Discovery of an Oral, Beyond-Rule-of-Five Mcl-1 Protein-Protein Interaction Modulator with the Potential of Treating Hematological Malignancies.

Avoidance of apoptosis is critical for the development and sustained growth of tumors. The pro-survival protein myeloid cell leukemia 1 (Mcl-1) is an anti-apoptotic member of the Bcl-2 family of proteins which is overexpressed in many cancers. Upregulation of Mcl-1 in human cancers is associated with high tumor grade, poor survival, and resistance to chemotherapy. Therefore, pharmacological inhibition of Mcl-1 is regarded as an attractive approach to treating relapsed or refractory malignancies. Herein, we disclose the design, synthesis, optimization, and early preclinical evaluation of a potent and selective small-molecule inhibitor of Mcl-1. Our exploratory design tactics focused on structural modifications which improve the potency and physicochemical properties of the inhibitor while minimizing the risk of functional cardiotoxicity. Despite being in the "non-Lipinski" beyond-Rule-of-Five property space, the developed compound benefits from exquisite oral bioavailability in vivo and induces potent pharmacodynamic inhibition of Mcl-1 in a mouse xenograft model.

Ontogeny and Cross-species Comparison of Pathways Involved in Drug Absorption, Distribution, Metabolism, and Excretion in Neonates (Review): Kidney.

The kidneys play an important role in many processes, including urine formation, water conservation, acid-base equilibrium, and elimination of waste. The anatomic and functional development of the kidney has different maturation time points in humans versus animals, with critical differences between species in maturation before and after birth. Absorption, distribution, metabolism, and excretion (ADME) of drugs vary depending on age and maturation, which will lead to differences in toxicity and efficacy. When neonate/juvenile laboratory animal studies are designed, a thorough knowledge of the differences in kidney development between newborns/children and laboratory animals is essential. The human and laboratory animal data must be combined to obtain a more complete picture of the development in the kidneys around the neonatal period and the complexity of ADME in newborns and children. This review examines the ontogeny and cross-species differences in ADME processes in the developing kidney in preterm and term laboratory animals and children. It provides an overview of insights into ADME functionality in the kidney by identifying what is currently known and which gaps still exist. Currently important renal function properties such as glomerular filtration rate, renal blood flow, and ability to concentrate are generally well known, while detailed knowledge about transporter and metabolism maturation is growing but is still lacking. Preclinical data in those properties is limited to rodents and generally covers only the expression levels of transporter or enzyme-encoding genes. More knowledge on a functional level is needed to predict the kinetics and toxicity in neonate/juvenile toxicity and efficacy studies. SIGNIFICANCE STATEMENT: This review provides insight in cross-species developmental differences of absorption, distribution, metabolism, and excretion properties in the kidney, which should be considered in neonate/juvenile study interpretation, hypotheses generation, and experimental design.

Translational safety biomarkers of colonic barrier integrity in the rat.

The intestinal barrier controls intestinal permeability, and its disruption has been associated with multiple diseases. Therefore, preclinical safety biomarkers monitoring barrier integrity are essential during the development of drugs targeting the intestines, particularly if starting treatment early after onset of disease. Classical toxicology endpoints are not sensitive enough and therefore our objective was to identify non-invasive markers enabling early in vivo detection of colonic barrier perturbation. Male Sprague-Dawley rats were dosed intracolonically via the rectum, using sodium caprate or ibuprofen as tool compounds to alter barrier integrity. Several potentially translational biomarkers and probe molecules related to permeability, inflammation or tissue damage were evaluated, using various analytical platforms, including immunoassays, targeted metabolomics and highly sensitive ultra-performance liquid chromatography-tandem mass spectrometry. Several markers were identified that allow early in vivo detection of colonic barrier integrity changes, before histopathological evidence of tissue damage. The most promising permeability markers identified were plasma fluorescein isothiocyanate-dextran 4000 and a lactulose/mannitol/sucralose mixture in urine. These markers showed maximum increases over 100-fold or approximately 10-50-fold, respectively. Intracolonic administration of the above probe molecules outperformed oral administration and inflammatory or other biomarkers, such as α2 -macroglobulin, calprotectin, cytokines, prostaglandins and a panel of metabolic molecules to identify early and subtle changes in barrier integrity. However, optimal timing of probe administration and sample collection is important for all markers evaluated. Inclusion of these probe molecules in preclinical toxicity studies might aid in risk assessment and the design of a clinical biomarker plan, as several of these markers have translational potential.

Early postnatal gentamicin and ceftazidime treatment in normal and food restricted neonatal wistar rats: Implications for kidney development.

BACKGROUND: Up to two-thirds of premature born neonates are treated for infections with aminoglycosides such as gentamicin. Although acute toxicities are well described, there is uncertainty on developmental changes after treatment of premature born neonates. We studied the effect of gentamicin and ceftazidime on kidney development in the rat. Additionally, we evaluated the modulating effect of extrauterine growth restriction. METHODS: On postnatal day (PND) 2, Wistar rats were cross-fostered into normal sized litters (12 pups) or large litters (20 pups) to create normal food (NF) or food restricted (FR) litters to simulate growth restriction and dosed daily intraperitoneally with placebo, 4 mg/kg of gentamicin or 50 mg/kg ceftazidime until PND 8. Gentamicin pharmacokinetics were studied in a separate group of animals. Kidneys were weighed. Renal expression of 18 developmental genes was evaluated by quantitative PCR on PND 8. On PND 35, glomerular number was assessed by stereology and glomerular generations were counted. RESULTS: Food restricted litters showed 22% less body weight compared with controls by day 35 (p < 0.001), 1.4- to 1.5-fold down regulation of Renin, Oat1, and Agtr1a (p < 0.05) expression and a 12% reduction in glomerular numbers (mean 30841 vs. 35187, p < 0.001), whereas glomerular generation count was unaffected. Gentamicin pharmacokinetic parameters were found to be in a human clinical range (mean maximum concentration in plasma of 4.88 mg/L and mean area under the plasma-concentration time curve up to the last measured concentration after 4 hr of 10.71 mg.h/L for sexes combined) and all endpoints were unaffected. Ceftazidime reduced Renin expression by 1.7-fold (p < 0.01). CONCLUSION: Our experiments showed that gentamicin at clinical levels did not disturb kidney development, ceftazidime can affect Renin expression, and extrauterine growth restriction impairs kidney development, but did not modulate potential drug toxicity. Birth Defects Research 109:1228-1235, 2017. © 2017 Wiley Periodicals, Inc.

The Effects of Early Postnatal Diuretics Treatment on Kidney Development and Long-Term Kidney Function in Wistar Rats.

BACKGROUND: Diuretics are administered to neonates to control fluid balance. We studied whether clinical doses affected kidney development and function and whether extrauterine growth retardation (EUGR) could be a modulator. METHODS: Wistar rats were cross-fostered in normal food or food restricted litters at postnatal day (PND) 2 and treated daily with 0.9% NaCl, 5 mg/kg furosemide or 5 mg/kg hydrochlorothiazide (HCTZ) up to PND 8. Kidneys were evaluated on proliferation, apoptosis and a set of mRNA target genes at PND 8, glomerular- and glomerular generation count at PND 35, clinical pathology parameters at 3- and 9 months, neutrophil gelatinase-associated lipocalin at PND 8, 3 and 6 months, monthly blood pressure from 3 months onward and histopathology at study end. RESULTS: Treatment with furosemide or HCTZ did not have relevant effects on measured parameters. EUGR resulted in lower body weight from day 3 onwards (-29% at weaning; p < 0.001, -10% at necropsy; p < 0.001), less glomerular generations (4.4 ± 0.32 vs. 5.0 ± 0.423; p = 0.025, males only), decreased glomerular numbers (27,861 ± 3,468 vs. 30,527 ± 4,096; p = 0.026), higher creatinine clearance (0.84 ± 0.1 vs. 0.77 ± 0.09 ml/min/kg; p = 0.047) at 3 months and lower plasma creatinine (25.7 ± 1.8 vs. 27.5 ± 2.8 µmol/l; p = 0.043) at 9 months. CONCLUSION: Furosemide and HCTZ did not influence kidney development or function when administered in a clinically relevant dose to rat pups at a stage of ongoing nephrogenesis. EUGR led to impaired kidney development but did not modify furosemide or HCTZ findings.

Impact of Early Postnatal NSAID Treatment on Nephrogenesis in Wistar Rats.

BACKGROUND: Prematurely born children with patent ductus arteriosus are treated with ibuprofen or indomethacin, which may inhibit kidney development. We determined whether clinical doses affected kidney development and function, with or without extrauterine growth retardation. METHODS: Wistar rats were cross-fostered in normal food (NF) or food restricted (FR) litters at postnatal day (PND) 2. On PND 3 to 4, three doses of 0.9% NaCl, 0.1 mg/kg indomethacin, or 10 mg/kg ibuprofen were administered via intraperitoneal injection with 12-hr intervals. Kidneys were evaluated for apoptosis, proliferation, and gene expression at PND 8; stereological assessment of nephron number at PND 35; and clinical pathology and neutrophil gelatinase-associated lipocalin at 4 and 9 months. Blood pressure was measured at the ages of 4, 6, and 9 months. RESULTS: NF and FR bodyweight differed from PND 3 onwards, ranging from 16.5 g at weaning (p < 0.001) to 39 g at necropsy (p = 0.019). Kidney proliferation/apoptosis ratios were 7:1 and 3:1 (p = 0.001), respectively and different expression of Wnt4 (0.7x), Oat1 (1.3x), Nphs1 (1.7x), and Aqp4 (1.3x) was noted (but its biological relevance doubted). Nephron numbers were decreased by 12% (p = 0.109) in the ibuprofen-NF group and 7.5% (p = 0.237) in FR groups. This coincided with a tendency to increased neutrophil gelatinase-associated lipocalin at 9 months. No differences were noted in electrolytes, creatinine, or urea clearance. No valid blood pressure results could be obtained. CONCLUSION: A clinical Ibuprofen dose showed potential to inhibit kidney development in neonatal rats. FR did not modulate these effects.

Antibiotics and renal branching morphogenesis: comparison of toxicities.

BACKGROUND: Many premature born neonates receive antibiotic drugs to treat infections, which are applied during active nephrogenesis. We studied the impact of clinical concentrations of gentamicin and alternatives, ceftazidime and meropenem, on ureteric branching. METHODS: Mice metanephroi were dissected at embryonic day 13 and cultured in media with or without various concentrations of gentamicin, ceftazidime, or meropenem. Zero and 24 h kidney size were assessed by surface area measurements, and the ureteric tree was visualized by whole mount staining and confocal microscopy. Branching was evaluated by counting and gene expression levels of Wt1, Sox9, Bmp7, Fgf8, and Gdnf were investigated. RESULTS: A concentration of 2,000 µmol/l ceftazidime impaired ureteric development. In addition, a 4.5-fold and a 2.5-fold downregulation was noted in Fgf8 and Gdnf, respectively. No adverse effects were noted after gentamicin or meropenem treatment. No relationship was noted between surface area expansion and ureteric bud formation, but surface area at explantation related to bud count after 24 h of culture. CONCLUSION: Ceftazidime, but not gentamicin or meropenem reduced ureteric branching in mice and suggest a role for Fgf8 and Gdnf in its mechanism. Metanephros surface area measurements can be used to reduce intra- and inter-litter variation.

The interplay between drugs and the kidney in premature neonates.

The kidney plays a central role in the clearance of drugs. However, renal drug handling entails more than glomerular filtration and includes tubular excretion and reabsorption, and intracellular metabolization by cellular enzyme systems, such as the Cytochrome P450 isoenzymes. All these processes show maturation from birth onwards, which is one of the reasons why drug dosing in children is not simply similar to dosing in small adults. As kidney development normally finishes around the 36th week of gestation, being born prematurely will result in even more immature renal drug handling. Environmental effects, such as extra-uterine growth restriction, sepsis, asphyxia, or drug treatments like caffeine, aminoglycosides, or non-steroidal anti-inflammatory drugs, may further hamper drug handling in the kidney. Dosing in preterm neonates is therefore dependent on many factors that need to be taken into account. Drug treatment may significantly hamper postnatal kidney development in preterm neonates, just like renal immaturity has an impact on drug handling. The restricted kidney development results in a lower number of nephrons that may have several long-term sequelae, such as hypertension, albuminuria, and renal failure. This review focuses on the interplay between drugs and the kidney in premature neonates.

Adult renal size is not a suitable marker for nephron numbers: an individual patient data meta-analysis.

BACKGROUND: Renal size is often used as a marker for nephron numbers as estimation of glomerular numbers is not yet possible in vivo. However, the validity of an association between the two is questionable. As a proper marker for nephron number in an individual is needed in clinical practice, this study was designed to assess the association between renal size and nephron numbers. METHODS: An individual patient data meta-analysis was performed on data retrieved with a PubMed and Embase search. Only studies were included that described individual human data on kidney size and nephron numbers determined by stereology, the gold standard methodology to estimate nephron numbers. As renal size increases until the end of puberty, and nephron numbers decline after the age of 60 years, only data from individuals aged 18-60 years without renal disease were included. RESULTS: Six papers were identified that provided data on renal weight and nephron numbers from 114 individuals. Backward linear regression identified kidney weight and race as the only 2 significant factors explaining nephron numbers (R square 0.085, p=0.007). Controlling for race, there was a significant correlation between nephron number and kidney weight (r=0.231, r square=0.053, p=0.01). CONCLUSION: These data indicate that only ∼5% of the variation in nephron numbers is explained by differences in renal size. Renal size in adulthood should not be used as a marker for nephron numbers in an individual.

Effect of NSAIDs and diuretics on nephrogenesis in an ex vivo embryogenic kidney model.

The kidney is one of the key organs in clearing foreign compounds. The effects of drugs on the developing kidney are relatively unknown. We studied the direct effect of furosemide, hydrochlorothiazide, ibuprofen, and indomethacin on kidney development in an ex vivo embryonic kidney model. At embryonic day 13, metanephroi were dissected from mice and cultured in control media or media supplemented with various clinically relevant concentrations of drugs. The ureteric tree was visualized by whole-mount staining and branching was evaluated by counting. Additionally, gene expression levels of Wt1, Sox9, Bmp7, Fgf8, and Gdnf were investigated. No distinct differences were noted on either ureteric tip development or gene expression analysis for each drug after 24 hr of exposure. Even though short-term exposure to clinically relevant concentrations seems not to disturb renal development, future research is needed to study prolonged or repeated exposures.

Effect of drugs on renal development.

Many nephrotoxic effects of drugs have been described, whereas the effect on renal development has received less attention. Nephrogenesis ceases at approximately 36 weeks of gestation, indicating that drugs administered to pregnant women and to preterm-born neonates may influence kidney development. Such an effect on renal development may lead to a wide spectrum of renal malformations (congenital anomalies of the kidney and urinary tract [CAKUT]), ranging from renal agenesis to a reduced nephron number. Any of these anomalies may have long-term sequelae, and CAKUT is the primary cause for renal replacement therapy in childhood. This review focuses on research into the effect of drug treatment during active nephrogenesis during pregnancy and in preterm-born infants. Because the effects of many widely used drugs have not been unraveled thus far, more research is needed to study the effect on renal development and long-term renal sequelae after drug treatment during nephrogenesis.