Evaluation of a Benzodiazepine Immunoassay for Urine Drug Testing in Clinical Specimens.

BACKGROUND: Benzodiazepines are commonly prescribed medications frequently linked to instances of abuse and overdose. Historically, FDA-cleared benzodiazepine urine immunoassays cross-react poorly with glucuronidated benzodiazepine metabolites, leading to false negatives. Clinical laboratories have addressed this deficiency by creating laboratory-developed tests (LDTs) that incorporate a beta-glucuronidase hydrolysis step to increase the clinical sensitivity of these assays. METHODS: Performance characteristics of 2 FDA-cleared benzodiazepine urine immunoassays (Benzodiazepines Plus, no glucuronidase and Benzodiazepines II, with glucuronidase; Roche Diagnostics) and a previously described benzodiazepine immunoassay LDT (with glucuronidase) were evaluated using 258 clinical urine specimens. The positive immunoassay cutoff was set at 200 ng/mL of nordiazepam and results were compared to an LC-MS/MS benzodiazepine LDT. Clinical sensitivity, specificity, precision, and immunoassay cross-reactivity were determined for all 3 immunoassays. RESULTS: The Benzodiazepines II and LDT immunoassays exhibited greater clinical sensitivity (100% and 95.2%) compared to the Benzodiazepines Plus assay (66.7%). Clinical specificity of 100% was observed for all 3 assays. Immunoassay response of the Benzodiazepines II assay was greater across the range of concentrations tested (100-1000 ng/mL) relative to the other immunoassays and was the most sensitive immunoassay for the detection of lorazepam glucuronide. CONCLUSIONS: The Benzodiazepines II immunoassay demonstrated the greatest clinical and analytical sensitivity compared to the Benzodiazepines Plus and LDT immunoassays. The incorporation of beta-glucuronidase was crucial, as the Benzodiazepines II and LDT immunoassays demonstrated superior clinical sensitivity when compared to the Benzodiazepines Plus immunoassay that does not incorporate a beta-glucuronidase hydrolysis step.

Evaluating the performance of the Roche FEN2 fentanyl immunoassay and its clinical implementation: The role of LDT-based mass spectrometry testing.

Introduction: While laboratory-developed tests (LDTs) using liquid chromatography tandem mass spectrometry (LC-MS/MS) are widely employed to support the development of FDA-cleared drug immunoassays, their significance in the clinical implementation and evaluation of such assays is often overlooked. This paper reports on the important role of LC-MS/MS LDTs in demonstrating improved performance of the Roche FEN2 fentanyl immunoassay compared with the Thermo DRI fentanyl immunoassay. Methods: The FEN2 assay was implemented according to the manufacturer's instructions and its performance was compared to the existing DRI assay using LC-MS/MS as a reference. Clinical sensitivity and specificity were determined using 250 consecutive random patient specimens. Spiking experiments were conducted to determine cross-reactivity with 31 fentanyl analogs. Select DRI false-positive samples were analyzed by the FEN2 assay via time-of-flight mass spectrometry method (LC-QTOF). Results: The FEN2 assay showed improved clinical sensitivity compared to the DRI (98% vs 61%) in 250 consecutive patient samples due to its ability to detect norfentanyl. It also showed better clinical specificity by correctly classifying select DRI false-positive results. Upon implementation in clinical practice, the FEN2 resulted in a higher screening positivity rate than the DRI (17.3% vs 13.3%) and a greater LC-MS/MS confirmation rate of immunoassay-positive samples (96.8% vs 88.8%, respectively). Conclusion: The use of LC-MS/MS LDTs demonstrated that the FEN2 assay has greater clinical sensitivity and is less prone to false-positives than the DRI assay. These findings support the use of FEN2 in routine clinical practice and emphasize the role of mass spectrometry-based LDTs in clinical toxicology testing.

Comparison of two highly sensitive benzodiazepine immunoassay lab developed tests for urine drug testing in clinical specimens.

Background: The VALID Act is a legislative effort that, if enacted, would alter the regulatory requirements of laboratory developed tests (LDTs) used for clinical testing in the United States. Benzodiazepines, which are primarily excreted into urine as glucuronidated metabolites such as lorazepam, cross-react poorly with FDA-cleared immunoassays, leading to false-negatives. This shortfall can be addressed with LDTs created by adding glucuronidase to the immunoassay reagents producing "high sensitivity" assays that detect glucuronidated metabolites. Methods: Precision and stability of two high-sensitivity (HS) benzodiazepine immunoassays from Roche and Thermo Scientific were evaluated using manufacturer-supplied quality control (QC) material and glucuronidated QC material. The immunoassays were directly compared to an LC-MS/MS LDT benzodiazepine assay to determine clinical sensitivity/specificity using urine specimens (n = 82 for Thermo Scientific; n = 265 for Roche). The clinical impact of the HS LDT immunoassay was determined by analyzing clinical testing results 60 days before and after its implementation. Results: The precision and clinical sensitivity/specificity of the HS-Thermo Scientific and HS-Roche benzodiazepine assays were acceptable. The reagent stability of the HS-Thermo Scientific immunoassay was poor, whereas the HS-Roche immunoassay was stable. After implementation of the HS-Roche benzodiazepine immunoassay as an LDT, there was a 30-fold increase (p-value: < 0.00001) in the percentage of lorazepam confirmations. Conclusions: We demonstrate the development and validation of an immunoassay LDT with improved sensitivity for glucuronidated benzodiazepines. This LDT can detect glucuronidated benzodiazepines in clinical urine specimens and is stable for 60 days. Importantly, we were able to validate the immunoassay as an LDT by utilizing an LC-MS/MS LDT.

SARS-CoV-2 Serology Status Detected by Commercialized Platforms Distinguishes Previous Infection and Vaccination Adaptive Immune Responses.

BACKGROUND: The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has infected over 110 million individuals and led to 2.5 million deaths worldwide. As more individuals are vaccinated, the clinical performance and utility of SARS-CoV-2 serology platforms needs to be evaluated. METHODS: The ability of 4 commercial SARS-CoV-2 serology platforms to detect previous infection or vaccination were evaluated using a cohort of 53 patients who were SARS-CoV-2 PCR positive, 89 SARS-CoV-2-vaccinated healthcare workers (Pfizer or Moderna), and 127 patients who were SARS-CoV-2 negative. Serology results were compared to a cell-based SARS-CoV-2 pseudovirus (PSV) neutralizing antibodies assay. RESULTS: The Roche S-(spike) antibody and Diazyme neutralizing antibodies (NAbs) assays detected adaptive immune response in 100.0% and 90.1% of vaccinated individuals who received 2 doses of vaccine (initial and booster), respectively. The Roche N-(nucleocapsid) antibody assay and Diazyme IgG assay did not detect adaptive immune response in vaccinated individuals. The Diazyme NAbs assay correlated with the PSV SARS-CoV-2 median infective dose (ID50) neutralization titers (R2 = 0.70), while correlation of the Roche S-antibody assay was weaker (R2 = 0.39). Median PSV SARS-CoV-2 ID50 titers more than doubled in vaccinated individuals who received 2 doses of the Moderna vaccine (ID50, 597) compared to individuals who received a single dose (ID50, 284). CONCLUSIONS: The Roche S-antibody and Diazyme NAbs assays robustly detected adaptive immune responses in SARS-CoV-2 vaccinated individuals and SARS-CoV-2 infected individuals. The Diazyme NAbs assay strongly correlates with the PSV SARS-CoV-2 NAbs in vaccinated individuals. Understanding the reactivity of commercially available serology platforms is important when distinguishing vaccination response versus natural infection.

SARS-CoV-2 Serology Status Detected by Commercialized Platforms Distinguishes Previous Infection and Vaccination Adaptive Immune Responses.

Background: The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has infected over 110 million individuals and led to 2.5 million deaths worldwide. As more individuals are vaccinated, the clinical performance and utility of SARS-CoV-2 serology platforms needs to be evaluated. Methods: The ability of four commercial SARS-CoV-2 serology platforms to detect previous infection or vaccination were evaluated using a cohort of 53 SARS-CoV-2 PCR-positive patients, 89 SARS-CoV-2-vaccinated healthcare workers (Pfizer or Moderna), and 127 SARS-CoV-2 negative patients. Serology results were compared to a cell based SARS-CoV-2 pseudovirus (PSV) neutralizing antibodies assay. Results: The Roche S-(spike) antibody and Diazyme neutralizing antibodies (NAbs) assays detected adaptive immune response in 100.0% and 90.1% of vaccinated individuals who received two-doses of vaccine (initial and booster), respectively. The Roche N-(nucleocapsid) antibody assay and Diazyme IgG assay did not detect adaptive immune response in vaccinated individuals. The Diazyme Nabs assay correlated with the PSV SARS-CoV-2 ID50 neutralization titers (R2= 0.70), while correlation of the Roche S-antibody assay was weaker (R2= 0.39). Median PSV SARS-CoV-2 ID50 titers more than doubled in vaccinated individuals who received two-doses of the Moderna vaccine (ID50: 597) compared to individuals that received a single dose (ID50: 284). Conclusions: The Roche S-antibody and Diazyme NAbs assays robustly detected adaptive immune responses in SARS-CoV-2 vaccinated individuals and SARS-CoV-2 infected individuals. The Diazyme NAbs assay strongly correlates with the PSV SARS-CoV-2 NAbs in vaccinated individuals. Understanding the reactivity of commercially available serology platforms is important when distinguishing vaccination response versus natural infection.

Commercial Serology Assays Predict Neutralization Activity against SARS-CoV-2.

BACKGROUND: It is unknown whether a positive serology result correlates with protective immunity against SARS-CoV-2. There are also concerns regarding the low positive predictive value of SARS-CoV-2 serology tests, especially when testing populations with low disease prevalence. METHODS: A neutralization assay was validated in a set of PCR-confirmed positive specimens and in a negative cohort. In addition, 9530 specimens were screened using the Diazyme SARS-CoV-2 IgG serology assay and all positive results (N = 164 individuals) were reanalyzed using the neutralization assay, the Roche total immunoglobin assay, and the Abbott IgG assay. The relationship between the magnitude of a positive SARS-CoV-2 serology result and neutralizing activity was determined. Neutralizing antibody titers (50% inhibitory dilution, ID50) were also longitudinally monitored in patients confirmed to have SARS-CoV-2 by PCR. RESULTS: The SARS-CoV-2 neutralization assay had a positive percentage agreement (PPA) of 96.6% with a SARS-CoV-2 PCR test and a negative percentage agreement (NPA) of 98.0% across 100 negative control individuals. ID50 neutralization titers positively correlated with all 3 clinical serology platforms. Longitudinal monitoring of hospitalized PCR-confirmed patients with COVID-19 demonstrated they made high neutralization titers against SARS-CoV-2. PPA between the Diazyme IgG assay alone and the neutralization assay was 50.6%, while combining the Diazyme IgG assay with either the Roche or Abbott platforms increased the PPA to 79.2 and 78.4%, respectively. CONCLUSIONS: These 3 clinical serology assays positively correlate with SARS-CoV-2 neutralization activity observed in patients with COVID-19. All patients confirmed SARS-CoV-2 positive by PCR develop neutralizing antibodies.

Multi-Platform Comparison of SARS-CoV-2 Serology Assays for the Detection of COVID-19.

BACKGROUND: COVID-19 is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel beta-coronavirus that is responsible for the 2019 coronavirus pandemic. Acute infections should be diagnosed by polymerase chain reaction (PCR) based tests, but serology tests can demonstrate previous exposure to the virus. METHODS: We compared the performance of the Diazyme, Roche, and Abbott SARS-CoV-2 serology assays using 179 negative participants to determine negative percentage agreement (NPA) and in 60 SARS-CoV-2 PCR-confirmed positive patients to determine positive percentage agreement (PPA) at 3 different time frames following a positive SARS-CoV-2 PCR result. RESULTS: At ≥15 days, the PPA (95% CI) was 100 (86.3-100)% for the Diazyme IgM/IgG panel, 96.0 (79.7-99.9)% for the Roche total Ig assay, and 100 (86.3-100)% for the Abbott IgG assay. The NPA (95% CI) was 98.3 (95.2-99.7)% for the Diazyme IgM/IgG panel, 99.4 (96.9-100)% for the Roche total Ig assay, and 98.9 (96.0-99.9)% for the Abbott IgG assay. When the Roche total Ig assay was combined with either the Diazyme IgM/IgG panel or the Abbott IgG assay, the positive predictive value was 100% while the negative predictive value remained greater than 99%. CONCLUSIONS: Our data demonstrates that the Diazyme, Roche, and Abbott SARS-CoV-2 serology assays have similar clinical performances. We demonstrated a low false-positive rate across all 3 platforms and observed that false positives observed on the Roche platform are unique compared to those observed on the Diazyme or Abbott assays. Using multiple platforms in tandem increases the PPVs, which is important when screening populations with low disease prevalence.

Longitudinal Monitoring of SARS-CoV-2 IgM and IgG Seropositivity to Detect COVID-19.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a novel beta-coronavirus that has recently emerged as the cause of the 2019 coronavirus pandemic (COVID-19). Polymerase chain reaction (PCR) based tests are optimal and recommended for the diagnosis of an acute SARS-CoV-2 infection. Serology tests for viral antibodies provide an important tool to diagnose previous exposure to the virus. Here we evaluate the analytical performance parameters of the Diazyme SARS-CoV-2 IgM/IgG serology assays and describe the kinetics of IgM and IgG seroconversion observed in patients with PCR-confirmed COVID-19 who were admitted to our hospital. METHODS: We validated the performance of the Diazyme assay in 235 presumed SARS-CoV-2 negative subjects to determine specificity. Subsequently, we evaluated the SARS-CoV-2 IgM and IgG seroconversion of 54 PCR-confirmed COVID-19 patients and determined sensitivity of the assay at three different timeframes. RESULT: Sensitivity and specificity for detecting seropositivity at ≥15 days following a positive SARS-CoV-2 PCR result, was 100.0% and 98.7% when assaying for the panel of IgM and IgG. The median time to seropositivity observed for a reactive IgM and IgG result from the date of a positive PCR was 5 days (IQR: 2.75-9 days) and 4 days (IQR: 2.75-6.75 days), respectively. CONCLUSIONS: Our data demonstrate that the Diazyme IgM/IgG assays are suited for the purpose of detecting SARS-CoV-2 IgG and IgM in patients with suspected SARS-CoV-2 infections. For the first time, we report longitudinal data showing the evolution of seroconversion for both IgG and IgM in a cohort of acutely ill patients in the United States. We also demonstrate a low false positive rate in patients who were presumed to be disease free.

A Dilution Method to Mitigate Biotin Interference in Cardiac Troponin T Testing.

BACKGROUND: Oral biotin supplementation is known to interfere with biotin-streptavidin-based immunoassays, including Roche's fifth-generation cardiac troponin T (cTnT) assay, which plays a critical role in the diagnosis of myocardial infarction (MI). The utility of dilution, a quick and easy method to detect and remove interferences, has not been published for biotin interference. METHODS: Concentrations of cTnT were measured in pooled serum from clinical samples. Serum samples were supplemented with biotin to known concentrations, then cTnT concentrations were remeasured to assess for biotin interference. Samples were then diluted to assess for effective removal of biotin interference. RESULTS: At cTnT values near the critical reporting range for our institution (100 ng/L) we observed significant interference in measured values with added biotin concentrations above 50 ng/mL. In specimens without added biotin, autodilution at a 1:10 ratio yielded a mean 157% capture of measured cTnT, precluding the use of autodilution for detecting and mitigating biotin interference. A 1:10 dilution with serum containing 20-30 ng/L cTnT yielded a mean capture of 107%, which was suitable for detecting underlying biotin interference in supplemented samples. CONCLUSIONS: Biotin interference, at supraphysiologic concentrations, may create an artifactual reduction in measured cTnT to levels that could lead to delayed detection of an MI. Dilution with serum of known cTnT concentration of 20-30 ng/L is a fast and effective method to mitigate the analytical consequences of biotin interference.

MGST1, a GSH transferase/peroxidase essential for development and hematopoietic stem cell differentiation.

We show for the first time that, in contrast to other glutathione transferases and peroxidases, deletion of microsomal glutathione transferase 1 (MGST1) in mice is embryonic lethal. To elucidate why, we used zebrafish development as a model system and found that knockdown of MGST1 produced impaired hematopoiesis. We show that MGST1 is expressed early during zebrafish development and plays an important role in hematopoiesis. High expression of MGST1 was detected in regions of active hematopoiesis and co-expressed with markers for hematopoietic stem cells. Further, morpholino-mediated knock-down of MGST1 led to a significant reduction of differentiated hematopoietic cells both from the myeloid and the lymphoid lineages. In fact, hemoglobin was virtually absent in the knock-down fish as revealed by diaminofluorene staining. The impact of MGST1 on hematopoiesis was also shown in hematopoietic stem/progenitor cells (HSPC) isolated from mice, where it was expressed at high levels. Upon promoting HSPC differentiation, lentiviral shRNA MGST1 knockdown significantly reduced differentiated, dedicated cells of the hematopoietic system. Further, MGST1 knockdown resulted in a significant lowering of mitochondrial metabolism and an induction of glycolytic enzymes, energetic states closely coupled to HSPC dynamics. Thus, the non-selenium, glutathione dependent redox regulatory enzyme MGST1 is crucial for embryonic development and for hematopoiesis in vertebrates.

Hydroxyurea derivatives of irofulven with improved antitumor efficacy.

Irofulven is a semi-synthetic derivative of Illudin S, a toxic sesquiterpene isolated from the mushroom Omphalotus illudens. Irofulven has displayed significant antitumor activity in various clinical trials but displayed a limited therapeutic index. A new derivative of irofulven was prepared by reacting hydroxyurea with irofulven under acidic conditions. Acetylation of this new compound with acetic anhydride produced a second derivative. Both of these new derivatives displayed significant antitumor activity in vitro and in vivo comparable to or exceeding that of irofulven.

Absence of MGST1 mRNA and protein expression in human neuroblastoma cell lines and primary tissue.

A recent study identified a haplotype on a small region of chromosome 12, between markers D12S1725 and D12S1596, shared by all patients with familial neuroblastoma (NB). We previously localized the human MGST1 gene, whose gene product protects against oxidative stress, to this very same chromosomal region (12p112.1-p13.33). Owing to the chromosomal location of MGST1; its roles in tumorigenesis, drug resistance, and oxidative stress; and the known sensitivity of NB cell lines to oxidative stress, we considered a role for MGST1 in NB development. Surprisingly there was no detectable MGST1 mRNA or protein in either NB cell lines or NB primary tumor tissue, although all other human tissues, cell lines, and primary tumor tissue examined to date express MGST1 at high levels. The mechanism behind the failure of NB cells and tissue to express MGST1 mRNA is unknown and involves the failure of MGST1 pre-mRNA expression, but does not involve chromosomal rearrangement or nucleotide variation in the promoter, exons, or 3' untranslated region of MGST1. MGST1 provides significant protection against oxidative stress and constitutes 4 to 6% of all protein in the outer membrane of the mitochondria. As NB cells are extremely sensitive to oxidative stress, and often used as a model system to investigate mitochondrial response to endogenous and exogenous stress, these findings may be due to the lack of expression MGST1 protein in NB. The significance of this finding to the development of neuroblastoma (familial or otherwise), however, is unknown and may even be incidental. Although our studies provide a molecular basis for previous work on the sensitivity of NB cells to oxidative stress, and possibly marked variations in NB mitochondrial homeostasis, they also imply that the results of these earlier studies using NB cells are not transferable to other tumor and cell types that express MGST1 at high concentrations.

Structure-activity studies of urea, carbamate, and sulfonamide derivatives of acylfulvene.

Illudin S and M (1, 2) are highly toxic sesquiterpenes found in the basidiomycete Omphalotus illudens. Illudins have a low therapeutic index, but acylfulvene derivatives display potent in vivo antitumor activity against a variety of multidrug resistant tumors. The lead acylfulvene (4), irofulven (5), in a randomized phase IIB clinical trial significantly increased overall survival in patients with metastatic hormone-refractory prostate cancer who failed prior treatment with two different standard chemotherapeutic regimens. Irofulven is unique, as the primary allylic hydroxyl group can undergo displacement with a variety of nucleophiles to produce analogues that retain key functional groups required for biological activity including the reactive cyclopropylmethyl carbinol and alpha,beta-unsaturated ketone. As described here, we synthesized a variety of urea, carbamate, and sulfonamide derivatives that retain key functional groups and display potent biological activity toward target solid tumor cells in vitro but are relatively nontoxic toward a nontarget B-cell derived cell line.

Stability of levothyroxine sodium 0.4 microg/mL in 0.9% sodium chloride injection.

CONTEXT: Intravenous levothyroxine therapy decreases vasopressor requirements and prevents cardiovascular collapse in hemodynamically unstable patients eligible for organ donation. The stability of levothyroxine when used in this manner is unknown. OBJECTIVE: To determine the stability of levothyroxine solution for intravenous use at a concentration of 0.4 microg/mL diluted in 0.9% sodium chloride. DESIGN: Triplicate sample sets were prepared by reconstituting levothyroxine 200 microg for injection with 5 mL of 0.9% sodium chloride with further dilution in 500 mL of 0.9% sodium chloride. One sample set was protected from light and the other was left unprotected. Both sample sets were stored at room temperature, and samples from each were analyzed for initial concentration and 4, 8, 12, and 24 hours later. CONCLUSIONS: Levothyroxine sodium 0.4 microg/mL in 500 mL 0.9% sodium chloride is stable for 24 hours at room temperature when protected from light.

Synergy of irofulven in combination with other DNA damaging agents: synergistic interaction with altretamine, alkylating, and platinum-derived agents in the MV522 lung tumor model.

PURPOSE: Irofulven (MGI 114, NSC 683863) is a semisynthetic derivative of illudin S, a natural product present in the Omphalotus illudins (Jack O'Lantern) mushroom. This novel agent produces DNA damage, that in contrast to other agents, is predominately ignored by the global genome repair pathway of the nucleotide excision repair (NER)(2) system. The aim of this study was to determine the antitumor activity of irofulven when administered in combination with 44 different DNA damaging agents, whose damage is in general detected and repaired by the genome repair pathway. METHODS: The human lung carcinoma MV522 cell line and its corresponding xenograft model were used to evaluate the activity of irofulven in combination with different DNA damaging agents. RESULTS: Two main classes of DNA damaging agents, platinum-derived agents, and select bifunctional alkylating agents, demonstrated in vivo synergistic or super-additive interaction with irofulven. DNA helicase inhibiting agents also demonstrated synergy in vitro, but an enhanced interaction with irofulven could not be demonstrated in vivo. There was no detectable synergistic activity between irofulven and agents capable of inducing DNA cleavage or intercalating into DNA. CONCLUSION: These results indicate that the antitumor activity of irofulven is enhanced when combined with platinum-derived agents, altretamine, and select alkylating agents such as melphalan or chlorambucil. A common factor between these agents appears to be the production of intrastrand DNA crosslinks. The synergistic interaction between irofulven and other agents may stem from the nucleotide excision repair system being selectively overwhelmed at two distinct points in the pathway, resulting in prolonged stalling of transcription forks, and subsequent initiation of apoptosis.

Synergy of Irofulven in combination with various anti-metabolites, enzyme inhibitors, and miscellaneous agents in MV522 lung carcinoma cells: marked interaction with gemcitabine and 5-fluorouracil.

The novel agent Irofulven (HMAF, NSC 683863) has demonstrated significant antitumor activity against solid tumors in various xenograft models and human clinical trials. The antitumor potential of combining irofulven with 72 different anti-metabolite, enzyme inhibiting, and miscellaneous agents was investigated in this study. The human lung carcinoma MV522 cell line and its corresponding xenograft model were used to evaluate the activity of irofulven in combination with these different agents. Irofulven in combination with select anti-metabolites, notably cytidine or adenine-derived agents, displayed strong synergistic activity in both in vitro and in vivo studies. Agents demonstrating strong synergistic interaction with irofulven included gemcitabine, cyclocytidine, cytarabine, fludarabine phosphate, cladribine, and 5-fluorouracil. Other anti-metabolites, enzyme inhibitors, and a variety of miscellaneous agents failed to interact beneficially when administered in combination with irofulven. The therapeutic activity of irofulven is enhanced considerably when irofulven is combined with select anti-metabolite agents, and further clinical evaluation of these combinations is warranted. The synergistic interaction with these combinations may stem from a variety of actions including inhibition of the nucleotide excision repair (NER) pathway, topoisomerase I activity, and caspase-dependent and independent induction of apoptosis.

Disruption of the ugt1 locus in mice resembles human Crigler-Najjar type I disease.

The 9 UDP-glucuronosyltranferases (UGTs) encoded by the UGT1 locus in humans are key enzymes in the metabolism of most drugs as well as endogenous substances such as bile acids, fatty acids, steroids, hormones, neurotransmitters, and bilirubin. Severe unconjugated hyperbilirubinemia in humans that suffer from Crigler-Najjar type I disease results from lesions in the UGT1A1 gene and is often fatal. To examine the physiological importance of the Ugt1 locus in mice, this locus was rendered non-functional by interrupting exon 4 to create Ugt1(-/-) mice. Because UGT1A1 in humans is responsible for 100% of the conjugated bilirubin, it followed that newborn Ugt1(-/-) mice developed serum levels of unconjugated bilirubin that were 40-60 times higher than Ugt1(+/-) or wild-type mice. The result of extreme unconjugated bilirubin in Ugt1(-/-) mice, comparable to the induced levels noted in patients with Crigler-Najjar type 1 disease, is fatal in neonatal Ugt1(-/-) mice within 2 weeks following birth. The extreme jaundice is present as a phenotype in skin color after 8 h. Neonatal Ugt1(-/-) mice exhibit no detectable UGT1A-specific RNA, which corresponds to a complete absence of UGT1A proteins in liver microsomes. Conserved glucuronidation activity attributed to the Ugt1 locus can be defined in Ugt1(-/-) mice, because UGT2-dependent glucuronidation activity is unaffected. Remarkably, the loss of UGT1A functionality in liver results in significant alterations in cellular metabolism as investigated through changes in gene expression. Thus, the loss of UGT1A function in Ugt1(-/-) mice leads to a metabolic syndrome that can serve as a model to further investigate the toxicities associated with unconjugated bilirubin and the impact of this disease in humans.

Synthesis and antitumor activity of amine analogs of irofulven.

Acylfulvenes, a class of semisynthetic analogs of Illudin S, show high toxicity toward prostate cancer cells. Here we probe the effect of changes in hydrophilic character of the analogs.

Phase I/II study of continuous-infusion troxacitabine in refractory acute myeloid leukemia.

PURPOSE: Troxacitabine is a non-natural nucleoside analog with unique structural and metabolic features. Bolus intravenous (IV) troxacitabine regimens have shown significant activity in patients with refractory acute myeloid leukemia (AML) and preclinical data suggest that administration via continuous infusion may result in enhanced antitumor activity. PATIENTS AND METHODS: Patients with refractory AML initially received troxacitabine 10.1 mg/m2 by continuous IV infusion (CIVI) for 48 hours. Infusion duration and daily dose were increased in subsequent patient cohorts. RESULTS: Forty-eight patients, median age 58 years (range, 21 to 81 years), were treated. Dose-limiting toxicities were mucositis and hand-foot syndrome, and 12.0 mg/m2/d for 5 days was established as the maximum-tolerated dose. Seven patients (15%) achieved complete remission (CR) or CR with incomplete platelet recovery (CRp), with a median survival among responders of 12 months. Steady-state concentrations of troxacitabine were found to be linearly and inversely proportionally related to calculated creatinine clearance at doses of 10.1 and 12.0 mg/m2/d. All patients responding to troxacitabine had steady-state serum drug concentration of more than approximately 80 ng/mL. In 27 patients achieving target troxacitabine plasma concentrations (ie, approximately 80 ng/mL) the CR + CRp rate was 26%. CONCLUSION: Troxacitabine administered as a CIVI allows a significant increase in dose-intensity in comparison to IV bolus regimens, has antileukemic activity, and warrants additional investigation in patients with refractory AML. The recommended phase II study dose is 12.0 mg/m2 daily CIVI for 5 days.

Structural organization of the murine microsomal glutathione S-transferase gene (MGST1) from the 129/SvJ strain: identification of the promoter region and a comprehensive examination of tissue expression.

The structure and regulation of the murine microsomal glutathione transferase gene (MGST1) from the 129/SvJ strain is described and demonstrates considerable difference in nucleotide sequence and consequently in restriction enzyme sites as compared to other mouse strains. A comparison of the amino acid sequence for MGST1 revealed one difference in exon 2 between the 129/SvJ strain (arginine at position 5) and the sequence previously reported for the Balb/c strain (lysine). The promoter region immediately upstream of the dominant first exon is functional, transcriptionally responds to oxidative stress, and is highly homologous to the human region. Oxidative stress also induced the production of endogenous MGST1 mRNA. The tissue-specific expression of MGST1 mRNA was studied, and as anticipated, was indeed highest in liver. There was, however, marked mRNA expression in several tissues not previously studied including smooth muscle, epidymus, ovaries, and endocrine glands in which the expression of various peroxidases is also very high (salivary and thyroid). Overall, there was a good agreement between the mRNA content detected and previous reports of MGST1 activity with the exception of brain tissue.