Scaffold-Hopping Approach To Discover Potent, Selective, and Efficacious Inhibitors of NF-κB Inducing Kinase.

NF-κB-inducing kinase (NIK) is a protein kinase central to the noncanonical NF-κB pathway downstream from multiple TNF receptor family members, including BAFF, which has been associated with B cell survival and maturation, dendritic cell activation, secondary lymphoid organ development, and bone metabolism. We report herein the discovery of lead chemical series of NIK inhibitors that were identified through a scaffold-hopping strategy using structure-based design. Electronic and steric properties of lead compounds were modified to address glutathione conjugation and amide hydrolysis. These highly potent compounds exhibited selective inhibition of LTßR-dependent p52 translocation and transcription of NF-κB2 related genes. Compound 4f is shown to have a favorable pharmacokinetic profile across species and to inhibit BAFF-induced B cell survival in vitro and reduce splenic marginal zone B cells in vivo.

NF-κB inducing kinase is a therapeutic target for systemic lupus erythematosus.

NF-κB-inducing kinase (NIK) mediates non-canonical NF-κB signaling downstream of multiple TNF family members, including BAFF, TWEAK, CD40, and OX40, which are implicated in the pathogenesis of systemic lupus erythematosus (SLE). Here, we show that experimental lupus in NZB/W F1 mice can be treated with a highly selective and potent NIK small molecule inhibitor. Both in vitro as well as in vivo, NIK inhibition recapitulates the pharmacological effects of BAFF blockade, which is clinically efficacious in SLE. Furthermore, NIK inhibition also affects T cell parameters in the spleen and proinflammatory gene expression in the kidney, which may be attributable to inhibition of OX40 and TWEAK signaling, respectively. As a consequence, NIK inhibition results in improved survival, reduced renal pathology, and lower proteinuria scores. Collectively, our data suggest that NIK inhibition is a potential therapeutic approach for SLE.

Exploration of PBPK Model-Calculation of Drug Time Course in Tissue Using IV Bolus Drug Plasma Concentration-Time Profile and the Physiological Parameters of the Organ.

An uncommon innovative consideration of the well-stirred linear physiologically based pharmacokinetic model and the drug plasma concentration-time profile, which is measured in routine intravenous bolus pharmacokinetic study, was applied for the calculation of the drug time course in human tissues. This cannot be obtained in the in vivo pharmacokinetic study. The physiological parameters of the organ such as organ tissue volume, organ blood flow rate, and its vascular volume were used in the calculation. The considered method was applied to calculate the time course of midazolam, alprazolam, quinidine, and diclofenac in human organs or tissues. The suggested method might be applied for the prediction of drug concentration-time profile in tissues and consequently the drug concentration level in the targeted tissue, as well as the possible undesirable toxic levels in other tissues.

On the accuracy of calculation of the mean residence time of drug in the body and its volumes of distribution based on the assumption of central elimination.

1. The steady state and terminal volumes of distribution, as well as the mean residence time of drug in the body (Vss, Vß, and MRT) are the common pharmacokinetic parameters calculated using the drug plasma concentration-time profile (Cp(t)) following intravenous (iv bolus or constant rate infusion) drug administration. 2. These traditional calculations are valid for the linear pharmacokinetic system with central elimination (i.e. elimination rate being proportional to drug concentration in plasma). The assumption of central elimination is not valid in general, so that the accuracy of the traditional calculation of these parameters is uncertain. 3. The comparison of Vss, Vß, and MRT calculated by the derived exact equations and by the commonly used ones was made considering a physiological model. It turned out that the difference between the exact and simplified calculations does not exceed 2%. 4. Thus the calculations of Vss, Vß, and MRT, which are based on the assumption of central elimination, may be considered as quite accurate. Consequently it can be used as the standard for comparisons with kinetic and in silico models.

Practical permeability-based hepatic clearance classification system (HepCCS) in drug discovery.

BACKGROUND: The use of liver microsomes and hepatocytes to predict total in vivo clearance is standard practice in the pharmaceutical industry; however, metabolic stability data alone cannot always predict in vivo clearance accurately. RESULTS: Apparent permeability generated from Mardin-Darby canine kidney cells and rat hepatocyte uptake for 33 discovery compounds were obtained. CONCLUSION: When there is underprediction of in vivo clearance, compounds with low apparent permeability (less than 3 × 10(-6) cm/s) all exhibited hepatic uptake. A systematic approach in the form of a classification system (hepatic clearance classification system) and decision tree that will help drug discovery scientists understand in vitro-in vivo clearance prediction disconnect early is proposed.

On the maintenance of hepatocyte intracellular pH 7.0 in the in-vitro metabolic stability assay.

The account of pH difference between hepatocytes (intracellular pH 7.0) and extracellular water (pH 7.4) leads to the novel equation for hepatic clearance (Berezhkovskiy, J Pharma Sci 100:1167-1683, 2011). The metabolic stability assay using hepatocytes is commonly performed in the incubation buffer of pH 7.4. If hepatocytes retain their physiological pH 7.0 in these conditions, then the assay would mimic the in vivo condition, that is pH 7.4 for plasma and extracellular water, and pH 7.0 in hepatocytes. In this case the rate of drug elimination, taken as proportional to unbound drug concentration in buffer, would correspond to the in vivo rate of drug elimination as proportional to the unbound drug concentration in the extracellular water. Consequently the commonly used PBPK equation for the rate of hepatic elimination, and the equation for hepatic clearance would be valid. However, the experiment designed to determine hepatocyte internal pH indicated that it was not maintained in the in vitro stability assay, so that hepatocytes acquire the same pH as the incubation buffer. Thus, the novel equations for hepatic clearance (that include an ionization factor) should be applied regardless if the intrinsic clearance was obtained either from microsomal or hepatocyte stability assay.

On the accuracy of a one-compartment approach for determination of drug terminal half-life.

The drug terminal half-life (t(1/2)) is commonly predicted by a simplified one-compartment approach (t(1/2) = ln 2V(ss)/CL), where V(ss) and CL are the steady-state volume of distribution and the total body clearance of drug, respectively. The analysis of the accuracy of this approach is provided. It turns out that most often a simplified one-compartment calculation underestimates t(1/2) by no more than 25% for human, 26% for dog, 20% for monkey, 19% for rat, and 23% for mouse. Thus, the application of a one-compartment calculation of t(1/2) is well justifiable, except for the rare cases of very high drug clearance (CL/(rQ) ≳ 0.5), where r is the equilibrium blood-plasma concentration ratio, and Q is the cardiac output.

Prediction of drug terminal half-life and terminal volume of distribution after intravenous dosing based on drug clearance, steady-state volume of distribution, and physiological parameters of the body.

The steady state, V(ss), terminal volume of distribution, V(ß), and the terminal half-life, t(1/2), are commonly obtained from the drug plasma concentration-time profile, C(p)(t), following intravenous dosing. Unlike V(ss) that can be calculated based on the physicochemical properties of drugs considering the equilibrium partitioning between plasma and organ tissues, t(1/2) and V(ß) cannot be calculated that way because they depend on the rates of drug transfer between blood and tissues. Considering the physiological pharmacokinetic model pertinent to the terminal phase of drug elimination, a novel equation that calculates t(1/2) (and consequently V(ß)) was derived. It turns out that V(ss), the total body clearance, Cl, equilibrium blood-plasma concentration ratio, r; and the physiological parameters of the body such as cardiac output, and blood and tissue volumes are sufficient for determination of terminal kinetics. Calculation of t(1/2) by the obtained equation appears to be in good agreement with the experimentally observed vales of this parameter in pharmacokinetic studies in rat, monkey, dog, and human. The equation for the determination of the pre-exponent of the terminal phase of C(p)(t) is also found. The obtained equation allows to predict t(1/2) in human assuming that V(ss) and Cl were either obtained by allometric scaling or, respectively, calculated in silico or based on in vitro drug stability measurements. For compounds that have high clearance, the derived equation may be applied to calculate r just using the routine data on Cl, V(ss), and t(1/2), rather than doing the in vitro assay to measure this parameter.

An alternative approach for quantitative bioanalysis using diluted blood to profile oral exposure of small molecule anticancer drugs in mice.

A quantitative bioanalytical method for pharmacokinetic studies using diluted whole blood from serially bled mice was developed. Oral exposure profiles in mice for five model anticancer compounds dacarbazine, gefitinib, gemcitabine, imatinib, and topotecan were determined following discrete and cassette (five-in-one) dosing. Six micro blood samples per animal were collected and added to a fixed amount of water. This dilution served several purposes: the red blood cells were lysed; an anticoagulant was unnecessary and the fluid volume of diluted sample was sufficient for bioanalytical assays. AUC values obtained from blood concentrations were within twofold for discrete and cassette dosing except for imatinib (2.1-fold difference) and in agreement with those obtained from plasma concentrations after discrete dosing. All compounds were stable in plasma and diluted blood samples for at least 2 weeks at approximately -80°C. Matrix and intermatrix effects were evaluated to ensure robustness and integrity of the bioanalytical assays. This method provides significant process improvement by enhancing efficiency for sample collection and processing and reducing resources (e.g., reduced compound, cost, and animal requirement) compared with conventional methods. Our study demonstrates the applicability of using diluted micro blood samples for small molecule quantitative bioanalysis to support mouse studies in drug discovery.

Conceptual underestimation of the total body clearance by the sum of clearances of individual organs in physiologically based pharmacokinetics.

It is commonly assumed for linear pharmacokinetics that the total body clearance (CL) is equal to the sum of clearances of individual elimination organs. This is not quite valid because, in general, the concentration of drug in arterial blood entering the elimination organ is not the same as the measured venous blood concentration that is used to calculate CL. Consideration of physiologically based pharmacokinetic model that differentiates between venous and arterial blood shows that CL exceeds the sum of clearances provided by individual organs. Assuming liver as the only elimination organ, it was found that the underestimation of CL by the sum of clearances of individual elimination organs would not exceed 35% for mammals. The underestimation of CL would be more pronounced for high extraction ratio drugs. Thus, for the case when in vivo measured CL considerably exceeds the in vitro predictions (assuming that they provide the organ clearances correctly), a possible reason for discrepancy could be the initial nonlinear phase of drug distribution and excretion. Probably, at this stage a substantial amount of drug is eliminated before distribution into the organs and tissues.

Determination of hepatic clearance with the account of drug-protein binding kinetics.

Binding of drugs to plasma proteins is commonly considered in pharmacokinetics as being in an instantaneous equilibrium. Although if the timescale of dissociation of drug-protein complex becomes comparable to the time that a drug molecule spends in blood while passing through the elimination organ, the kinetics of protein binding may influence the organ clearance. This appears possible for the compounds that have large dissociation energy from protein. Typically, the dissociation of drug-protein complex is fast. However, the longest experimentally observed average bound time of drug to human albumin was as much as 11 min, whereas the time that a drug molecule spends in blood while passing through the liver is around 19 s. The equations for the calculation of hepatic clearance (Cl(h)) with the account of protein binding kinetics are derived for the well-stirred and parallel-tube models. It turns out that for drugs with very low extraction ratio, the influence of protein binding kinetics on Cl(h) is negligible; however, for drugs with high extraction ratio, it may lead to substantially smaller values (possibly by an order of magnitude) of Cl(h) compared with that provided by the common calculations.

On the accuracy of determination of unbound drug fraction in tissue using diluted tissue homogenate.

The unbound drug fraction in tissue, f(ut) , is commonly measured in vitro using the diluted tissue homogenate. An appraisal of the calculation procedure that is routinely applied to obtain f(ut) is presented. An accurate detailed calculation that takes into account the drug protein binding in tissue extracellular water and the pH difference between extra- and intracellular water is considered. It turns out that for neutral compounds, the routine calculation provides f(ut) quite accurately. Though for acidic compounds, the routine calculation can considerably underestimate f(ut) up to 1.8-fold for monoprotic and up to 2.4-fold for diprotic ones, whereas for basic compounds, f(ut) can be substantially overestimated up to 1.9-fold for monoprotic and up to 4.3-fold for diprotic ones.

Consistency of the novel equations for determination of hepatic clearance and drug time course in liver that account for the difference in drug ionization in extracellular and intracellular tissue water.

Intrinsic clearances of seven diverse compounds in rat liver microsomes were measured at intracellular pH 7.0 and extracellular pH 7.4. The obtained values were quite close for each compound. These results confirm the validity of the recently published novel equations for calculation of hepatic clearance and drug time course in liver that account for pH differences in extracellular water and hepatocytes.

Preclinical stereoselective disposition and toxicokinetics of two novel MET inhibitors.

The R- and S-enantiomer of N-(4-(3-(1-ethyl-3,3-difluoropiperidin-4-ylamino)-1H-pyrazolo[3,4-b]pyridin-4-yloxy)-3-fluorophenyl)-2-(4-fluorophenyl)-3-oxo-2,3-dihydropyridazine-4-carboxamide are novel MET kinase inhibitors that have been investigated as potential anticancer agents. The effect of the chirality of these compounds on preclinical in vivo pharmacokinetics and toxicity was studied. The plasma clearance for the S-enantiomer was low in mice and monkeys (23.7 and 7.8 mL min(-1) kg(-1), respectively) and high in rats (79.2 mL min(-1) kg(-1)). The R/S enantiomer clearance ratio was 1.5 except in rats (0.49). After oral single-dose administration at 5 mg kg(-1) the R/S enantiomer ratio of AUC(inf) was 0.95, 1.9 and 0.41 in mice, rats and monkeys, respectively. In an oral single-dose dose-ranging study at 200 and 500 mg kg(-1) and multi-dose toxicity study in mice plasma AUC exposure was approximately 2- to 3-fold higher for the R-enantiomer compared to the S-enantiomer. Greater toxicity of the S-enantiomer was observed which appeared to be due to high plasma C(min) values and tissue concentrations approximately 24 h after the final dose. Both enantiomers showed low to moderate permeability in MDCKI cells with no significant efflux, no preferential distribution into red blood cells and similar plasma protein binding in vitro. Overall, the differences between the enantiomers with respect to low dose pharmacokinetics and in vitro properties were relatively modest. However, toxicity results warrant further development of the R-enantiomer over the S-enantiomer.

The influence of hepatic transport on the distribution volumes and mean residence time of drug in the body and the accuracy of estimating these parameters by the traditional pharmacokinetic calculations.

The influence of hepatic uptake and efflux, which includes passive diffusion and transporter-mediated component, on drug distribution volumes [steady-state volume of distribution (V(ss)) and terminal volume of distribution (V(ß))], mean residence time (MRT), clearance, and terminal half-life is considered using a simplified physiologically based pharmacokinetic model. To account for hepatic uptake, liver is treated as two-compartmental unit with drug transfer from extracellular water into hepatocytes. The exactly calculated distribution volumes and MRT are compared with that obtained by the traditional equations based on the assumption of central elimination. It was found that V(ss) may increase more than 10-fold and V(ß) more than 100-fold due to the contribution of transporter-mediated uptake. The terminal half-life may be substantially shortened (more than 100-fold) due to transporters. It may also decrease significantly due to the increase of intrinsic hepatic clearance (CL(int)), whereas hepatic clearance has already reached saturation (and stays close to the possible maximum value). It is shown that in case of transporter-mediated uptake of compound into hepatocytes, in the absence of efflux and passive diffusion (unidirectional uptake), hepatic clearance is independent of CL(int) and is determined by hepatic blood flow and uptake rate constant. The effects of transporter-mediated uptake are mostly pronounced for hydrophilic acidic compounds and moderately lipophilic neutral compounds. For basic compounds and lipophilic neutral compounds the change of distribution volumes due to transporters is rather unlikely. It was found that the traditional equations provide very accurate values of V(ss), V(ß), and MRT in the absence of transporter action even for very low rates of passive diffusion. On the other hand, the traditional equations fail to provide the correct values of these parameters when the increase of distribution volumes due to transporters takes place, and actually yield the values substantially smaller than the true ones (up to an order of magnitude for V(ss) and MRT, and three orders of magnitude for V(ß)).

A convenient method to measure blood-plasma concentration ratio using routine plasma collection in in vivo pharmacokinetic studies.

A practical time-saving method of determination of equilibrium blood-plasma concentration ratio is described. The method is based on the analysis of compound plasma concentrations in regular blood sample and the blood sample diluted with blank plasma. Since only plasma concentrations are analyzed, the method can be conveniently applied in routine pharmacokinetic studies with minimal additional work for obtaining blood-plasma ratio. The method can also be easily used in in vitro experiment. The results obtained by suggested method are in good agreement with that obtained by common in vitro measurements of blood-plasma ratio.

The corrected traditional equations for calculation of hepatic clearance that account for the difference in drug ionization in extracellular and intracellular tissue water and the corresponding corrected PBPK equation.

The estimation of hepatic clearance, Clh, using in vitro data on metabolic stability of compound, its protein binding and blood­plasma equilibrium concentration ratio is commonly performed using well-stirred, parallel tube or dispersion models. It appears that for ionizable drugs there is a difference of the steady-state concentrations in extracelluar and intracellular water (at hepatocytes), where metabolism takes place. This occurs due to the different pH of extra- and intracellular water (7.4 and 7.0, respectively). The account of this fact leads to the novel equations for Clh . These equations include the additional parameter named ionization factor, FI, which is the ratio of the unionized drug fractions in plasma and intracellular tissue water (or the ratio of the unbound drug concentrations in intracellular tissue water and plasma at equilibrium). For neutral drugs FI = 1 and the novel equations coincide with the traditional ones. It is shown that the account of this factor may yield the calculated Clh up to 6.3-fold greater than that obtained by the traditional equations for the strong diprotic basic compounds, and up to 6.3-fold smaller for the strong diprotic acidic compounds. For triprotic acids and bases the difference could be as much as 15-fold. The account of pH difference between extra- and intracellular water also results in the change of the term commonly used to describe drug metabolic elimination rate in physiologically based pharmacokinetic (PBPK) equation. This consequently may lead to a noticeable change of drug concentration-time profiles in plasma and tissues. The effect of ionization factor is especially pronounced for the low-extraction ratio drugs. The examples of significant improvement in the prediction of hepatic clearance due to the account of ionization factor are provided. A more general equation for hepatic clearance, which accounts for ionization factor and possible drug uptake and efflux, is obtained.

On the accuracy of estimation of basic pharmacokinetic parameters by the traditional noncompartmental equations and the prediction of the steady-state volume of distribution in obese patients based upon data derived from normal subjects.

The steady-state and terminal volumes of distribution, as well as the mean residence time of drug in the body (V(ss), V(ß), and MRT) are the common pharmacokinetic parameters calculated using the drug plasma concentration-time profile C(p) (t) following intravenous (i.v. bolus or constant rate infusion) drug administration. These calculations are valid for the linear pharmacokinetic system with central elimination (i.e., elimination rate being proportional to drug concentration in plasma). Formally, the assumption of central elimination is not normally met because the rate of drug elimination is proportional to the unbound drug concentration at elimination site, although equilibration between systemic circulation and the site of clearance for majority of small molecule drugs is fast. Thus, the assumption of central elimination is practically quite adequate. It appears reasonable to estimate the extent of possible errors in determination of these pharmacokinetic parameters due to the absence of central elimination. The comparison of V(ss), V(ß), and MRT calculated by exact equations and the commonly used ones was made considering a simplified physiologically based pharmacokinetic model. It was found that if the drug plasma concentration profile is detected accurately, determination of drug distribution volumes and MRT using the traditional noncompartmental calculations of these parameters from C(p) (t) yields the values very close to that obtained from exact equations. Though in practice, the accurate measurement of C(p) (t), especially its terminal phase, may not always be possible. This is particularly applicable for obtaining the distribution volumes of lipophilic compounds in obese subjects, when the possibility of late terminal phase at low drug concentration is quite likely, specifically for compounds with high clearance. An accurate determination of V(ss) is much needed in clinical practice because it is critical for the proper selection of drug treatment regimen. For that reason, we developed a convenient method for calculation of V(ss) in obese (or underweight) subjects. It is based on using the V(ss) values obtained from pharmacokinetic studies in normal subjects and the physicochemical properties of drug molecule. A simple criterion that determines either the increase or decrease of V(ss) (per unit body weight) due to obesity is obtained. The accurate determination of adipose tissue-plasma partition coefficient is crucial for the practical application of suggested method.

Preclinical absorption, distribution, metabolism, excretion, and pharmacokinetic-pharmacodynamic modelling of N-(4-(3-((3S,4R)-1-ethyl-3-fluoropiperidine-4-ylamino)-1H-pyrazolo[3,4-b]pyridin-4-yloxy)-3-fluorophenyl)-2-(4-fluorophenyl)-3-oxo-2,3-dihydropyridazine-4-carboxamide, a novel MET kinase inhibitor.

GNE-A (AR00451896; N-(4-(3-((3S,4R)-1-ethyl-3-fluoropiperidine-4-ylamino)-1H-pyrazolo[3,4-b]pyridin-4-yloxy)-3-fluorophenyl)-2-(4-fluorophenyl)-3-oxo-2,3-dihydropyridazine-4-carboxamide) is a potent, selective MET kinase inhibitor being developed as a potential drug for the treatment of human cancers. Plasma clearance was low in mice and dogs (15.8 and 2.44 mL/min/kg, respectively) and moderate in rats and monkeys (36.6 and 13.9 mL/min/kg, respectively). The volume of distribution ranged from 2.1 to 9.0 L/kg. The mean terminal elimination half-life ranged from 1.67 h in rats to 16.3 h in dogs. Oral bioavailability in rats, mice, monkeys, and dogs were 11.2%, 88.0%, 72.4%, and 55.8%, respectively. Allometric scaling predicted a clearance of 1.3-7.4 mL/min/kg and a volume of distribution of 4.8-11 L/kg in human. Plasma protein binding was high (96.7-99.0% bound). Blood-to-plasma concentration ratios (0.78-1.46) indicated that GNE-A did not preferentially distribute into red blood cells. Transporter studies in MDCKI-MDR1 and MDCKII-Bcrp1 cells suggested that GNE-A is likely a substrate for MDR1 and BCRP. Pharmacokinetic-pharmacodynamic modelling of tumour growth inhibition in MET-amplified EBC-1 human non-small cell lung carcinoma tumour xenograft mice projected oral doses of 5.6 and 13 mg/kg/day for 50% and 90% tumour growth inhibition, respectively. Overall, GNE-A exhibited favourable preclinical properties and projected human dose estimates.

On the possibility of self-induction of drug protein binding.

The equilibrium unbound drug fraction (f(u)) is an important pharmacokinetic parameter, which influences drug elimination and distribution in the body. Commonly the drug plasma concentration is substantially less then that of drug binding proteins, so that f(u) can be assumed constant independent of drug concentration. A general consideration of protein binding based on the mass-action law provides that the unbound drug fraction increases with the increase of drug concentration, which is also a usual experimental observation. For several drugs, though, a seemingly unusual sharp decrease of the unbound drug fraction with the increase of total drug concentration (R(o)) in the interval 0 < R(o) less, similar 5 microM was experimentally observed. A possible explanation of this apparently strange phenomenon is presented. The explanation is based on the consideration of a two-step mechanism of drug protein binding. The first step occurs as a drug binding to the site with relatively low affinity. Consequently this binding leads to the activation of a high affinity site, which otherwise is not available for binding. The suggested binding scheme yields the curves for f(u) dependence on the total drug concentration that are in good agreement with experimental measurements. The interpretation of pharmacokinetic data for the drugs with such unusual concentration dependence of f(u) appears to be a formidable problem.