydroxy-(S)-mephenytoin product, using HPLC coupled to mass spectrometric detection. All of the steps of the new assay, namely incubation, product separation, and radioactivity counting, are performed in a 96-well format and can be automated. This assay represents a non-HPLC, high-throughput version of the classic (S)-mephenytoin 4'-hydroxylation assay, which is the most widely used method to assess the potential for CYP2C19 inhibition of new chemical entities.