David Brott

Characterization of renal biomarkers for use in clinical trials: biomarker evaluation in healthy volunteers.

Background Several preclinical urinary biomarkers have been qualified and accepted by the health authorities (US Food and Drug Administration, European Medicines Agency, and Pharmaceuticals and Medical Devices Agency) for detecting drug-induced kidney injury during preclinical toxicologic testing. Validated human assays for many of these biomarkers have become commercially available, and this study was designed to characterize some of the novel clinical renal biomarkers. The objective of this study was to evaluate clinical renal biomarkers in a typical Phase I healthy volunteer population to determine confidence intervals (pilot reference intervals), intersubject and intrasubject variability, effects of food intake, effect of sex, and vendor assay comparisons. Methods Spot urine samples from 20 male and 19 female healthy volunteers collected on multiple days were analyzed using single analyte and multiplex assays. The following analytes were measured: α-1-microglobulin, β-2-microglobulin, calbindin, clusterin, connective tissue growth factor, creatinine, cystatin C, glutathione S-transferase-α, kidney injury marker-1, microalbumin, N-acetyl-β-(D) glucosaminidase, neutrophil gelatinase-associated lipocalin, osteopontin, Tamm-Horsfall urinary glycoprotein, tissue inhibitor of metalloproteinase 1, trefoil factor 3, and vascular endothelial growth factor. Results Confidence intervals were determined from the single analyte and multiplex assays. Intersubject and intrasubject variability ranged from 38% to 299% and from 29% to 82% for biomarker concentration, and from 24% to 331% and from 10% to 67% for biomarker concentration normalized to creatinine, respectively. There was no major effect of food intake or sex. Single analyte and multiplex assays correlated with r2≥0.700 for five of six biomarkers when evaluating biomarker concentration, but for only two biomarkers when evaluating concentration normalized to creatinine. Conclusion Confidence intervals as well as intersubject and intrasubject variability were determined for novel clinical renal biomarkers/assays, which should be considered for evaluation in the next steps of the qualification process.

Combining radio telemetry and automated blood sampling: a novel approach for integrative pharmacology and toxicology studies.

INTRODUCTION A novel automated blood sampling and telemetry (ABST) system was developed to integrate pharmacokinetic (PK), pharmacodynamic (PD) and toxicology studies. The goals of this investigation were to determine: 1) optimal feeding conditions and minimal acclimation times for recording PD parameters (blood pressure, heart rate, and temperature) after animals arrived on-site; 2) stress hormone levels in ABST-housed rats; 3) the feasibility of simultaneously recording cardiovascular parameters with electroencephalogram (EEG); 4) the equivalence of renal endpoints from ABST-housed rats with those in the metabolic cage, and 5) the cardiovascular responses to baclofen. METHODS Body weight, blood pressure, temperature, stress biomarkers, urine chemistries, renal biomarkers and responses to vehicle or baclofen (10mg/kg) were compared in awake and freely moving rats housed in the ABST system, home cage (HC) or metabolic cage. RESULTS Fasted rats lost 5+/-1% and 7+/-1% body weight when housed in ABST and metabolic cages, respectively. Weight loss was reversed by supplementing regular diet with hydration and nutritional supplements. Based on PD parameters, the minimum acclimation time required for both ABST and HC rats was 3days. The feasibility of simultaneously measuring multiple parameters, such as EEG with cardiovascular parameters in ABST was demonstrated. Renal function and biomarkers in rats continuously housed in the ABST and metabolic cages were equivalent (p>0.05) on days 1, 3, and 7. Baclofen-induced quantitatively and qualitatively similar (p>0.05) PK, mean arterial pressure, heart rate and temperature in ABST- and HC-housed rats. DISCUSSION These studies demonstrate for the first time that drug-induced PD responses can be recorded simultaneously with time-matched pharmacokinetic, biochemical and metabolic parameters in the same animal. The ABST system has the added advantage of blood sampling without the need for satellite PK animals. ABST is a useful and novel tool for establishing efficacy and safety margins using an in vivo integrative pharmacology approach.

Evidence for the Nitric Oxide Pathway as a Potential Mode of Action in Fenoldopam-induced Vascular Injury

Fenoldopam, a dopaminergic DA1 agonist, induces vasodilatation via nitric oxide (NO), and this may be associated with mesenteric arterial injury. NO is produced from the enzymatic action of nitric oxide synthase (NOS), which is regulated by the shear-stress mediating protein caveolin-1. Profound vasodilatation and accompanied decreased shear are early events that could initiate vascular injury. Therefore, it is of interest to determine the role of caveolin-1 and the NO pathway in fenoldopam-induced vascular injury. At sites of fenoldopam-induced mesenteric arterial injury, decreased caveolin-1 expression and apoptosis were prominent immunohistochemical findings. An additional finding at these sites of injury were loss and/or reduced expression of caveolin-1 regulated structural proteins, connexin-43, (gap junction) ZO-1, and claudin (tight junctions). Because functional loss of caveolin-1 is associated with increased NOS activity and vasodilatation via NO, studies were conducted to show a NO donor produced vascular lesions in the mesenteric arteries morphologically similar to those induced by fenoldopam. Moreover, the incidence and severity of fenoldopam-induced vascular injury were reduced when an NOS inhibitor or a scavenger of NO-generated free radicals were coadministered with fenoldopam. Collectively, these data suggest that caveolin-1 and its regulated NO pathway may play an important role in vasodilatory drug-induced vascular injury.

Characterization of renal biomarkers for use in clinical trials: effect of preanalytical processing and qualification using samples from subjects with diabetes

Background Identifying the potential for drug-induced kidney injury is essential for the successful research and development of new drugs. Newer and more sensitive preclinical drug-induced kidney injury biomarkers are now qualified for use in rat toxicology studies, but biomarkers for clinical studies are still undergoing qualification. The current studies investigated biomarkers in healthy volunteer (HV) urine samples with and without the addition of stabilizer as well as in urine from patients with normoalbuminuric diabetes mellitus (P-DM). Methods Urine samples from 20 male HV with stabilizer, 69 male HV without stabilizer, and 95 male DM without stabilizer (39 type 1 and 56 type 2) were analyzed for the following bio-markers using multiplex assays: α-1-microglobulin (A1M), β-2-microglobulin, calbindin, clus-terin, connective tissue growth factor (CTGF), creatinine, cystatin-C, glutathione S-transferase α (GSTα), kidney injury marker-1 (KIM-1), microalbumin, neutrophil gelatinase-associated lipocalin, osteopontin, Tamm–Horsfall urinary glycoprotein (THP), tissue inhibitor of metalloproteinase 1, trefoil factor 3 (TFF3), and vascular endothelial growth factor. Results CTGF and GSTα assays on nonstabilized urine were deemed nonoptimal (>50% of values below assay lower limits of quantification). “Expected values” were determined for HV with stabilizer, HV without stabilizer, and P-DM without stabilizer. There was a statistically significant difference between HV with stabilizer compared to HV without stabilizer for A1M, CTGF, GSTα, and THP. DM urine samples differed from HV (without stabilizer) for A1M CTGF, GSTα, KIM-1, microalbumin, osteopontin, and TFF3. A1M also correctly identified HV and DM with an accuracy of 89.0%. Summary These studies: 1) determined that nonstabilized urine can be used for assays under qualification; and 2) documented that A1M, CTGF, GSTα, KIM-1, microalbumin, osteopontin, and TFF3 were significantly increased in urine from P-DM. In addition, the 89.0% accuracy of A1M in distinguishing P-DM from HV may allow this biomarker to be used to monitor efficacy of potential renal protective agents.

Translation Strategy for the Qualification of Drug-induced Vascular Injury Biomarkers

Drug-induced vascular injury (DIVI) is a common preclinical toxicity usually characterized by hemorrhage, vascular endothelial and smooth muscle damage, and inflammation. DIVI findings can cause delays or termination of drug candidates due to low safety margins. The situation is complicated by the absence of sensitive, noninvasive biomarkers for monitoring vascular injury and the uncertain relevance to humans. The Safer And Faster Evidence-based Translation (SAFE-T) consortium is a public–private partnership funded within the European Commission’s Innovative Medicines Initiative (IMI) aiming to accelerate drug development by qualifying biomarkers for drug-induced organ injuries, including DIVI. The group is using patients with vascular diseases that have key histomorphologic features (endothelial damage, smooth muscle damage, and inflammation) in common with those observed in DIVI, and has selected candidate biomarkers associated with these features. Studied populations include healthy volunteers, patients with spontaneous vasculitides and other vascular disorders. Initial results from studies with healthy volunteers and patients with vasculitides show that a panel of biomarkers can successfully discriminate the population groups. The SAFE-T group plans to seek endorsement from health authorities (European Medicines Agency and Food and Drug Administration) to qualify the biomarkers for use in regulatory decision-making processes.

A peripherally restricted P2Y12 receptor antagonist altered rat tumor incidences with no human relevance: Mode of action consistent with dopamine agonism

Background Ticagrelor is an orally available, direct acting and reversible P2Y12 receptor antagonist approved for treatment of acute coronary syndrome. The objectives of these studies were to (1) evaluate the Ticagrelor 2-year rat carcinogenicity bioassay data; (2) investigate potential mode of action (MOA) and (3) interpret human relevance. Methods The following studies were done (1) rat two-year carcinogenicity study in male and female rats, (2) in vitro and in vivo genotoxicity assays, (3) quantitative whole body autoradiography (QWBA; male and female rats), (4) in vitro pharmacological profiling for more than 300 assays, and (5) in vivo ovariectomized rat assay. Results The carcinogenicity study indicated Ticagrelor increased uterine tumor incidence while decreasing mammary and pituitary tumors/hyperplasia incidences in only high dose female rats. However, this altered tumor incidences were not P2Y12 target related since marketed non-reversible P2Y12 receptor antagonists were not associated with alter tumor incidences. MOA studies determined Ticagrelor exposure in the anterior pituitary and Ticagrelor was (1) non-genotoxic, (2) peripherally-restricted, (3) a dopamine transport (DAT) inhibitor with an IC50 lower than systemic free exposure in the rat carcinogenic study and more than a log higher than the free systemic exposure seen in clinical trials and (4) an inhibitor of estradiol-induced prolactin secretion. Discussion Similar to Ticagrelor, centrally active dopamine agonists induce the same altered tumor incidence patterns that according to literature do not translate into the clinical setting, with a MOA involving decreased prolactin secretion. The Ticagrelor MOA data and literature suggest that altered dopamine levels in the hypophyseal part of the hypothalamus–hypophyseal axis (by Ticagrelor) will result in similar altered tumor incidences in rat that do not translate into the clinical setting, based on qualitative species differences. In conclusion Ticagrelor increased uterine tumors in the rat carcinogenesis study by a MOA consistent with reduced dopamine inhibition of prolactin, which is not a patient safety risk.

Evaluation of von Willebrand Factor and von Willebrand Factor Propeptide in Models of Vascular Endothelial Cell Activation, Perturbation, and/or Injury

Pharmacologically, vasoactive agents targeting endothelial and/or smooth muscle cells (SMC) are known to cause acute drug-induced vascular injury (DIVI) and the resulting pathology is due to endothelial cell (EC) perturbation, activation, and/or injury. Alteration in EC structure and/or function may be a critical event in vascular injury and, therefore, evaluation of the circulatory kinetic profile and secretory pattern of EC-specific proteins such as VWF and VWFpp could serve as acute vascular injury biomarkers. In rat and dog models of DIVI, this profile was determined using pharmacologically diverse agents associated with functional stimulation/perturbation (DDAVP), pathological activation (lipopolysaccharide [LPS]/endotoxin), and structural damage (fenoldopam [FD], dopamine [DA], and potassium channel opener (PCO) ZD6169). In rats, FD caused moderate DIVI and time-related increase in plasma VWF levels ∼33% while in control rats VWF increased ∼5%. In dogs, VWF levels transiently increased ∼30% when there was morphologic evidence of DIVI by DA or ZD6169. However, in dogs, VWFpp increased >60-fold (LPS) and >6-fold (DDAVP), respectively. This was in comparison to smaller dynamic 1.38-fold (LPS) and 0.54-fold (DDAVP) increases seen in plasma VWF. Furthermore, DA was associated with a dose-dependent increase in plasma VWFpp. In summary, VWF and VWFpp can discriminate between physiological and pathological perturbation, activation, and injury to ECs.

Inhibition of Oestradiol-induced Prolactin Release in a Dual-Cannulated Ovariectomized Rat Model by Carmoxirole, a Peripherally Restricted Dopamine Agonist

Centrally acting dopamine agonists (e.g. bromocriptine) and dopamine transport inhibitors (e.g. GBR12909) are known to inhibit oestradiol‐induced prolactin release. The capacity of peripherally restricted compounds to do likewise, however, is unknown. Here, the effects of the peripherally restricted dopamine receptor agonist carmoxirole on oestradiol‐induced prolactin release were investigated. Dual‐cannulated ovariectomized rats were used, so that a robust, reproducible response to exogenous oestrogen could be induced and sequential blood samples were taken with minimal stress. Carmoxirole (15 mg/kg) inhibited oestradiol‐induced prolactin release, similar to bromocriptine and GBR12909. However, carmoxirole also induced a rapid, transient, oestradiol‐independent release of prolactin. These data show that peripherally restricted dopamine receptor agonists are sufficient to inhibit oestradiol‐induced prolactin release. Like centrally acting compounds, they may therefore be expected to affect the incidence of prolactin‐dependent tumours in rat carcinogenesis studies without inducing central‐mediated side effects.

An acute rat in vivo screening model to predict compounds that alter blood glucose and/or insulin regulation

INTRODUCTION Drug-induced glucose dysregulation and insulin resistance have been associated with weight gain and potential induction and/or exacerbation of diabetes mellitus in the clinic suggesting they may be safety biomarkers when developing antipsychotics. Glucose and insulin have also been suggested as potential efficacy biomarkers for some oncology compounds. The objective of this study was to qualify a medium throughput rat in vivo acute Intravenous Glucose Tolerance Test (IVGTT) for predicting compounds that will induce altered blood glucose and/or insulin levels. METHODS Acute and sub-chronic studies were performed to qualify an acute IVGTT model. Double cannulated male rats (Han-Wistar and Sprague-Dawley) were administered vehicle, olanzapine, aripiprazole or other compounds at t=-44min for acute studies and at time=-44min on the last day of dosing for sub-chronic studies, treated with dextrose (time=0min; i.v.) and blood collected using an automated Culex® system for glucose and insulin analysis (time=-45, -1, 2, 10, 15, 30, 45, 60, 75, 90, 120, 150 and 180min). RESULTS Olanzapine significantly increased glucose and insulin area under the curve (AUC) values while aripiprazole AUC values were similar to control, in both acute and sub-chronic studies. All atypical antipsychotics evaluated were consistent with literature references of clinical weight gain. As efficacy biomarkers, insulin AUC but not glucose AUC values were increased with a compound known to have insulin growth factor-1 (IGF-1) activity, compared to control treatment. DISCUSSION These studies qualified the medium throughput acute IVGTT model to more quickly screen compounds for 1) safety - the potential to elicit glucose dysregulation and/or insulin resistance and 2) efficacy - as a surrogate for compounds affecting the glucose and/or insulin regulatory pathways. These data demonstrate that the same in vivo rat model and assays can be used to predict both clinical safety and efficacy of compounds.

Renal biomarker changes associated with hyaline droplet nephropathy in rats are time and potentially compound dependent.

Alpha 2u-globulin mediated hyaline droplet nephropathy (HDN) is a male rat specific lesion induced when a compound or metabolite binds to alpha 2u-globulin. The objective of this study was to investigate if the newer and more sensitive renal biomarkers would be altered with HDN as well as be able to distinguish between HDN and oxidative stress-induced kidney injury. Rats were dosed orally for 7 days to determine (1) if HDN (induced by 2-propanol or D-limonene) altered the newer renal biomarkers and not BUN or creatinine, (2) if renal biomarkers could distinguish between HDN and oxidative stress-induced kidney injury (induced by potassium bromate), (3) sensitivity of HDN-induced renal biomarker changes relative to D-limonene dose, and (4) reversibility of HDN and renal biomarkers, using vehicle or 300 mg/kg/day D-limonene with 7 days of dosing and necropsies scheduled over the period of Days 8-85. HDN-induced renal biomarker changes in male rats were potentially compound specific: (1) 2-propanol induced mild HDN without increased renal biomarkers, (2) potassium bromate induced moderate HDN with increased clusterin, and (3) D-limonene induced marked HDN with increased αGST, μGST and albumin. Administration of potassium bromate did not result in oxidative stress-induced kidney injury, based on histopathology and renal biomarkers creatinine and BUN. The compound D-limonene induced a dose dependent increase in HDN severity and renal biomarker changes without altering BUN, creatinine or NAG: (1) minimal induction of HDN and no altered biomarkers at 10 mg/kg/day, (2) mild induction of HDN with increased αGST and μGST at 50 mg/kg/day and (3) marked induction of HDN with increased αGST, μGST and albumin at 300 mg/kg/day. HDN induced by D-limonene was reversible, but with a variable renal biomarker pattern over time: Day 8 there was increased αGST, μGST and albumin; on Day 15 increased clusterin, albumin and Kim-1. In summary, HDN altered the newer and more sensitive renal biomarkers in a time and possibly compound dependent manner.