Use of the Cassette-Dosing Approach to Assess Brain Penetration in Drug Discovery

ABSTRACT: The objective of the present study was to examine the cassette dosing method in determination of brain-to-plasma concentration ratio (area under the concentration-time profiles for plasma/area under the concentration-time profiles for brain, K p ). Eleven model compounds, amprenavir, citalopram, digoxin, elacridar, imatinib, (3S,6S,12aS)-1,2,3,4,6,7,12,12a-octahydro-9-methoxy-6-(2-methylpropyl)-1,4-dioxopyrazino[1,2:1,6]pyrido[3,4-b]indole-3-propanoic acid 1,1-dimethylethyl ester (Ko143), loperamide, prazosin, quinidine, sulfasalazine, and verapamil, were selected to compare their K p determined from discrete dosing in wild-type mice and their K p from cassette dosing in wild-type, Mdr1a/1b(؊/؊), Bcrp1(؊/؊), and Mdr1a/1b(؊/؊)/Bcrp1(؊/؊) mice at 1 to 3 mg/kg. The mice brain and plasma were collected at 0.25, 1, and 3 h and were analyzed using high-performance liquid chromatography-tandem mass spectrometry methods. The K p determined from discrete dosing versus cassette dosing in the wild-type mice were within 2-fold for all the compounds except sulfasalazine and Ko143. The brain concentrations of sulfasalazine and Ko143 and the plasma concentrations of Ko143 were below the lower limit of quantitation. In addition, the K p values estimated by mass spectrometry responses, namely the ratio of compound peak area to internal standard peak area, were within 2-fold of the K p observed from the actual concentrations. Furthermore, the ratios of K p in Mdr1a/1b(؊/؊), Bcrp1(؊/؊), and Mdr1a/1b(؊/؊)/ Bcrp1(؊/؊) mice versus the K p in the wild-type mice from cassette dosing were consistent with the ones reported in the literature where the compounds were dosed discretely. These results demonstrate that drug-drug interactions at the blood-brain barrier are unlikely at a subcutaneous dose of 1 to 3 mg/kg and support the use of the cassette dosing approach to assess brain penetration in drug discovery

GluN2A NMDA Receptor Enhancement Improves Brain Oscillations, Synchrony, and Cognitive Functions in Dravet Syndrome and Alzheimer's Disease Models.

NMDA receptors (NMDARs) play subunit-specific roles in synaptic function and are implicated in neuropsychiatric and neurodegenerative disorders. However, the in vivo consequences and therapeutic potential of pharmacologically enhancing NMDAR function via allosteric modulation are largely unknown. We examine the in vivo effects of GNE-0723, a positive allosteric modulator of GluN2A-subunit-containing NMDARs, on brain network and cognitive functions in mouse models of Dravet syndrome (DS) and Alzheimer's disease (AD). GNE-0723 use dependently potentiates synaptic NMDA receptor currents and reduces brain oscillation power with a predominant effect on low-frequency (12-20 Hz) oscillations. Interestingly, DS and AD mouse models display aberrant low-frequency oscillatory power that is tightly correlated with network hypersynchrony. GNE-0723 treatment reduces aberrant low-frequency oscillations and epileptiform discharges and improves cognitive functions in DS and AD mouse models. GluN2A-subunit-containing NMDAR enhancers may have therapeutic benefits in brain disorders with network hypersynchrony and cognitive impairments

Comparison of in vitro – in vivo extrapolation of biliary clearance using an empirical scaling factor versus transport‐based scaling factors in sandwich‐cultured rat hepatocytes

Biliary clearance (CL b ) is often underestimated by in vitro – in vivo extrapolation from sandwich‐cultured hepatocytes (SCHs). The objective of this study was to compare the performance of a universal correction factor with transporter‐based correction factors in correcting underestimation of CL b . The apparent in vitro CL b of a training set of 21 compounds was determined using the SCH model and extrapolated to apparent in vivo CL b (CL b, app ). A universal correction factor (10.2) was obtained by a linear regression of the predicted CL b, app and observed in vivo CL b of training set compounds and applied to an independent test set ( n = 20); the corrected CL b predictions of 13 compounds were within twofold error of observed values. Furthermore, two transporter‐based correction factors (Organic anion transporting polypeptides/multidrug‐resistance‐related protein 2 and diffusion/P‐glycoprotein) were estimated by linear regression analysis of training set compounds. The applications of the two correction factors to the test set resulted in improved prediction precision. In conclusion, both the universal correction factor and transporter‐based correction factors provided reasonable corrections of CL b values, which are often underestimated by the SCH model. The use of transporter‐based correction factors resulted in an even greater improvement of predictions for compounds with intermediate‐to‐high CL b values. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:2837–2850, 2013Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/99050/1/23620_ftp.pd

Dependence of Tumor Cell Lines and Patient-Derived Tumors on the NAD Salvage Pathway Renders Them Sensitive to NAMPT Inhibition with GNE-618

Nicotinamide adenine dinucleotide (NAD) is a critical metabolite that is required for a range of cellular reactions. A key enzyme in the NAD salvage pathway is nicotinamide phosphoribosyl transferase (NAMPT), and here, we describe GNE-618, an NAMPT inhibitor that depletes NAD and induces cell death in vitro and in vivo. While cells proficient for nicotinic acid phosphoribosyl transferase (NAPRT1) can be protected from NAMPT inhibition as they convert nicotinic acid (NA) to NAD independent of the salvage pathway, this protection only occurs if NA is added before NAD depletion. We also demonstrate that tumor cells are unable to generate NAD by de novo synthesis as they lack expression of key enzymes in this pathway, thus providing a mechanistic rationale for the reliance of tumor cells on the NAD salvage pathway. Identifying tumors that are sensitive to NAMPT inhibition is one potential way to enhance the therapeutic effectiveness of an NAMPT inhibitor, and here, we show that NAMPT, but not NAPRT1, mRNA and protein levels inversely correlate with sensitivity to GNE-618 across a panel of 53 non-small cell lung carcinoma cell lines. Finally, we demonstrate that GNE-618 reduced tumor growth in a patient-derived model, which is thought to more closely represent heterogeneous primary patient tumors. Thus, we show that dependence of tumor cells on the NAD salvage pathway renders them sensitive to GNE-618 in vitro and in vivo, and our data support further evaluation of the use of NAMPT mRNA and protein levels as predictors of overall sensitivity

Supplementation of Nicotinic Acid with NAMPT Inhibitors Results in Loss of In Vivo Efficacy in NAPRT1-Deficient Tumor Models

Nicotinamide adenine dinucleotide (NAD) is a metabolite essential for cell survival and generated de novo from tryptophan or recycled from nicotinamide (NAM) through the nicotinamide phosphoribosyltransferase (NAMPT)-dependent salvage pathway. Alternatively, nicotinic acid (NA) is metabolized to NAD through the nicotinic acid phosphoribosyltransferase domain containing 1 (NAPRT1)-dependent salvage pathway. Tumor cells are more reliant on the NAMPT salvage pathway making this enzyme an attractive therapeutic target. Moreover, the therapeutic index of NAMPT inhibitors may be increased by in NAPRT-deficient tumors by NA supplementation as normal tissues may regenerate NAD through NAPRT1. To confirm the latter, we tested novel NAMPT inhibitors, GNE-617 and GNE-618, in cell culture- and patient-derived tumor models. While NA did not protect NAPRT1-deficient tumor cell lines from NAMPT inhibition in vitro, it rescued efficacy of GNE-617 and GNE-618 in cell culture- and patient-derived tumor xenografts in vivo. NA co-treatment increased NAD and NAM levels in NAPRT1-deficient tumors to levels that sustained growth in vivo. Furthermore, NAM co-administration with GNE-617 led to increased tumor NAD levels and rescued in vivo efficacy as well. Importantly, tumor xenografts remained NAPRT1-deficient in the presence of NA, indicating that the NAPRT1-dependent pathway is not reactivated. Protection of NAPRT1-deficient tumors in vivo may be due to increased circulating levels of metabolites generated by mouse liver, in response to NA or through competitive reactivation of NAMPT by NAM. Our results have important implications for the development of NAMPT inhibitors when considering NA co-treatment as a rescue strategy