Warren E. Glaab

Plasma MicroRNAs as sensitive and specific biomarkers of tissue injury.

BACKGROUND MicroRNAs (miRNAs) are endogenous, small noncoding RNAs. Because of their size, abundance, tissue specificity, and relative stability in plasma, miRNAs hold promise as unique accessible biomarkers to monitor tissue injury. METHODS We investigated the use of liver-, muscle- and brain-specific miRNAs as circulating biomarkers of tissue injury. We used a highly sensitive quantitative PCR assay to measure specific miRNAs (miR-122, miR-133a, and miR-124) in plasma samples from rats treated with liver or muscle toxicants and from a rat surgical model of stroke. RESULTS We observed increases in plasma concentrations of miR-122, miR-133a, and miR-124 corresponding to injuries in liver, muscle, and brain, respectively. miR-122 and miR-133a illustrated specificity for liver and muscle toxicity, respectively, because they were not detectable in the plasma of animals with toxicity to the other organ. This result contrasted with the results for alanine aminotransferase (ALT) and aspartate aminotransferase, which were both increased with either organ toxicity. Furthermore, miR-122 exhibited a diagnostic sensitivity superior to that of ALT when the results were correlated to the liver histopathologic results. The miR-124 concentration increased in the plasma of rats 8 h after surgery to produce brain injury and peaked at 24 h, while the miR-122 and miR-133a concentrations remained at baseline values. CONCLUSIONS These results demonstrate that tissue-specific miRNAs may serve as diagnostically sensitive plasma biomarkers of tissue injury.

A rapid urine test for early detection of kidney injury

Kidney injury molecule-1 (Kim-1) has been qualified by the Food and Drug Administration and European Medicines Agency as a highly sensitive and specific urinary biomarker to monitor drug-induced kidney injury in preclinical studies and on a case-by-case basis in clinical trials. Here we report the development and evaluation of a rapid direct immunochromatographic lateral flow 15-min assay for detection of urinary Kim-1 (rat) or KIM-1 (human). The urinary Kim-1 band intensity using the rat Kim-1 dipstick significantly correlated with levels of Kim-1 as measured by a microbead-based assay, histopathological damage, and immunohistochemical assessment of renal Kim-1 in a dose- and time-dependent manner. Kim-1 was detected following kidney injury induced in rats by cadmium, gentamicin, or bilateral renal ischemia/reperfusion. In humans, the urinary KIM-1 band intensity was significantly greater in urine from patients with acute kidney injury than in urine from healthy volunteers. The KIM-1 dipstick also enabled temporal evaluation of kidney injury and recovery in two patients who developed postoperative acute kidney injury following cytoreductive surgery for malignant mesothelioma with intraoperative local cisplatin administration. We hope that future, more extensive studies will confirm the utility of these results, which show that the Kim-1/KIM-1 dipsticks can provide a sensitive and accurate detection of Kim-1/KIM-1, thereby providing a rapid diagnostic assay for kidney damage and facilitating the rapid and early detection of kidney injury in preclinical and clinical studies.

A panel of urinary biomarkers to monitor reversibility of renal injury and a serum marker with improved potential to assess renal function

The Predictive Safety Testing Consortiums first regulatory submission to qualify kidney safety biomarkers revealed two deficiencies. To address the need for biomarkers that monitor recovery from agent-induced renal damage, we scored changes in the levels of urinary biomarkers in rats during recovery from renal injury induced by exposure to carbapenem A or gentamicin. All biomarkers responded to histologic tubular toxicities to varied degrees and with different kinetics. After a recovery period, all biomarkers returned to levels approaching those observed in uninjured animals. We next addressed the need for a serum biomarker that reflects general kidney function regardless of the exact site of renal injury. Our assay for serum cystatin C is more sensitive and specific than serum creatinine (SCr) or blood urea nitrogen (BUN) in monitoring generalized renal function after exposure of rats to eight nephrotoxicants and two hepatotoxicants. This sensitive serum biomarker will enable testing of renal function in animal studies that do not involve urine collection.

Specificity of mutations induced by methyl methanesulfonate in mismatch repair-deficient human cancer cell lines.

Recently, we showed that the cytotoxic and mutagenic response in human cells to the model SN2 alkylating agent methyl methanesulfonate (MMS) can be modulated by the mismatch repair (MMR) pathway. That is, human cancer cell lines defective in MMR are more resistant to the cytotoxic effects of MMS exposure and suffer more induced mutations at the HPRT locus than MMR-proficient cell lines. Since MMS produces little O6-methylguanine (O6-meG), the observed hypermutability and resistance to cytotoxicity in MMR-defective cells likely results from lesions other than O6-meG. MMS produces a high yield of N7-methylguanine (N7-meG) and N3-methyladenine (N3-meA), which can lead to the formation of promutagenic abasic sites, and these lesions may be responsible for the observed cytotoxic and/or mutagenic effects of MMS. To further investigate the mechanism of MMS mutagenesis, two MMR-defective human cancer cell lines were treated with MMS and the frequency and the types of mutations produced at the HPRT locus were determined. MMS treatment (1.5 mM) produced a 1.6- and a 2.2-fold increase in mutations above spontaneous levels in HCT116 and DLD-1 cell lines, respectively. An average 3.7-fold increase in transversion mutations was observed, which accounted for greater than one-third of all induced mutations in both cell lines. In contrast, an average 1.6-fold increase was seen among transition mutations (the class expected from O-alkylation products). Since transversion mutations are not produced by O6-meG, these findings suggest that abasic sites may be the lesion responsible for a large proportion of MMS mutagenicity in MMR-defective cells. Furthermore, these data suggest the MMS-induced damage, either abasic site-inducing base alterations (i.e., N7-meG and N3-meA) or the resulting abasic sites themselves, may be substrates for recognition and/or repair by MMR proteins.

Evaluation of the Relative Performance of 12 Urinary Biomarkers for Renal Safety Across 22 Rat Sensitivity and Specificity Studies

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, β2-microglobulin, cystatin C, retinol binding protein 4, total protein, and N-acetyl-β-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.

A performance evaluation of three drug-induced liver injury biomarkers in the rat: alpha-glutathione S-transferase, arginase 1, and 4-hydroxyphenyl-pyruvate dioxygenase.

Alanine aminotransferase (ALT) activity is the most frequently relied upon reference standard for monitoring liver injury in humans and nonclinical species. However, limitations of ALT include a lack of specificity for diagnosing liver injury (e.g., present in muscle and the gastrointestinal tract), its inability to monitor certain types of hepatic injury (e.g., biliary injury), and ambiguity with respect to interpretation of modest or transient elevations (< 3× upper limit of normal). As an initial step to both understand and qualify additional biomarkers of hepatotoxicity that may add value to ALT, three novel candidates have been evaluated in 34 acute toxicity rat studies: (1) alpha-glutathione S-transferase (GSTA), (2) arginase 1 (ARG1), and (3) 4-hydroxyphenylpyruvate dioxygenase (HPD). The performance of each biomarker was assessed for its diagnostic ability to accurately detect hepatocellular injury (i.e., microscopic histopathology), singularly or in combination with ALT. All three biomarkers, either alone or in combination with ALT, improved specificity when compared with ALT alone. Hepatocellular necrosis and/or degeneration were detected by all three biomarkers in the majority of animals. ARG1 and HPD were also sensitive in detecting single-cell necrosis in the absence of more extensive hepatocellular necrosis/degeneration. ARG1 showed the best sensitivity for detecting biliary injury with or without ALT. All the biomarkers were able to detect biliary injury with single-cell necrosis. Taken together, these novel liver toxicity biomarkers, GSTA, ARG1, and HPD, add value (both enhanced specificity and sensitivity) to the measurement of ALT alone for monitoring drug-induced liver injury in rat.

Analysis of sequence alterations in a defined DNA region: comparison of temperature-modulated heteroduplex analysis and denaturing gradient gel electrophoresis.

The ability to detect DNA sequence heterogeneity quickly and reliably is becoming increasingly important as more genes involved in disease processes are discovered. We have assessed the ability of a high pressure liquid chromatography technique (HPLC) termed temperature-modulated heteroduplex analysis (TMHA) to detect a collection of 20 point mutations distributed throughout a 279 base pair fragment spanning the exon 8 region of the human HPRT gene. All mutant/wild type heteroduplexes formed from mutations in the lowest temperature melting domain of the fragment were easily resolved from the corresponding mutant and wt homoduplexes, while those generated from mutants in the next higher melting domain barely resolved from their parental homoduplexes. For comparison, identical heteroduplex samples were subjected to denaturing gradient gel electrophoresis (DGGE). Heteroduplexes in the lowest temperature melting domain were easily resolved, while no resolution was achieved with those in the next higher melting domain. These results suggest that TMHA and DGGE are measuring similar melting characteristics in heteroduplex molecules. TMHA appears to be a robust approach for detecting and/or purifying a wide variety of mutations in a defined region of DNA, provided that the melting characteristics of the fragment under study are carefully considered.

Evaluation of the Relative Performance of Drug-Induced Skeletal Muscle Injury Biomarkers in Rats

Novel skeletal muscle (SKM) injury biomarkers that have recently been identified may outperform or add value to the conventional SKM injury biomarkers aspartate transaminase (AST) and creatine kinase (CK). The relative performance of these novel biomarkers of SKM injury including skeletal troponin I (sTnI), myosin light chain 3 (Myl3), CK M Isoform (Ckm), and fatty acid binding protein 3 (Fabp3) was assessed in 34 rat studies including both SKM toxicants and compounds with toxicities in tissues other than SKM. sTnI, Myl3, Ckm, and Fabp3 all outperformed CK or AST and/or added value for the diagnosis of drug-induced SKM injury (ie, myocyte degeneration/necrosis). In addition, when used in conjunction with CK and AST, sTnI, Myl3, CKm, and Fabp3 individually and collectively improved diagnostic sensitivity and specificity, as well as diagnostic certainty, for SKM injury and responded in a sensitive manner to low levels of SKM degeneration/necrosis in rats. These findings support the proposal that sTnI, Myl3, Ckm, and Fabp3 are suitable for voluntary use, in conjunction with CK and AST, in regulatory safety studies in rats to monitor drug-induced SKM injury and the potential translational use of these exploratory biomarkers in early clinical trials to ensure patient safety.

Toxicogenomics in drug development: a match made in heaven?

Compound toxicity accounts for approximately half of all drug failures during development. Currently accepted preclinical studies for drug safety evaluation are time, resource, and animal intensive with often limited clinical predictivity. It is thus highly desirable to develop more efficient and predictive tools for early detection and assessment of potential compound liabilities. The emergence of genomics technologies over the last two decades promised to provide a solution. The premise of toxicogenomics (TGx) is straight forward: compounds with similar toxicity mechanisms and outcomes should perturb the transcriptome similarly and these perturbations could be used as more efficient and/or more predictive biomarkers of downstream toxicity outcome. This concept was reinforced by a number of pioneering studies demonstrating, for example, strong correlations between histopathology, clinical chemistry, and gene expression when different hepatocellular injuries were induced by chemical agents as reviewed in.[1,2] With such early advances, TGx was poised for earlier detection of a vast variety of drug-related outcomes, covering histopathologies across various organs, carcinogenicity, reproductive toxicity, etc., while deciphering mechanisms of action to create a more predictive and resource-sparing battery of tests for hazard identification, risk assessment, toxicity monitoring, and problem-solving across the drug development pipeline. This paradigm shift was anticipated to liberate the pharmaceutical and chemical industries from the current burden of toxicity liabilities, by enabling faster development of clinically safer compounds while reducing cost, infrastructure, and animal requirements.[1–3] TGx and drug discovery/development was expected to be a match made in heaven.

Response of Novel Skeletal Muscle Biomarkers in Dogs to Drug-Induced Skeletal Muscle Injury or Sustained Endurance Exercise

The skeletal muscle (SKM) injury biomarkers, skeletal troponin I (sTnI), myosin light chain 3 (Myl3), and creatine kinase muscle isoform (Ckm) have been shown recently to be more sensitive and specific for monitoring drug-induced SKM injury than the conventional biomarkers, aspartate transaminase (AST) and creatine kinase (CK) enzymatic assays in rat toxicology studies. To evaluate the utility of these SKM biomarkers across species, they were assessed in 2 dog models: a drug-induced injury study in Beagle dogs and a 160 km endurance exercise run completed by Alaskan sled dogs. In the drug-induced injury model, mean sTnI and Myl3 plasma levels were 6- and 18-fold, respectively, compared with baseline as early as Study Day (SD) 15, while mean plasma AST and CK levels did not increase, and biopsy samples were non-remarkable for histopathology prior to SD 29 when degeneration was first noted. Peak group mean plasma responses over baseline for sTnI, Myl3, and Ckm biomarkers were 96-, 103-, and 11-fold, respectively, compared with 2.5-fold for AST and 3.8-fold for CK-enzymatic (CK-enz) assay. In the sled dog sustained exercise model, the peak response for all biomarkers was observed at the first sampling (2 h) after the completion of the run. The sTnI, Myl3, and Ckm mean fold peak values compared with baseline were 170-, 120-, and 150-fold, respectively, while AST increased 7-fold and CK-enz increased 29-fold. These findings support the conclusion that sTnI, Myl3, and Ckm are sensitive early tissue leakage biomarkers for monitoring SKM injury and effects of exercise in dog, extending their utility across preclinical species beyond the rat, and provide further support to investigate their translational utility to clinical trial settings to monitor for drug-induced SKM injury and ensure patient safety.