ABSTRACT
Emerging urinary kidney safety biomarkers have been evaluated in recent years and have been shown to be superior to the serum parameters blood urea nitrogen (BUN) and creatinine (sCr) for monitoring kidney injury in the proximal tubule. However, their potential application in differentiating the location of the initial kidney injury (eg, glomerulus vs tubule) has not been fully explored. Here, we assessed the performance of two algorithms that were constructed using either an empirical or a mathematical model to predict the site of kidney injury using a data set consisting of 22 rat kidney toxicity studies with known urine biomarker and histopathologic outcomes. Two kidney safety biomarkers used in both models, kidney injury molecule 1 (KIM-1) and albumin (ALB), were the best performers to differentiate glomerular injury from tubular injury. The performance of algorithms using these two biomarkers against the gold standard of kidney histopathologic examination showed high sensitivity in differentiating the location of the kidney damage to either the glomerulus or the proximal tubules. These data support the exploration of such an approach for use in clinical settings, leveraging urinary biomarker data to aid in the diagnosis of either glomerular or tubular injury where histopathologic assessments are not conducted.
Subject(s)
Biomarkers , Kidney Glomerulus , Animals , Rats , Kidney Glomerulus/pathology , Biomarkers/urine , Biomarkers/blood , Albuminuria , Male , Kidney Tubules/pathology , Algorithms , Rats, Sprague-Dawley , Albumins/metabolism , Albumins/analysis , Hepatitis A Virus Cellular Receptor 1/analysis , Acute Kidney Injury/urine , Acute Kidney Injury/pathology , Cell Adhesion MoleculesABSTRACT
Activating mutations of the leucine-rich repeat kinase 2 (LRRK2) gene are associated with Parkinson disease (PD), prompting development of LRRK2 inhibitors as potential treatment for PD. However, kidney safety concerns have surfaced from LRRK2 knockout (KO) mice and rats and from repeat-dose studies in rodents administered LRRK2 inhibitors. To support drug development of this therapeutic target, we conducted a study of 26 weeks' duration in 2-month-old wild-type and LRRK2 KO Long-Evans Hooded rats to systematically examine the performance of urinary safety biomarkers and to characterize the nature of the morphological changes in the kidneys by light microscopy and by ultrastructural evaluation. Our data reveal the time course of early-onsetâ¯albuminuria at 3 and 4 months in LRRK2 KO female and male rats, respectively. The increases in urine albumin were not accompanied by concurrent increases in serum creatinine, blood urea nitrogen, or renal safety biomarkers such as kidney injury molecule 1 or clusterin, although morphological alterations in both glomerular and tubular structure were identified by light and transmission electron microscopy at 8 months of age. Diet optimization with controlled food intake attenuated the progression of albuminuria and associated renal changes.
Subject(s)
Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 , Parkinson Disease , Protein Serine-Threonine Kinases , Animals , Female , Male , Mice , Rats , Albuminuria/pathology , Biomarkers , Kidney/pathology , Leucine , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/genetics , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/metabolism , Mice, Knockout , Mutation , Parkinson Disease/drug therapy , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Rats, Long-EvansABSTRACT
Kidney biopsies are used sparingly to diagnose kidney injury in the clinic. Here we have conducted a small exploratory study to directly compare the low-grade kidney injury monitoring performance of serum safety biomarkers, novel urine safety biomarkers, microscopic histopathology and targeted gene expression alterations in kidney biopsy specimens in rhesus monkeys treated with tobramycin. Targeted gene expression increases were observed in the kidney biopsy samples and whole kidney sections for kidney injury molecule 1 (KIM-1), clusterin (CLU), osteopontin (OPN) messenger RNA transcripts. In addition, increases of the urinary kidney safety protein biomarkers including KIM-1, CLU, OPN were also observed. These increases in gene expression and urinary protein end point were in concordance with the eventual low-grade kidney lesions seen in terminal tissue sections. In contrast, conventional serum biomarkers blood urea nitrogen and serum creatinine were not as sensitive in monitoring kidney injury. Although these data do not support routinely adding kidney biopsies to regular toxicology studies, they provide evidence on the value and limitations of incorporating gene expression profiling on kidney biopsy specimens, further underscore the value of urinary kidney safety biomarkers for improved low-grade kidney injury monitoring, and open the door for future definitive studies.
Subject(s)
Acute Kidney Injury , Tobramycin , Acute Kidney Injury/diagnosis , Animals , Biomarkers , Biopsy , Gene Expression Profiling , Kidney/pathology , Macaca mulatta , Tobramycin/metabolismABSTRACT
Novel urinary protein biomarkers have recently been identified and qualified in rats for the early detection of renal injury in drug development studies. However, there are few reports on the utility of these renal biomarkers in mice, another important and widely used preclinical animal species for drug development studies. The purpose of this study was to assess the value of these recently qualified biomarkers for the early detection of drug-induced kidney injury (DIKI) in different strains of mice using multiple assay panels. To this end, we evaluated biomarker response to kidney injury induced by several nephrotoxic agents including amphotericin B, compound X, and compound Y. Several of the biomarkers were shown to be sensitive to DIKI in mice. When measured, urinary albumin and neutrophil gelatinase-associated lipocalin were highly sensitive to renal tubular injury, regardless of the assay platforms, mouse strain, and nephrotoxic agents. Depending on the type of renal tubular injury, kidney injury molecule-1 was also highly sensitive, regardless of the assay platforms and mouse strain. Osteopontin and cystatin C were modestly to highly sensitive to renal tubular injury, but the assay type and/or the mouse strain should be considered before using these biomarkers. Calbindin D28 was highly sensitive to injury to the distal nephron in mice. To our knowledge, this is the first report that demonstrates the utility of novel urinary biomarkers evaluated across multiple assay platforms and nephrotoxicants in different mice strains with DIKI. These results will help drug developers make informed decisions when selecting urinary biomarkers for monitoring DIKI in mice for toxicology studies.
Subject(s)
Acute Kidney Injury/chemically induced , Acute Kidney Injury/diagnosis , Amphotericin B/toxicity , Biomarkers/urine , Drug Development/methods , Animals , Disease Models, Animal , Humans , Male , Mice , Mice, Inbred C57BL , Predictive Value of TestsABSTRACT
Drug-induced kidney injury (DIKI) is a frequent occurrence in nonclinical drug development. It is well established that novel urine kidney safety biomarkers will outperform urea nitrogen (BUN) and serum creatinine (sCr) for monitoring direct drug injury to the kidney across numerous compounds spanning diverse mechanisms and efforts are underway for a formal regulatory clinical qualification. However, it remains unclear how these novel biomarkers will perform under prerenal azotemia when BUN and sCr are elevated but no intra-renal injury is suspected. This lack of knowledge is largely due to the dearth of such nonclinical animal models. We report here that treatment of dogs with a potent antihypertensive compound MK-5478 at a suprapharmacologic dose for up to 9 days results in the development of prerenal azotemia and, in some dogs, kidney toxicity through the dual sustained effects of MK-5478 as a nitric oxide donor and an angiotensin II receptor blocker (ARB). While conventional serum biomarkers BUN, and often sCr as well, were highly elevated in these dogs with or without kidney damage, urine kidney biomarkers clusterin (CLU) and neutrophil gelatinase-associated lipocalin (NGAL) showed increases only in dogs with kidney histopathologic changes following the sustained period of prerenal azotemia. Urine albumin (ALB) and total protein also tracked with kidney lesions but with less sensitivity. Thus, we present evidence for the first time that urine kidney safety biomarkers used together with BUN and sCr can distinguish intra-renal injury among dogs with prerenal azotemia while the conventional serum biomarkers alone are ambiguous, either being interpreted as false positives of kidney injury, or dismissed under circumstances as benign without appreciation for a threshold of impending injury.
Subject(s)
Acute Kidney Injury/urine , Azotemia/chemically induced , Azotemia/urine , Biomarkers/urine , Albuminuria/urine , Angiotensin II Type 1 Receptor Blockers/toxicity , Animals , Antihypertensive Agents/toxicity , Blood Urea Nitrogen , Clusterin/urine , Creatinine/blood , Dogs , Female , Lipocalin-2/urine , Male , Nitric Oxide Donors/toxicityABSTRACT
MK-7680, a cyclic nucleotide prodrug, caused significant kidney tubule injury in female rats when administered orally at 1000 mg/kg/day for 2 weeks using 10% Polysorbate 80 as vehicle. However, kidney injury was absent when MK-7680 was administered at the same dose regimen using 100% Polyethylene Glycol 200 (PEG 200) as the vehicle. Subsequent investigations revealed that MK-7680 triphosphate concentrations in kidney were much lower in rats treated with MK-7680 using PEG 200 compared with 10% Polysorbate 80 vehicle, whereas plasma exposures of MK-7680 prodrug were similar. In vitro studies demonstrated that PEG 200 is an inhibitor of human renal uptake transporter organic anion transporter 3 (OAT3), of which MK-7680 is a substrate. Furthermore, PEG 200 and PEG 400 were found to interfere in vitro with human renal transporters OAT3, organic cation transporter (OCT) 2, multidrug resistance-associated protein (MRP) 2 and 4, and multidrug and toxin extrusion protein (MATE) 1 and 2K, but not OAT1. These results support a conclusion that PEG 200 may prevent MK-7680-induced kidney injury by inhibiting its active uptake into proximal tubular cells by OAT3. Caution should be exercised therefore when using PEGs as vehicles for toxicity assessment for compounds that are substrates of renal transporters.
ABSTRACT
Newer urinary protein kidney safety biomarkers can outperform the conventional kidney functional biomarkers blood urea nitrogen (BUN) and serum creatinine (SCr) in rats. However, there is far less experience with the relative performance of these biomarkers in dogs and nonhuman primates. Here, we report urine protein biomarker performance in tenofovir-treated cynomolgus monkeys and beagle dogs. Tenofovir intravenous daily dosing in monkeys for 2 or 4 weeks at 30 mg/kg/day resulted in minimal to moderate tubular degeneration and regeneration, and tenofovir disoproxil fumarate oral dosing in dogs for 10 days at 45 mg/kg/day resulted in mild to marked tubular degeneration, necrosis, and regeneration. Among biomarkers tested, kidney injury molecule 1 (Kim-1) and clusterin (CLU) clearly outperformed BUN and SCr and were the most reliable in detecting the onset and progression of tenofovir-induced tubular injury. Cystatin C, retinol binding protein 4, ß2-microglobulin, neutrophil gelatinase-associated lipocalin, albumin, and total protein also performed better than BUN and SCr and added value when considered together with Kim-1 and CLU. These findings demonstrate the promising utility of these urinary safety biomarkers in monkeys and dogs and support their further evaluation in human to improve early detection of renal tubular injury.
Subject(s)
Acute Kidney Injury/urine , Biomarkers/urine , Kidney Tubules/drug effects , Tenofovir/toxicity , Acute Kidney Injury/chemically induced , Acute Kidney Injury/pathology , Administration, Oral , Animals , Biomarkers/blood , Dogs , Female , Injections, Intravenous , Kidney Tubules/pathology , Macaca fascicularis , Male , Sensitivity and Specificity , Species SpecificityABSTRACT
Traditional kidney biomarkers are insensitive indicators of acute kidney injury, with meaningful changes occurring late in the course of injury. The aim of this work was to demonstrate the diagnostic potential of urinary osteopontin (OPN) and neutrophil gelatinase-associated lipocalin (NGAL) for drug-induced kidney injury (DIKI) in rats using data from a recent regulatory qualification submission of translational DIKI biomarkers and to compare performance of NGAL and OPN to five previously qualified DIKI urinary biomarkers. Data were compiled from 15 studies of 11 different pharmaceuticals contributed by Critical Path Institute's Predictive Safety Testing Consortium (PSTC) Nephrotoxicity Working Group (NWG). Rats were given doses known to cause DIKI or other target organ toxicity, and urinary levels of the candidate biomarkers were assessed relative to kidney histopathology and serum creatinine (sCr) and blood urea nitrogen (BUN).OPN and NGAL outperformed sCr and BUN in identifying DIKI manifested as renal tubular epithelial degeneration or necrosis. In addition, urinary OPN and NGAL, when used with sCr and BUN, increased the ability to detect renal tubular epithelial degeneration or necrosis. NGAL and OPN had comparable or improved performance relative to Kim-1, clusterin, albumin, total protein, and beta-2 microglobulin. Given these data, both urinary OPN and NGAL are appropriate for use with current methods for assessing nephrotoxicity to identify and monitor DIKI in regulatory toxicology studies in rats. These data also support exploratory use of urinary OPN and NGAL in safety monitoring strategies of early clinical trials to aid in the assurance of patient safety.
Subject(s)
Acute Kidney Injury/diagnosis , Acute-Phase Proteins/urine , Lipocalins/urine , Osteopontin/urine , Proto-Oncogene Proteins/urine , Acute Kidney Injury/blood , Acute Kidney Injury/chemically induced , Acute Kidney Injury/urine , Animals , Area Under Curve , Biomarkers/blood , Biomarkers/urine , Blood Urea Nitrogen , Creatinine/blood , Disease Models, Animal , Lipocalin-2 , Predictive Value of Tests , ROC Curve , Rats , Reproducibility of Results , UrinalysisABSTRACT
Novel urinary kidney safety biomarkers have been identified recently that may outperform or add value to the conventional renal function biomarkers, blood urea nitrogen (BUN) and serum creatinine (SCr). To assess the relative performance of the growing list of novel biomarkers, a comprehensive evaluation was conducted for 12 urinary biomarkers in 22 rat studies including 12 kidney toxicants and 10 compounds with toxicities observed in organs other than kidney. The kidney toxicity studies included kidney tubular toxicants and glomerular toxicants. The 12 urinary biomarkers evaluated included Kim-1, clusterin, osteopontin, osteoactivin, albumin, lipocalin-2, GST-α, ß2-microglobulin, cystatin C, retinol binding protein 4, total protein, and N-acetyl-ß-D-glucosaminidase. Receiver operator characteristic (ROC) curves were generated for each biomarker and for BUN and SCr to compare the relative performance of the 12 biomarkers in individual animals against the microscopic histomorphologic changes observed in the kidney. Among the kidney toxicity biomarkers analyzed, Kim-1, clusterin, and albumin showed the highest overall performance for detecting drug-induced renal tubular injury in the rat in a sensitive and specific manner, whereas albumin showed the highest performance in detecting drug-induced glomerular injury. Although most of the evaluated kidney biomarkers were more sensitive in detecting kidney toxicity compared with BUN and SCr, all biomarkers demonstrated some lack of specificity, most notably NGAL and osteopontin, illustrating the need for caution when interpreting urinary biomarker increases in rat samples when organ toxicity is unknown.
Subject(s)
Biomarkers/urine , Kidney Diseases/chemically induced , Kidney Diseases/urine , Kidney/drug effects , Toxicity Tests , Animals , Enzyme-Linked Immunosorbent Assay , Female , Kidney/pathology , Kidney Diseases/blood , Kidney Diseases/pathology , Limit of Detection , Male , ROC Curve , Rats , Rats, Sprague-Dawley , Rats, WistarABSTRACT
OBJECTIVE: To identify biomarkers that distinguish between active antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) and remission in a manner superior or complementary to established markers of systemic inflammation. METHODS: Markers of vascular injury and angiogenesis were measured before and after treatment in a large clinical trial in AAV: 163 subjects enrolled in the Rituximab in ANCA-Associated Vasculitis trial were screened for the present study. Serum levels of E-selectin, intercellular adhesion molecule 3 matrix metalloproteinase protein 1 (MMP-1), MMP-3, MMP-9, P-selectin, thrombomodulin, and vascular endothelial growth factor were measured at study screening (time of active disease) and at month 6. Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) levels had been measured at the time of the clinical visit. The primary outcome measure was the difference in marker level between screening and month 6 among patients whose disease was in remission (Birmingham Vasculitis Activity Score for Wegener's granulomatosis [BVAS/WG] score of 0) at month 6. RESULTS: All patients had severe active vasculitis at screening (mean ± SD BVAS/WG score 8.6 ± 3.2). Among the 123 patients whose disease was clinically in remission at month 6, levels of all markers except E-selectin showed significant declines. MMP-3 levels were also higher among the 23 patients with active disease at month 6 than among the 123 patients whose disease was in remission. MMP-3 levels correlated weakly with ESR and CRP levels. CONCLUSION: Many markers of vascular injury and angiogenesis are elevated in severe active AAV and decline with treatment, but MMP-3 appears to distinguish active AAV from remission better than the other markers studied. Further study of MMP-3 is warranted to determine its clinical utility in combination with conventional markers of inflammation and ANCA titers.
Subject(s)
Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/blood , Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/physiopathology , E-Selectin/blood , Matrix Metalloproteinase 3/blood , Neovascularization, Pathologic/blood , Vascular System Injuries/blood , Adult , Aged , Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/drug therapy , Antibodies, Monoclonal, Murine-Derived/therapeutic use , Antirheumatic Agents/therapeutic use , Biomarkers/blood , Blood Sedimentation , C-Reactive Protein/metabolism , Case-Control Studies , Diagnosis, Differential , Female , Follow-Up Studies , Humans , Male , Middle Aged , Neovascularization, Pathologic/physiopathology , Remission Induction , Rituximab , Vascular System Injuries/physiopathologyABSTRACT
BACKGROUND: The effects of histamine H1 antagonist chlorcyclizine on rat palate development were characterized following in utero exposure. METHODS: To identify the optimum dose for inducing cleft palate, pregnant rats were administered 30, 60, or 90 mg/kg chlorcyclizine on Gestation Days 11 to 14. Fetal palate gene expression was also assessed after 90 mg/kg chlorcyclizine at 8, 15 and 30 hours post-dose on Gestation Day 14 using microarray and qRT-PCR. RESULTS: Rats in the 60- and 90-mg/kg groups exhibited adverse clinical signs and body weight loss. Rats in the 90-mg/kg group also demonstrated increases in late resorptions and decreases in fetal weight. Effects in the low-dose group were limited to decreases in body weight gain. Fetal assessment on Gestation Day 21 revealed that findings were limited to the 60- and 90-mg/kg groups, and included cleft palate (80% of litters for both groups), high arched palate, small nose, micrognathia, high domed head, digits shortened/absent and small limb. The fetal incidence of cleft palate was higher at 90 mg/kg, thus this dose was selected to assess palate gene expression. The altered genes associated with chlorcyclizine-induced cleft palate included Wnt5a, Bmp2, Bmp4, Fgf10, Fgfr2, Msx1, and Insig1 but the magnitude of the change was relatively small (1.5- to 2-fold). CONCLUSIONS: Expression of several genes involved in palate, limb and digit development was altered in the fetal palate following in utero exposure to chlorcyclizine. The subtle perturbation and interplay of these genes may have profound effects on the dynamics of fetal palate development.
Subject(s)
Cleft Palate/chemically induced , Embryo, Mammalian/drug effects , Histamine H1 Antagonists/toxicity , Palate/drug effects , Piperazines/toxicity , Animals , Biomarkers/metabolism , Cleft Palate/genetics , Cleft Palate/pathology , Embryo, Mammalian/abnormalities , Female , Fetal Resorption/chemically induced , Fetal Weight/drug effects , Gene Expression Profiling , Gene Expression Regulation, Developmental/drug effects , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Maternal Exposure , Microarray Analysis , Palate/abnormalities , Palate/metabolism , Pregnancy , RNA, Messenger/metabolism , Rats , Rats, Inbred Strains , Weight GainABSTRACT
Chromatin modifications are now widely accepted as being essential steps involved in activation, repression, and poising of the expression of a large number of genes within the genome. Not only does understanding the role of such changes provide an opportunity to elucidate mechanisms controlling gene expression but in parallel offers the ability to develop novel indicative and predictive biomarkers of disease and toxicity. In the current study, we have applied the chromatin immunoprecipitation assay to investigate putative changes in the chromatin environment associated with the kidney injury molecule 1 (Kim1) gene upon exposure of rats to the nephrotoxicant, gentamicin. Chromatin was isolated from the kidneys of both control and gentamicin-treated animals and interrogated using specific antibodies recognizing two modifications of histone H3, acetylation of lysine 9, and trimethylation of lysine 4, along with RNA polymerase II. Enriched chromatin fractions were analyzed by quantitative PCR using tiled primer pairs covering 4 kb of the Kim1 gene (spanning -2 kb to + 2 kb, relative to the transcription start site). The results demonstrate a substantial increase in the presence of RNA polymerase II and both histone modifications following gentamicin treatment in regions downstream but not upstream of the transcriptional start site of the Kim1 gene. These changes were associated with a marked increase in messenger RNA coding for the Kim1 protein. Together these data suggest, for the first time, that the Kim1 gene is regulated in an epigenetic fashion under conditions of nephrotoxicity.
Subject(s)
Anti-Bacterial Agents/therapeutic use , Cell Adhesion Molecules/genetics , Gene Silencing/drug effects , Gentamicins/toxicity , Kidney Diseases/chemically induced , Kidney/drug effects , Acetylation , Animals , Cell Adhesion Molecules/metabolism , Chromatin/chemistry , Chromatin/drug effects , Gene Expression/drug effects , Histones/metabolism , Kidney/metabolism , Kidney/pathology , Kidney Diseases/genetics , Kidney Diseases/pathology , Male , Methylation , RNA, Messenger/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
Treatment with drugs from multiple classes induces vascular injury with medial necrosis, hemorrhage, endothelial damage, and inflammation. Previous research has suggested early events might be occurring well in advance of the full lesions that appear forty-eight to seventy-two hours after dosing with SCH 351591, a PDE IV inhibitor. This study was performed to study early events in detail. Rats were dosed with 20 mg/kg of drug by gavage and sacrificed at times between fifteen and 240 minutes after dosing. Tissues were collected for histopathological analysis and gene expression studies. Serum was collected for biomarker analysis. The data from biomarker analysis showed a three-part response with an early phase that was maximal at fifteen to thirty minutes, a second phase from forty-five to 180 minutes, and the third phase that was starting to rise at four hours. The first phase included increases in lymphocytes, serum histamine, and serum nitrite. The second phase shows continued elevation of serum nitrite. The third phase was marked by an increase in serum GRO/CINC-1. At fifteen minutes, histopathology showed activation of mast cells, but not degranulation. Increases in endothelial activation and perivascular inflammatory cells were first apparent at thirty minutes and increased through 240 minutes.
Subject(s)
Cyclic N-Oxides/toxicity , Inflammation/chemically induced , Phosphodiesterase 4 Inhibitors/toxicity , Quinolines/toxicity , Vascular System Injuries/chemically induced , Animals , Biomarkers/analysis , Biomarkers/blood , Gene Expression/drug effects , Inflammation/pathology , Mesentery/blood supply , Mesentery/pathology , Rats , Rats, Sprague-Dawley , Time , Vascular System Injuries/blood , Vascular System Injuries/pathologyABSTRACT
Application of any new biomarker to support safety-related decisions during regulated phases of drug development requires provision of a substantial data set that critically assesses analytical and biological performance of that biomarker. Such an approach enables stakeholders from industry and regulatory bodies to objectively evaluate whether superior standards of performance have been met and whether specific claims of fit-for-purpose use are supported. It is therefore important during the biomarker evaluation process that stakeholders seek agreement on which critical experiments are needed to test that a biomarker meets specific performance claims, how new biomarker and traditional comparators will be measured and how the resulting data will be merged, analyzed and interpreted.
Subject(s)
Biomarkers , Drug Discovery , Pharmaceutical Preparations , Animals , Drug Discovery/legislation & jurisprudence , Drug Discovery/methods , Drug-Related Side Effects and Adverse Reactions , Humans , Pharmaceutical Preparations/standardsABSTRACT
The first formal qualification of safety biomarkers for regulatory decision making marks a milestone in the application of biomarkers to drug development. Following submission of drug toxicity studies and analyses of biomarker performance to the Food and Drug Administration (FDA) and European Medicines Agency (EMEA) by the Predictive Safety Testing Consortium's (PSTC) Nephrotoxicity Working Group, seven renal safety biomarkers have been qualified for limited use in nonclinical and clinical drug development to help guide safety assessments. This was a pilot process, and the experience gained will both facilitate better understanding of how the qualification process will probably evolve and clarify the minimal requirements necessary to evaluate the performance of biomarkers of organ injury within specific contexts.
Subject(s)
Biomarkers, Pharmacological , Drug Approval/legislation & jurisprudence , Kidney , Animals , Drug-Related Side Effects and Adverse Reactions , Europe , Humans , Kidney/drug effects , Kidney/injuries , Pharmaceutical Preparations/standards , United States , United States Food and Drug AdministrationABSTRACT
The induction of dog CYP3A12 and CYP3A26 mRNA levels was evaluated in liver slices after treatment with 22 xenobiotics. Eleven of the 22 xenobiotics increased 3A12 mRNA by more than four-fold, while nine did the same for 3A26 mRNA. A four-fold increase in the mRNA level was used as the cut-off for indication of induction based on the noise level of the real time-PCR. A good correlation was found between the mRNA levels for 3A12 and 3A26 after treatment with compounds, suggesting that these two CYPs may be co-induced. Induction of CYP3A4 in human hepatocytes was evaluated after treatment with the same 22 compounds. Thirteen out of the 22 compounds increased the 3A4 mRNA levels by more than four-fold. When the mRNA levels of 3A4 and 3A12 were compared after treatment with compounds, no correlation was found. The regulation of CYP3A expression has been demonstrated to be controlled by pregnane X receptor (PXR). Upon examination of the sequence homology and the three-dimensional structures of human PXR and a dog PXR model, only two different amino acids (met323/val and arg410/lys) were found in the ligand-binding domain. This finding suggests that these two amino acids may play a role in the binding specificity of ligands.
Subject(s)
Cytochrome P-450 Enzyme System/biosynthesis , Enzyme Induction/drug effects , Liver/drug effects , Liver/enzymology , Xenobiotics/pharmacology , Animals , Cytochrome P-450 CYP3A/biosynthesis , Dogs , Hepatocytes/drug effects , Hepatocytes/enzymology , Humans , In Vitro Techniques , Male , Models, Chemical , Models, Molecular , Pregnane X Receptor , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Receptors, Steroid/biosynthesis , Receptors, Steroid/drug effects , Reverse Transcriptase Polymerase Chain Reaction , Species SpecificityABSTRACT
Assessment of cytochrome P450 (CYP) induction at the mRNA level in preclinical rodent studies has gained interest in recent years, but there are still concerns regarding correlations between the mRNA and the enzyme activity levels, especially in mice. The purpose of the present study was to systematically evaluate patterns of temporal changes of CYPs 1a1, 1a2, 2b10, 3a11, and 4a10 at mRNA, protein, and activity levels in order to determine to what extent mRNA levels could be used either qualitatively or quantitatively for the assessment of CYP enzyme induction. In this study, livers from male CD-1 mice treated daily with beta-naphthoflavone, phenobarbital, dexamethasone, clofibrate, and control vehicles were collected for RNA and microsomal analysis after 0.5, 1, 2, 4, and 8 days of daily dose. The results revealed a good correlation among mRNA, protein, and enzyme activity levels, with the best correlation at the time points between Days 2 and 8, suggesting that the appropriate time to monitor CYP mRNA may be beyond Day 2 of chemical treatments. Based on these results, we concluded that the mRNA approach is a useful tool to monitor CYP induction in mice, particularly when treatment duration is beyond 2 days.
Subject(s)
Cytochrome P-450 Enzyme System/biosynthesis , Drug Evaluation, Preclinical/methods , Liver/drug effects , RNA, Messenger/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction , Animals , Clofibrate/pharmacology , Cytochrome P-450 Enzyme System/genetics , Dexamethasone/pharmacology , Enzyme Induction/drug effects , Feasibility Studies , Isoenzymes , Liver/enzymology , Male , Mice , Phenobarbital/pharmacology , Reproducibility of Results , Time Factors , beta-Naphthoflavone/pharmacologyABSTRACT
Drug-associated vascular injury can be caused by phosphodiesterase (PDE) IV inhibitors and drugs from several other classes. The pathogenesis is poorly understood, but it appears to include vascular and innate immunological components. This research was undertaken to identify changes in peripheral blood associated with vascular injury caused by PDE IV inhibitors. We evaluated twelve proteins, serum nitrite, and leukocyte populations in peripheral blood of rats treated with experimental PDE IV inhibitors. We found that these compounds produced histological microvascular injury in a dose- and time-dependent manner. Measurement of these serum proteins showed changes in eight of the twelve examined. Changes were seen in the levels of: tissue inhibitor of metalloproteinase-1, alpha1-acid glycoprotein, GRO/CINC-1, vascular endothelial growth factor, C-reactive protein, haptoglobin, thrombomodulin, and interleukin-6. No changes were seen in levels of tumor necrosis factor-alpha, hepatocyte growth factor, nerve growth factor, and granulocyte-monocyte colony stimulating factor. Serum levels of nitrite were also increased. Circulating granulocyte numbers were increased, and lymphocyte numbers were decreased. The changes in these parameters showed both a dose- and time-dependent association with histopathologic changes. These biomarkers could provide an additional tool for the nonclinical and clinical evaluation of investigational compounds.
Subject(s)
Biomarkers/blood , Blood Vessels/drug effects , Cyclic N-Oxides/toxicity , Phosphodiesterase 4 Inhibitors , Phosphodiesterase Inhibitors/toxicity , Quinolines/toxicity , Vascular Diseases/chemically induced , Animals , Clinical Chemistry Tests , Dose-Response Relationship, Drug , Immunohistochemistry , Leukocyte Count , Mesenteric Arteries/pathology , Nitrates/blood , Nitrites/blood , Rats , Rats, Sprague-Dawley , Vascular Diseases/blood , Vascular Diseases/pathologyABSTRACT
Drug-induced renal injury is a common finding in the early preclinical phase of drug development. But the specific genes responding to renal injury remain poorly defined. Identification of drug-induced gene changes is critical to provide insights into molecular mechanisms and detection of renal damage. To identify genes associated with the development of drug-induced nephrotoxicity, a literature survey was conducted and a panel of 48 genes was selected based on gene expression changes in multiple published studies. Male Sprague-Dawley rats were dosed daily for 1, 3 or 5 days to the known nephrotoxicants gentamicin, bacitracin, vancomycin and cisplatin, or the known hepatotoxicants ketoconazole, 1-naphthyl isothiocyanate and 4,4-diaminodiphenylmethane. Histopathological evaluation and clinical chemistry revealed renal proximal tubular necrosis in rats treated with the nephrotoxicants, but not from those treated with the hepatotoxicants. RNA was extracted from the kidney, and RT-PCR was performed to evaluate expression profiles of the selected genes. Among the genes examined, 24 genes are confirmed to be highly induced or repressed in rats treated with nephrotoxicants; further investigation identified that 5 of the 24 genes were also altered by hepatotoxicants. These data led to the identification of a set of genomic biomarker candidates whose expression in kidney is selectively regulated only by nephrotoxicants. Among those genes displaying the highest expression changes specifically in nephrotoxicant-treated rats were kidney injury molecule 1 (Kim1), lipocalin 2 (Lcn2), and osteopontin (Spp1). The establishment of such a genomic marker set offers a new tool in our ongoing quest to monitor nephrotoxicity.
Subject(s)
Anti-Bacterial Agents/toxicity , Antineoplastic Agents/toxicity , Genetic Markers , Kidney Diseases/chemically induced , Kidney Diseases/genetics , Kidney/drug effects , Animals , Bacitracin/toxicity , Cisplatin/toxicity , Gene Expression/genetics , Gene Expression Profiling , Gentamicins/toxicity , Kidney/metabolism , Kidney/pathology , Kidney Diseases/metabolism , Kidney Diseases/pathology , Liver/drug effects , Liver/metabolism , Liver/pathology , Male , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Toxicogenetics , Vancomycin/toxicityABSTRACT
Phospholipidosis (PLD) is characterized by the excessive intracellular accumulation of phospholipids. It is well established that a large number of cationic amphiphilic drugs have the potential to induce PLD. In the present study, we describe two facile in vitro methods to determine the PLD-inducing potential of a molecule. The first approach is based on a recent study by (Sawada et al., 2005, Toxicol. Sci. 83, 282-292) in which 17 genes were identified as potential biomarkers of PLD in HepG2 cells. To confirm the utility of this gene panel, we treated HepG2 cells with PLD-positive and -negative compounds and then analyzed gene expression using real-time PCR. Our initial analysis, which used a single dose of each drug, correctly identified five of eight positive compounds and four of four negative compounds. We then increased the doses of the three false negatives (amiodarone, tamoxifen, and loratadine) and found that the changes in gene expression became large enough to correctly identify them as PLD-inducing drugs. Our results suggest that a range of concentrations should be used to increase the accuracy of prediction in this assay. Our second approach utilized a fluorescently labeled phospholipid (LipidTox) which was added to the media of growing HepG2 cells along with compounds positive and negative for PLD. Phospholipid accumulation was determined using confocal microscopy and, more quantitatively, using a 96-well plate assay and a fluorescent plate reader. Using an expanded set of compounds, we show that this assay correctly identified 100% of PLD-positive and -negative compounds. Dose-dependent increases in intracellular fluorescent phospholipid accumulation were observed. We found that this assay was less time consuming, more sensitive, and higher throughput than gene expression analysis. To our knowledge, this study represents the first validation of the use of LipidTox in identifying drugs that can induce PLD.