Rapid and Definitive Identification of Cyclic Peptide Soft Spots by Isotope-Labeled Reductive Dimethylation and Mass Spectrometry Fragmentation.

Cyclic peptides are an emerging therapeutic modality over the past few decades. To identify drug candidates with sufficient proteolytic stability for oral administration, it is critical to pinpoint the amide bond hydrolysis sites, or soft spots, to better understand their metabolism and provide guidance on further structure optimization. However, the unambiguous characterization of cyclic peptide soft spots remains a significant challenge during early stage discovery studies, as amide bond hydrolysis forms a linearized isobaric sequence with the addition of a water molecule, regardless of the amide hydrolysis location. In this study, an innovative strategy was developed to enable the rapid and definitive identification of cyclic peptide soft spots by isotope-labeled reductive dimethylation and mass spectrometry fragmentation. The dimethylated immonium ion with enhanced MS signal at a distinctive m/z in MS/MS fragmentation spectra reveals the N-terminal amino acid on a linearized peptide sequence definitively and, thus, significantly simplifies the soft spot identification workflow. This approach has been evaluated to demonstrate the potential of isotope-labeled dimethylation to be a powerful analytical tool in cyclic peptide drug discovery and development.

The application of cytokeratin-18 as a biomarker for drug-induced liver injury.

Drug-induced liver injury (DILI) is a frequent and dangerous adverse effect faced during preclinical and clinical drug therapy. DILI is a leading cause of candidate drug attrition, withdrawal and in clinic, is the primary cause of acute liver failure. Traditional diagnostic markers for DILI include alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP). Yet, these routinely used diagnostic markers have several noteworthy limitations, restricting their sensitivity, specificity and accuracy in diagnosing DILI. Consequently, new biomarkers for DILI need to be identified.A potential biomarker for DILI is cytokeratin-18 (CK18), an intermediate filament protein highly abundant in hepatocytes and cholangiocytes. Extensively researched in a variety of clinical settings, both full length and cleaved forms of CK18 can diagnose early-stage DILI and provide insight into the mechanism of hepatocellular injury compared to traditionally used diagnostic markers. However, relatively little research has been conducted on CK18 in preclinical models of DILI. In particular, CK18 and its relationship with DILI is yet to be characterised in an in vivo rat model. Such characterization of CK18 and ccCK18 responses may enable their use as translational biomarkers for hepatotoxicity and facilitate management of clinical DILI risk in drug development. The aim of this review is to discuss the application of CK18 as a biomarker for DILI. Specifically, this review will highlight the properties of CK18, summarise clinical research that utilised CK18 to diagnose DILI and examine the current challenges preventing the characterisation of CK18 in an in vivo rat model of DILI.

Can Galactose Be Converted to Glucose in HepG2 Cells? Improving the in Vitro Mitochondrial Toxicity Assay for the Assessment of Drug Induced Liver Injury.

Human hepatocellular carcinoma cells, HepG2, are often used for drug mediated mitochondrial toxicity assessments. Glucose in HepG2 culture media is replaced by galactose to reveal drug-induced mitochondrial toxicity as a marked shift of drug IC50 values for the reduction of cellular ATP. It has been postulated that galactose sensitizes HepG2 mitochondria by the additional ATP consumption demand in the Leloir pathway. However, our NMR metabolomics analysis of HepG2 cells and culture media showed very limited galactose metabolism. To clarify the role of galactose in HepG2 cellular metabolism, U-13C6-galactose or U-13C6-glucose was added to HepG2 culture media to help specifically track the metabolism of those two sugars. Conversion to U-13C3-lactate was hardly detected when HepG2 cells were incubated with U-13C6-galactose, while an abundance of U-13C3-lactate was produced when HepG2 cells were incubated with U-13C6-glucose. In the absence of glucose, HepG2 cells increased glutamine consumption as a bioenergetics source. The requirement of additional glutamine almost matched the amount of glucose needed to maintain a similar level of cellular ATP in HepG2 cells. This improved understanding of galactose and glutamine metabolism in HepG2 cells helped optimize the ATP-based mitochondrial toxicity assay. The modified assay showed 96% sensitivity and 97% specificity in correctly discriminating compounds known to cause mitochondrial toxicity from those with prior evidence of not being mitochondrial toxicants. The greatest significance of the modified assay was its improved sensitivity in detecting the inhibition of mitochondrial fatty acid ß-oxidation (FAO) when glutamine was withheld. Use of this improved assay for an empirical prediction of the likely contribution of mitochondrial toxicity to human DILI (drug induced liver injury) was attempted. According to testing of 65 DILI positive compounds representing numerous mechanisms of DILI together with 55 DILI negative compounds, the overall prediction of mitochondrial mechanism-related DILI showed 25% sensitivity and 95% specificity.

Vascular Imaging of Matrix Metalloproteinase Activity as an Informative Preclinical Biomarker of Drug-induced Vascular Injury.

Lack of biomarkers specific to and either predictive or diagnostic of drug-induced vascular injury (DIVI) continues to be a major obstacle during drug development. Biomarkers derived from physiologic responses to vessel injury, such as inflammation and vascular remodeling, could make good candidates; however, they characteristically lack specificity for vasculature. We evaluated whether vascular remodeling-associated protease activity, as well as changes to vessel permeability resulting from DIVI, could be visualized ex vivo in affected vessels, thereby allowing for visual monitoring of the pathology to address specificity. We found that visualization of matrix metalloproteinase activation accompanied by increased vascular leakage in the mesentery of rats treated with agents known to induce vascular injury correlated well with incidence and severity of histopathological findings and associated inflammation as well as with circulating levels of tissue inhibitors of metalloproteinase 1 and neutrophil gelatinase-associated lipocalin. The weight of evidence approach reported here shows promise as a composite DIVI preclinical tool by means of complementing noninvasive monitoring of circulating biomarkers of inflammation with direct imaging of affected vasculature and thus lending specificity to its interpretation. These findings are supportive of a potential strategy that relies on translational imaging tools in conjunction with circulating biomarker data for high-specificity monitoring of VI both preclinically and clinically.

Optical imaging biomarkers of drug-induced vascular injury.

Drug-induced vascular injury (DIVI), defined as arterial medial degeneration/necrosis usually associated with perivascular inflammation, is frequently observed in the mesenteric arteries of rats but the relevance to humans remains a hurdle for drug development. Here, we describe the evaluation of commercially available optical imaging biomarkers using a rat DIVI model. Male Sprague Dawley rats were administered 10 mg/kg/day of a proprietary soluble guanylate cyclase activator (sGCa). Optical agents, AngioSense for the detection of vessel permeability, MMPSense for the detection of activated matrix metalloproteinases (MMPs), and IntegriSense for the detection of αvß3 integrin, were injected via tail vein 24 hours before fluorescence (FL) ex vivo imaging. Imaging found a statistically significant difference in FL from all optical agents between treated and vehicle groups (p < .05). Mesenteric arteries were further analyzed by histopathology, flow cytometry, and immunohistochemistry. Histopathology confirmed perivascular inflammation and/or arterial medial degeneration in the sGCa-treated animals. Flow cytometry of digested arteries revealed myeloid cells as a main source of MMPSense signal. Immunohistochemical analysis further identified elevated MMP-9 expression within arterial walls and surrounding tissue of treated animals. Together, these data demonstrate that MMPSense and AngioSense are sensitive optical imaging biomarkers for the quantification of DIVI in rat mesenteric arteries.

Nonclinical safety biomarkers of drug-induced vascular injury: current status and blueprint for the future.

Better biomarkers are needed to identify, characterize, and/or monitor drug-induced vascular injury (DIVI) in nonclinical species and patients. The Predictive Safety Testing Consortium (PSTC), a precompetitive collaboration of pharmaceutical companies and the U.S. Food and Drug Administration (FDA), formed the Vascular Injury Working Group (VIWG) to develop and qualify translatable biomarkers of DIVI. The VIWG focused its research on acute DIVI because early detection for clinical and nonclinical safety monitoring is desirable. The VIWG developed a strategy based on the premise that biomarkers of DIVI in rat would be translatable to humans due to the morphologic similarity of vascular injury between species regardless of mechanism. The histomorphologic lexicon for DIVI in rat defines degenerative and adaptive findings of the vascular endothelium and smooth muscles, and characterizes inflammatory components. We describe the mechanisms of these changes and their associations with candidate biomarkers for which advanced analytical method validation was completed. Further development is recommended for circulating microRNAs, endothelial microparticles, and imaging techniques. Recommendations for sample collection and processing, analytical methods, and confirmation of target localization using immunohistochemistry and in situ hybridization are described. The methods described are anticipated to aid in the identification and qualification of translational biomarkers for DIVI.

Universal Accessible Biomarkers of Drug-Induced Tissue Injury and Systemic Inflammation in Rat: Performance Assessment of TIMP-1, A2M, AGP, NGAL, and Albumin.

The ability to monitor for general drug-induced tissue injury (DITI) or systemic inflammation in any tissue using blood-based accessible biomarkers would provide a valuable tool in early exploratory animal studies to understand potential drug liabilities. Here we describe the evaluation of 4 biomarkers of tissue remodeling and inflammation (α2-macroglobulin [A2M], α1-acid glycoprotein [AGP], neutrophil gelatinase-associated lipocalin [NGAL], and tissue inhibitor of metalloproteinases [TIMP-1]) as well as the traditional serum parameter albumin as potential blood-based biomarkers of DITI and systemic inflammatory response (SIR). Biomarker performance was assessed in 51 short-term rat in vivo studies with various end-organ toxicities or SIR and receiver operating characteristic curves were generated to compare relative performances. All 4 biomarkers performed well in their ability to detect DITI and SIR with an area under the curve (AUC) of 0.82-0.78, however TIMP-1 achieved the best sensitivity (at 95% specificity) of 61%; AGP, NGAL, and A2M sensitivity was 51%-52%. AUC for albumin was 0.72 with sensitivity of 39%. A2M was the best performer in studies with only SIR (AUC 0.91). In the subset of studies with drug-induced vascular injury, TIMP-1 performed best with an AUC of 0.96. Poor performance of all tested biomarkers was observed in samples with CNS toxicity. In summary, TIMP-1, A2M, AGP, and NGAL demonstrated performance as sensitive accessible biomarkers of DITI and SIR, supporting their potential application as universal accessible tissue toxicity biomarkers to quickly identify dose levels associated with drug-induced injury in early exploratory rat safety and other studies.

Use of a Bile Salt Export Pump Knockdown Rat Susceptibility Model to Interrogate Mechanism of Drug-Induced Liver Toxicity.

Inhibition of the bile salt export pump (BSEP) may be associated with clinical drug-induced liver injury, but is poorly predicted by preclinical animal models. Here we present the development of a novel rat model using siRNA knockdown (KD) of Bsep that displayed differentially enhanced hepatotoxicity to 8 Bsep inhibitors and not to 3 Bsep noninhibitors when administered at maximally tolerated doses for 7 days. Bsep KD alone resulted in 3- and 4.5-fold increases in liver and plasma levels, respectively, of the sum of the 3 most prevalent taurine conjugated bile acids (T3-BA), approximately 90% decrease in plasma and liver glycocholic acid, and a distinct bile acid regulating gene expression pattern, without resulting in hepatotoxicity. Among the Bsep inhibitors, only asunaprevir and TAK-875 resulted in serum transaminase and total bilirubin increases associated with increases in plasma T3-BA that were enhanced by Bsep KD. Benzbromarone, lopinavir, and simeprevir caused smaller increases in plasma T3-BA, but did not result in hepatotoxicity in Bsep KD rats. Bosentan, cyclosporine A, and ritonavir, however, showed no enhancement of T3-BA in plasma in Bsep KD rats, as well as Bsep noninhibitors acetaminophen, MK-0974, or clarithromycin. T3-BA findings were further strengthened through monitoring TCA-d4 converted from cholic acid-d4 overcoming interanimal variability in endogenous bile acids. Bsep KD also altered liver and/or plasma levels of asunaprevir, TAK-875, TAK-875 acyl-glucuronide, benzbromarone, and bosentan. The Bsep KD rat model has revealed differences in the effects on bile acid homeostasis among Bsep inhibitors that can best be monitored using measures of T3-BA and TCA-d4 in plasma. However, the phenotype caused by Bsep inhibition is complex due to the involvement of several compensatory mechanisms.

Expression and activities of several drug-metabolizing enzymes in LLC-PK1 cells.

LLC-PK1 cells are frequently used in toxicology research, but little information is available concerning the capacity of these cells to metabolize xenobiotics. We examined the expression and activities of cytochromes P450 (P450) 1A1/1A2 (CYP 1A1/1A2), 2E1 (CYP 2E1), flavin monooxygenase (FMO), 5-lipoxygenase (5-LO) and prostaglandin H synthase (PHS)-associated cyclooxygenase-1 (COX-1). We prepared S9 fractions from LLC-PK1 cells, rat liver, and rat kidney, and measured enzyme activities using ethoxyresorufin O-deethylation (EROD) for CYP 1A1/1A2 and ethoxycoumarin O-deethylation (ECOD) for CYP 2E1, benzydamine N-oxidation (BNO) for FMO, leukotriene B(4) (LTB(4)) formation for 5-LO, and thromboxane B(2) (TXB(2)) formation for COX-1 activities. To assure that product formation was due to enzymatic activity, we used the following inhibitors: 1-aminobenzotriazole (ABT) for P450, methimazole for FMO, caffeic acid for 5-LO and acetylsalicylic acid (ASA) for COX-1. We also performed Western blot analysis to confirm our observations. All five enzyme activities were demonstrable in rat liver at much greater levels than in rat kidney S9 fractions. Activities in LLC-PK1 cells were significantly lower than activities in rat liver S9 fraction and generally less than activities in rat kidney S9 fraction. Enzyme inhibitors decreased product formation in all three tissues and Western blot analysis supported our observations of low enzyme activity in LLC-PK1 cells. These results indicate that LLC-PK1 cells have very low content of relevant drug-metabolizing enzyme activities.

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.