Higher Urine bis(Monoacylglycerol)Phosphate Levels in LRRK2 G2019S Mutation Carriers: Implications for Therapeutic Development.

BACKGROUND: LRRK2 mutations are a common cause of dominantly inherited PD. Previous studies showed decreases in urine levels of didocohexaenoyl (22:6) bis(monoacylglycerol)phosphate in LRRK2-knockout mice and in non-human primates treated with LRRK2 kinase inhibitors. We hypothesized that urine levels of bis(monoacylglycerol)phosphate isoforms will be higher in individuals with a PD-causing gain-of-kinase function mutation, LRRK2 G2019S. The objective of this study was to investigate alterations in urinary phospholipids as biomarkers of LRRK2 mutations and Parkinson's disease status/phenotypes. METHODS: Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to assess 54 bioactive phospholipids in urine from the LRRK2 Cohort Consortium (n = 80). To confirm and extend the findings, urine from an independent LRRK2 cohort from Columbia University Irving Medical Center (n = 116) was used. Both cohorts were composed of LRRK2 G2019S carriers and non-carriers with and without PD. RESULTS: In each cohort, 4 bis(monoacylglycerol)phosphate isoforms (di-18:1-bis[monoacylglycerol]phosphate, didocohexaenoyl [22:6] bis[monoacylglycerol] phosphate, 2,2'-di-22:6-bis[monoacylglycerol]phosphate, and 2,2'-di-18:1-bis[monoacylglycerol]phosphate) were significantly higher (2.5- to 4.3-fold) in G2019S carriers compared with non-carriers. Interestingly, 2,2'-di-18:1-bis(monoacylglycerol)phosphate levels were marginally higher in LRRK2 carriers with PD than in those without PD (P = 0.045). Moreover, increased 2,2' and total di-22:6-bis(monoacylglycerol)phosphate were associated with worse cognitive status assessed by the Montreal Cognitive Assessment (P = 0.0033 and 0.0144, respectively). CONCLUSIONS: The observed association of bis(monoacylglycerol)phosphate isoforms with LRRK2 G2019S mutation, PD status among G2019S carriers, and correlation with cognitive decline suggest the potential use of urinary bis(monoacylglycerol)phosphate isoforms as biomarkers for clinical trials of LRRK2-targeted therapies. © 2019 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.

Validation of a multiplexed LC-MS/MS clinical assay to quantify insulin-like growth factor-binding proteins in human serum and its application in a clinical study.

Circulating insulin-like growth factor-binding proteins (IGFBPs) continue to gain attention as biomarkers of drug activities on insulin like growth factor (IGF)/IGF receptor signaling pathways. A multiplexed LC-MS/MS method was validated for the absolute quantitation of IGFBPs in human serum. The method was used to measure screening concentrations of IGFBPs in spinal and bulbar muscular atrophy (SBMA) patients in a phase 2 clinical trial. Concentrations of IGFBP 1, 2, 3, and 5 were simultaneously determined based on representative signature peptides derived from an optimized trypsin digestion procedure. Signature peptide levels were absolutely quantitated using a sensitive/specific targeted LC-MS/MS method. Corresponding mass-shifted, stable isotope-labeled peptides were employed as internal standards. A true blank matrix for the quantitation of IGFBPs was not available since they are endogenous proteins in human serum. In this method, calibration standards/curves were prepared using authentic synthetic peptides spiked into a surrogate matrix. The surrogate matrix was generated from human serum treated in the same way as the study samples, but using iodoacetic acid instead of iodoacetamide as the alkylation reagent. This surrogate matrix approach allowed for the direct and sensitive/specific quantification of IGFBP 1, 2, 3, and 5 due to the lack of any endogenous background. Equivalent matrix effect and recovery of analytes was achieved for the authentic and surrogate matrices. The fully validated LC-MS/MS assay will allow further evaluation of the utility of IGFBP biomarkers in clinical trials.

A multiplexed UPLC-MS/MS assay for the simultaneous measurement of urinary safety biomarkers of drug-induced kidney injury and phospholipidosis.

Drug-induced kidney injury (DIKI) is a major concern in drug risk assessment given its clinical importance and the absence of a sensitive/specific method of diagnosis. Pharmaceutical regulatory agencies have qualified and issued letters of support for new biomarkers to better evaluate DIKI in nonclinical toxicity and clinical studies. Additional efforts have focused on drug-induced phospholipidosis (DIPL) and its potential link with collateral renal damage. The combined use of urinary biomarkers is an efficient way to evaluate renal safety in nonclinical and clinical studies. Eight FDA/EMA/PMDA qualified (or supported) urinary biomarkers, including kidney injury molecule-1 (KIM-1), ß2-microglobulin (B2M), clusterin (CLU), cystatin C (CysC), trefoil factor 3 (TFF3), neutrophil gelatinase-associated lipocalin (NGAL), osteopontin (OPN), and alpha-glutathione S-transferase (α-GST), were quantified by multiplex UPLC-MS/MS in a repeat dose study of gentamicin in rats. Rats administered gentamicin at 100 mg/kg/day for 2 weeks developed renal lesions detected by histopathology. Biomarkers of tubular damage (CLU, KIM-1, OPN) increased 9.8, 34.7, and 35.6-fold (relative to concurrent controls), respectively, after 2 weeks of dosing. Biomarkers of glomerular damage and/or impairment of tubular reabsorption (CysC, B2M) increased 11.7 and 22.6-fold. NGAL and α-GST increased <3-fold after 2 weeks of dosing. TFF3 was comparable to concurrent controls. The elevated biomarker concentrations met PSTC threshold criteria and were consistent with mechanisms of gentamicin nephrotoxicity. Increased urinary di-22:6-BMP indicated concomitant DIPL as confirmed by TEM. This work provides evidence supporting the combined use of the DIKI biomarker panel and di-22:6-BMP as a biomarker of DIPL in drug risk assessment.

Di-22:6-bis(monoacylglycerol)phosphate: A clinical biomarker of drug-induced phospholipidosis for drug development and safety assessment.

The inability to routinely monitor drug-induced phospholipidosis (DIPL) presents a challenge in pharmaceutical drug development and in the clinic. Several nonclinical studies have shown di-docosahexaenoyl (22:6) bis(monoacylglycerol) phosphate (di-22:6-BMP) to be a reliable biomarker of tissue DIPL that can be monitored in the plasma/serum and urine. The aim of this study was to show the relevance of di-22:6-BMP as a DIPL biomarker for drug development and safety assessment in humans. DIPL shares many similarities with the inherited lysosomal storage disorder Niemann-Pick type C (NPC) disease. DIPL and NPC result in similar changes in lysosomal function and cholesterol status that lead to the accumulation of multi-lamellar bodies (myeloid bodies) in cells and tissues. To validate di-22:6-BMP as a biomarker of DIPL for clinical studies, NPC patients and healthy donors were classified by receiver operator curve analysis based on urinary di-22:6-BMP concentrations. By showing 96.7-specificity and 100-sensitivity to identify NPC disease, di-22:6-BMP can be used to assess DIPL in human studies. The mean concentration of di-22:6-BMP in the urine of NPC patients was 51.4-fold (p ≤ 0.05) above the healthy baseline range. Additionally, baseline levels of di-22:6-BMP were assessed in healthy non-medicated laboratory animals (rats, mice, dogs, and monkeys) and human subjects to define normal reference ranges for nonclinical/clinical studies. The baseline ranges of di-22:6-BMP in the plasma, serum, and urine of humans and laboratory animals were species dependent. The results of this study support the role of di-22:6-BMP as a biomarker of DIPL for pharmaceutical drug development and health care settings.

Elevated urine BMP phospholipids in LRRK2 and VPS35 mutation carriers with and without Parkinson's disease.

Elevated urine bis(monoacylglycerol)phosphate (BMP) levels have been found in gain-of-kinase function LRRK2 G2019S mutation carriers. Here, we have expanded urine BMP analysis to other Parkinson's disease (PD) associated mutations and found them to be consistently elevated in carriers of LRRK2 G2019S and R1441G/C as well as VPS35 D620N mutations. Urine BMP levels are promising biomarkers for patient stratification and potentially target engagement in clinical trials of emerging targeted PD therapies.

Elucidation of potential bortezomib response markers in mutliple myeloma patients.

Liquid chromatography coupled to mass spectrometry (LC/MS) was used to elucidate early biomarkers of bortezomib response in multiple myeloma patients. The change in serum myeloma M-protein level, maintained for a minimum of 6 weeks, is used as one of the main criteria to evaluate patient clinical response to therapy. The objective of this study was to identify biomarkers using LC/MS in order to predict patient response to bortezomib sooner and more accurately compared to serum M-protein levels. The plasma LC/MS biomolecular/biochemical profiles, comprised of thousands of endogenous small molecules, peptides and proteins, were determined for 10 multiple myeloma patients at predose and 24 h after initial dosing with bortezomib. The comparative analysis of the metabolic profiles of non-responders and partial responders provided an opportunity to investigate mechanisms related to disease progression and identify biomarkers related to drug response. The plasma levels of two potential efficacy response markers were significantly more abundant in the non-responsive patients compared to the responders at 24-h postdose. The potential response biomarkers, apolipoprotein C-I and apolipoprotein C-I', were identified by mass spectral analyses and confirmed by authentic protein standards based on MALDI-TOF MS/MS sequencing of proteolytic peptides.

Bis(monoacylglycerol)phosphate as a non-invasive biomarker to monitor the onset and time-course of phospholipidosis with drug-induced toxicities.

IMPORTANCE TO THE FIELD: Drug-induced phospholipidosis (PL) is a phospholipid storage disorder characterized by the accumulation of multi-lamellar bodies (myeloid bodies) in tissues. A major unanswered question is whether PL represents a benign adaptive response, symptom or early event in drug toxicity. The absence of a non-invasive biomarker to monitor tissue PL has made it difficult to determine the prevalence and implications of PL in the clinic. As a result, the interpretation of PL in risk assessment remains uncertain in preclinical and clinical drug development. AREAS COVERED IN THIS REVIEW: This review describes the rationale for bis(monoacylglycerol)phosphate (BMP) as a biomarker of PL and explores the potential links between PL and the toxicities of drugs. WHAT THE READER WILL GAIN: The similarities between the hypothesized roles of BMP in PL and Niemann-Pick type C disease are discussed. The potential implications of PL for cellular function are described in the context of drug-induced QT prolongation, myopathy and renal toxicity. TAKE HOME MESSAGE: A specific species of BMP, di-docosahexaenoyl-BMP, should be investigated further as a non-invasive biomarker to monitor the onset and time course of PL and to better understand the functional consequences which could contribute to the toxicities of drugs.

Toxicological protein biomarker analysis--an investigative one-week single dose intravenous infusion toxicity and toxicokinetic study in cynomolgus monkeys using an antibody-cytotoxic conjugate against ovarian cancer.

INTRODUCTION: Antibody-cytotoxic conjugates are complex novel therapeutic agents whose toxicological properties are not presently well understood. The objective of this study was to identify toxicological markers in serum that correlate with MLN8866 (an antibody-cytotoxic conjugate) exposure and related pathological events in monkeys. MATERIALS AND METHODS: Cynomolgus monkeys were treated once with 5, 15, or 30 mg/kg MLN8866 via a 20 min intravenous infusion. MLN8866 exposure (Cmax and AUCO-4 day) was determined by quantifying MLN8866 levels in serum. RESULTS: The increase in MLN8866 exposure was approximately dose proportional. Two acute phase proteins in serum (serum amyloid A and haptoglobin) were correlated with MLN8866 exposure and toxicological outcomes (e.g., erythropoiesis and leucopoiesis).

Drug safety evaluation through biomarker analysis--a toxicity study in the cynomolgus monkey using an antibody-cytotoxic conjugate against ovarian cancer.

Antibody-cytotoxin conjugates are complex novel therapeutic agents whose toxicological properties are not presently well understood. The objective of this study was to identify serum biomarkers that correlate with MLN8866 (an Antibody-Cytotoxic Conjugate, mAb8866-CT) pathological events in monkeys and to predict the maximal tolerated dose (MTD) level using biomarkers. Cynomolgus monkeys were administered a single dose MLN8666 (5, 15 or 30 mg/kg) by intravenous infusion and evaluated over a 7-day period. Exposure levels were determined by quantifying MLN8866 levels (Cmax and AUC(0-96 h)) in serum. The increase in MLN8866 Cmax and AUC(0-96 h) was approximately dose proportional. Two biomarkers in serum (m/z 316 and m/z 368) were identified to be correlated with MLN8866 toxicological outcomes. The predicted MTD, 11.4 mg/kg, was within the MTD range set by pathology results (5-15 mg/kg). Administration of MLN8866 at 15 mg/kg and 30 mg/kg dose levels resulted in changes in hematology parameters associated with impaired hematopoiesis and bone marrow toxicity. The projected MLN8866 MTD exposure level was integrated with toxicokinetic analysis and showed Cmax=236 microg/mL and AUC(0-96 h)=7246 h mg/mL. The safety of three different MLN8866 dosing regimens with three dosing schedules was explored with pharmacokinetic modeling.