The serine/threonine kinase IL-1R-associated kinase (IRAK)4 is a critical regulator of innate immunity. We have identified BMS-986126, a potent, highly selective inhibitor of IRAK4 kinase activity ...that demonstrates equipotent activity against multiple MyD88-dependent responses both in vitro and in vivo. BMS-986126 failed to inhibit assays downstream of MyD88-independent receptors, including the TNF receptor and TLR3. Very little activity was seen downstream of TLR4, which can also activate an MyD88-independent pathway. In mice, the compound inhibited cytokine production induced by injection of several different TLR agonists, including those for TLR2, TLR7, and TLR9. The compound also significantly suppressed skin inflammation induced by topical administration of the TLR7 agonist imiquimod. BMS-986126 demonstrated robust activity in the MRL/lpr and NZB/NZW models of lupus, inhibiting multiple pathogenic responses. In the MRL/lpr model, robust activity was observed with the combination of suboptimal doses of BMS-986126 and prednisolone, suggesting the potential for steroid sparing activity. BMS-986126 also demonstrated synergy with prednisolone in assays of TLR7- and TLR9-induced IFN target gene expression using human PBMCs. Lastly, BMS-986126 inhibited TLR7- and TLR9-dependent responses using cells derived from lupus patients, suggesting that inhibition of IRAK4 has the potential for therapeutic benefit in treating lupus.
In a preclinical setting, plasma or whole tissue drug concentrations are often correlated with pharmacodynamics, although according to the free drug hypothesis, unbound drug concentration should be ...more pharmacologically relevant. Alternatively, blood concentrations may be a good surrogate for tissue concentration for passively permeable compounds. However, for a large number of compounds that are substrates for uptake and/or efflux transporters expressed at the tissue level, significant discrepancies are expected between unbound concentrations in blood and those in tissues. Consequently, attempts have been made to measure tissue unbound drug concentrations using tissue homogenates, slices and microdialysis. Mathematical expressions for calculating the rate and extent of drug distribution into tissues have also been established. For example, a ratio of unbound concentration in the tissue to that in plasma (K(p,uu)) is the best indicator of the extent of tissue distribution. Despite these technical advances, however, very few examples demonstrate a focus on tissue unbound drug concentrations in a preclinical setting. This review will illustrate various techniques to estimate tissue unbound drug concentrations, relevant parameters to calculate the rate and extent of tissue distribution and different factors affecting tissue unbound concentration. The review will also highlight various examples from the literature where tissue unbound drug concentrations have demonstrated a superior correlation with efficacy. The impact of tissue unbound drug concentrations on the projection of human efficacious dose is also discussed.
Expression and functional changes in the organic anion transporting polypeptide (OATP)-multidrug resistance-associated protein (MRP) axis of transporters are well reported in patients with ...nonalcoholic steatohepatitis (NASH). These changes can impact plasma and tissue disposition of endo- and exogenous compounds. The transporter alterations are often assessed by administration of a xenobiotic or by transporter proteomic analysis from liver biopsies. Using gene expression, proteomics, and endogenous biomarkers, we show that the gene expression and activity of OATP and MRP transporters are associated with disease progression and recovery in humans and in preclinical animal models of NASH. Decreased OATP and increased MRP3/4 gene expression in two cohorts of patients with steatosis and NASH, as well as gene and protein expression in multiple NASH rodent models, have been established. Coproporphyrin I and III (CP I and III) were established as substrates of MRP4. CP I plasma concentration increased significantly in four animal models of NASH, indicating the transporter changes. Up to a 60-fold increase in CP I plasma concentration was observed in the mouse bile duct-ligated model compared with sham controls. In the choline-deficient amino acid-defined high-fat diet (CDAHFD) model, CP I plasma concentrations increased by >3-fold compared with chow diet-fed mice. In contrast, CP III plasma concentrations remain unaltered in the CDAHFD model, although they increased in the other three NASH models. These results suggest that tracking CP I plasma concentrations can provide transporter modulation information at a functional level in NASH animal models and in patients. SIGNIFICANCE STATEMENT: Our analysis demonstrates that multidrug resistance-associated protein 4 (MRP4) transporter gene expression tracks with nonalcoholic steatohepatitis (NASH) progression and intervention in patients. Additionally, we show that coproporphyrin I and III (CP I and III) are substrates of MRP4. CP I plasma and liver concentrations increase in different diet- and surgery-induced rodent NASH models, likely explained by both gene- and protein-level changes in transporters. CP I and III are therefore potential plasma-based biomarkers that can track NASH progression in preclinical models and in humans.
Inside-out-oriented membrane vesicles are useful tools to investigate whether a compound can be an inhibitor of efflux transporters such as multidrug resistance-associated protein 2 (MRP2). However, ...because of technical limitations of substrate diffusion and low dynamic uptake windows for interacting drugs used in the clinic, estradiol-17
-glucuronide (E17
G) remains the probe substrate that is frequently used in MRP2 inhibition assays. Here we recapitulated the sigmoidal kinetics of MRP2-mediated transport of E17
G, with apparent Michaelis-Menten constant (
) and
values of 170 ±17
M and 1447 ± 137 pmol/mg protein/min, respectively. The Hill coefficient (2.05 ± 0.1) suggests multiple substrate binding sites for E17
G transport with cooperative interactions. Using E17
G as a probe substrate, 51 of 97 compounds tested (53%) showed up to 6-fold stimulatory effects. Here, we demonstrate for the first time that coproporphyrin-I (CP-I) is a MRP2 substrate in membrane vesicles. The uptake of CP-I followed a hyperbolic relationship, adequately described by the standard Michaelis-Menten equation (apparent
and
values were 7.7 ± 0.7
M and 48 ± 11 pmol/mg protein/min, respectively), suggesting the involvement of a single binding site. Of the 47 compounds tested, 30 compounds were inhibitors of human MRP2 and 8 compounds (17%) stimulated MRP2-mediated CP-I transport. The stimulators were found to share the basic backbone structure of the physiologic steroids, which suggests a potential in vivo relevance of in vitro stimulation of MRP2 transport. We concluded that CP-I could be an alternative in vitro probe substrate replacing E17
G for appreciating MRP2 interactions while minimizing potential false-negative results for MRP2 inhibition due to stimulatory effects.
As an expansion investigation of drug-drug interaction (DDI) from previous clinical trials, additional plasma endogenous metabolites were quantitated in the same subjects to further identify the ...potential biomarkers of organic anion transporter (OAT) 1/3 inhibition. In the single dose, open label, three-phase with fixed order of treatments study, 14 healthy human volunteers orally received 1000 mg probenecid alone, or 40 mg furosemide alone, or 40 mg furosemide at 1 hour after receiving 1000 mg probenecid on days 1, 8, and 15, respectively. Endogenous metabolites including kynurenic acid, xanthurenic acid, indo-3-acetic acid, pantothenic acid,
-cresol sulfate, and bile acids in the plasma were measured by liquid chromatography-tandem mass spectrometry. The C
of kynurenic acids was significantly increased about 3.3- and 3.7-fold over the baseline values at predose followed by the treatment of probenecid alone or in combination with furosemide respectively. In comparison with the furosemide-alone group, the C
and area under the plasma concentration-time curve (AUC) up to 12 hours of kynurenic acid were significantly increased about 2.4- and 2.5-fold by probenecid alone, and 2.7- and 2.9-fold by probenecid plus furosemide, respectively. The increases in C
and AUC of plasma kynurenic acid by probenecid are comparable to the increases of furosemide C
and AUC reported previously. Additionally, the plasma concentrations of xanthurenic acid, indo-3-acetic acid, pantothenic acid, and
-cresol sulfate, but not bile acids, were also significantly elevated by probenecid treatments. The magnitude of effect size analysis for known potential endogenous biomarkers demonstrated that kynurenic acid in the plasma offers promise as a superior addition for early DDI assessment involving OAT1/3 inhibition. SIGNIFICANCE STATEMENT: This article reports that probenecid, an organic anion transporter (OAT) 1 and OAT3 inhibitor, significantly increased the plasma concentrations of kynurenic acid and several uremic acids in human subjects. Of those, the increases of plasma kynurenic acid exposure are comparable to the increases of furosemide by OAT1/3 inhibition. Effect size analysis for known potential endogenous biomarkers revealed that plasma kynurenic acid is a superior addition for early drug-drug interaction assessment involving OAT1/3 inhibition.
This commentary presents contributions and accomplishments of Professor Saranjit Singh, National Institute of Pharmaceutical Education and Research (NIPER), SAS Nagar, India, to pharmaceutical ...research and education. Prof. Singh completed his successful tenure in October 2021. Over his 40+ years of illustrious academic career, he trained 147 Masters and 15 PhD students in the fields of drug stability testing, degradation chemistry, impurity and metabolite characterization, and advanced analytical technologies. He has published ∼250 research articles, reviews, editorials, patent, book, and book chapters, and received numerous awards, including the Professor M.L. Khorana Memorial Lecture Award from the Indian Pharmaceutical Association (IPA) and the Outstanding Analyst and Eminent Analyst awards from the Indian Drug Manufacturers' Association (IDMA). This commentary highlights Prof. Singh's inspiring personal and renowned professional journey, including early life, education, career, accomplishments, as well as his services to academia, industry, and regulatory. By sharing the contributions and accomplishments of Prof. Singh, we strongly believe that his story will inspire the next generation of scientists to continue his legacy to advance the field.
The unbound concentration in plasma drives the transport of the drug into the brain, and the unbound drug concentration in the central nervous system (CNS) drives the interaction with the target ...eliciting the pharmacological effect. Delivery of the drug to the CNS is a challenge because of the unique neurovascular unit, which restricts the passage of drugs into the brain. The efflux transporters especially P-glycoprotein (P-gp) present at the blood-brain barrier (BBB) act as one of the major detractors for keeping drugs outside the CNS. The cerebrospinal fluid (CSF) drug concentration has been used as a surrogate for unbound brain concentrations and has proven to be a good indicator to relate to CNS activity. Herein, we have established a serial CSF sampling technique in rats, which allowed CSF sampling from a single animal and reduced the number of animals required, as well as the interanimal variance associated with a composite/terminal study design. Concentrations in the CSF sampled from the cisterna magna serially from the same rat were compared with the concentrations obtained from discrete CSF sampling and with brain concentrations. The serial CSF sampling technique was also authenticated by ensuring no change in the barrier without any indication of damage caused by the repeated puncture of cisterna magna. This technique was corroborated using three passively permeable compounds (carbamazepine, theophylline, and propranolol), three P-gp substrates (quinidine, verapamil, and digoxin), and one l-amino acid uptake transporter substrate (gabapentin). The P-gp substrates were also used in separate studies with the P-gp inhibitor elacridar to assess the effect on CSF concentration versus brain concentration on P-gp inhibition. The CSF concentration and unbound brain concentration were comparable (within 3-fold) for all compounds, including P-gp substrates even in the presence of elacridar. Therefore, this technique can prove to be beneficial for predicting the unbound drug concentrations in the brain from the CSF concentrations and reduce the cost incurred in preclinical animal models. Chemical inhibition by elacridar and prediction of the brain unbound concentrations from the serial CSF sampling of P-gp substrates in the rat may be an attractive alternative to the use of genetically knocked out rodents.
Previously we reported that coproporphyrin-I (CP-I) is an optimal probe substrate for multidrug resistance-associated protein 2 (MRP2), and stimulation of MRP2-mediated transport is probe ...substrate-dependent. In the present investigation, we assessed if the in vitro stimulation is physiologically relevant. Similar to human MRP2 transport, CP-I was transported by rat Mrp2 in a typical Michaelis-Menten kinetics with apparent K
and V
values of 15 ± 6 µM and 161 ± 20 pmol/min/mg protein, respectively. In vivo Mrp2 functions were monitored by biliary and renal secretion of CP-I and its isomer CP-III, in bile-duct cannulated rats before and after treatment with mitoxantrone, progesterone, and verapamil. These compounds stimulated Mrp2-mediated CP-I transport in vitro. No significant increase in biliary or renal clearances, as well as in the cumulative amount of CP-I or CP-III eliminated in bile, were detected following treatment with the in vitro stimulators, indicating an in vitro to in vivo disconnect. In presence of 10 µM bilirubin, the in vitro stimulation was suppressed. We concluded that the in vitro stimulation of CP-I transport mediated by Mrp2 is not translatable in vivo, and proposed that the presence of endogenous compounds such as bilirubin in the liver may contribute to the in vitro to in vivo disconnect.
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