Characterization of an Amphetamine Interference from Gabapentin in an LC-HRMS Method.

An amphetamine interference was observed during the development of an liquid chromatography-high-resolution mass spectrometry (LC-HRMS) multi-class confirmation method for the determination of 47 drugs and metabolites in urine. The interference passed all qualitative criteria for amphetamine leading to potential false-positive results. Upon investigation, it was found that the amphetamine interference was correlated with the presence of high levels of gabapentin. Gabapentin is routinely detected in patient urine specimens at levels in excess of 1 mg/mL as it is widely prescribed at high doses and does not undergo significant metabolism. The source of the interference was identified as a gabapentin in-source fragment isomeric with protonated amphetamine. Here we describe the characterization of this interference and how its effect was mitigated in the LC-HRMS method.

Identification of Novel Opioid Interferences using High-Resolution Mass Spectrometry.

Novel opioid interferences were observed during the development of a high-resolution liquid chromatography-mass spectrometry urine drug testing method for 47 analytes from multiple drug classes. The interferences affected both analytes and internal standards and were only observed when the method was challenged with patient samples. Some interferences were attributable to isomeric opioid metabolites not previously reported while others were due to interference from in-source dissociations or 13C isotopic contributions from known opioid metabolites not typically monitored as analytes. Based on patient drug profiles, known and inferred metabolism, accurate mass, retention time and MS/MS spectrum, the putative identity of each interference was assigned and later confirmed, when possible, using an authentic standard. Opioids are some of the most frequently monitored analytes in urine drug testing laboratories. Because of the potential for co-purification, co-chromatography and spectral similarity, it is anticipated that the reported opioid metabolite interferences could be present with other method conditions and instrument platforms. The objectives of this work are to raise awareness of these interferences and emphasize the importance of evaluating patient samples for potential interferences during method development.

CYP2D7-2D6 hybrid tandems: identification of novel CYP2D6 duplication arrangements and implications for phenotype prediction.

AIMS: Allelic variants of cytochrome P450 CYP2D6 (CYP2D6), such as gene deletion, duplication, multiplication and conversion, contribute to the wide range of CYP2D6 activity. Novel gene arrangements were discovered and characterized. MATERIALS & METHODS: DNA from 32 Caucasian and 59 African-American duplication-positive subjects were analyzed by long-range PCR and genotyping to detect CYP2D7-2D6 hybrid tandem alleles. Novel allelic variants were sequenced and a strategy for the detection and analysis of hybrid genes was refined. RESULTS: CYP2D7-2D6 hybrid tandem alleles were identified in one African-American and four Caucasian subjects. Three novel hybrid genes were found on CYP2D6*1 and CYP2D6*2 duplication backgrounds and designated CYP2D6*76, *77 and *78. CYP2D7 to 2D6 conversion occurred in introns 1 and 4, and exon 9. All carried a T-insertion in exon 1 abolishing activity. In Caucasians, four out of 33 (12%) of the duplication-positive alleles were hybrid tandems, three CYP2D6*77 + *2 and one CYP2D6*78 + *2. By contrast, in African-Americans only one of 60 duplication-positive alleles was identified as a hybrid tandem. This allele was designated CYP2D6*76 + *1. CONCLUSION: Hybrid tandem alleles occur infrequently (<0.25%) in Caucasians, but may explain why not every subject with a CYP2D6 duplication presents with an ultrarapid metabolizer phenotype.

Presence of a peptide component of thymosin fraction-5 manifesting discrete cytostatic properties in HL-60 human promyelocytic leukemia cells.

Thymosin fraction-5 (TF5), an array of small molecular weight peptides present in crude extracts of the adult bovine thymus, contains numerous constituents with demonstrable biological activity. Because TF5 generally enhances immune reactivity in a variety of settings, and additionally restricts proliferation of certain neoplasms, we examined the effects of TF5 on proliferative capacity in the human promyelocytic leukemia cell line HL-60. Vital dye-exclusion, oxidative metabolism of chromogenic dyes, and clonogenic growth profiles were monitored to assess rates of cellular proliferation; our results demonstrate that TF5 restricted HL-60 cell growth, an influence that exhibited comparable potency and efficacy among all three indices. This antiproliferative activity was labile, insofar as medium conditioned in HL-60 cells for 24 h became devoid of the initial growth-suppressive activity after 24-h culture when subsequently administered to naive cultures. Review of cytoarchitectural traits, chromatin staining by TUNEL, and fluorescent cytometric analyses demonstrated that TF5 failed to elicit apoptosis, however, suggesting that this material instead drove treated cells into growth arrest and an unanticipated cytostasis. Qualitatively similar responses were noted in the human monoblastic leukemia cell line U937. Partial purification of TF5 by FPLC yielded a component containing an antiproliferative activity associated with the approximately 1000-Da fraction. These results demonstrate that TF5 contains a sub-fraction possessing a growth-suppressive activity capable of restraining normal proliferation of human myeloid neoplasms via the apparent induction of true cytostasis.

Intrinsic cytotoxicity and chemomodulatory actions of novel phenethylisothiocyanate sphingoid base derivatives in HL-60 human promyelocytic leukemia cells.

The protein kinase C (PKC) isoenzyme superfamily represents a popular target in pharmacological interventions designed to elicit apoptosis directly in tumor cells or to potentiate the lethal effects of antineoplastic agents. Numerous observations support the clinical utility of PKC inhibition by experimental sphingolipid derivatives such as safingol. The present studies document the cytotoxicity and chemomodulatory capacity of phenethylisothiocyanate derivatives of sphinganine and sphingosine (PEITC-Sa and PEITC-So) in the human myeloid leukemia cell line HL-60. The biological actions of these novel derivatives were compared directly with those of the parent compounds sphinganine and sphingosine. Exposure to natural and modified sphingoid bases promoted extensive apoptotic cell death. The PEITC-sphingoid base derivatives exhibited higher cytotoxicity than their natural counterparts and were also distinctly superior to the clinically relevant sphingoid base analog safingol. In each instance, lethality was shown to correlate with inhibition of conventional and novel PKC isoforms and downstream loss of extracellular signal-regulated kinase (ERK)1/ERK2. The involvement of these signaling systems in potentiating the lethal actions of 1-(beta-D-arabinofuranosyl)cytosine (araC) was also examined with regard to the differential actions of PEITC-Sa and PEITC-So to that of the parent compounds as well as safingol. Exposure to araC alone rapidly increased PKC activity. In the presence of PEITC-Sa or PEITC-So, the therapeutic efficacy of araC increased markedly; moreover, potentiation was directly related to the loss of araC-stimulated PKC activity. These findings demonstrate that PEITC-substituted sphingoid base analogs exert potent antineoplastic effects in human leukemia cells. We suggest that these synthetic lipids represent potentially useful agents in the development of conventional PKC/novel PKC-directed chemotherapeutic strategies.

Specific inhibition of Stat5a/b promotes apoptosis of IL-2-responsive primary and tumor-derived lymphoid cells.

Stat5a/b exhibits 96% homology and are required for normal immune function. The present studies examined Stat5a/b function in lymphoid cells by specific and simultaneous disruption of both proteins using novel phosphorothioate-2'-O-methoxyethyl antisense oligodeoxynucleotides (asODN). Efficient delivery was confirmed by the presence of fluorescent TAMRA-labeled ODN in >or=55 and 95% in human primary and tumor cell lines, respectively. Acute asODN administration reduced levels of Stat5a (90%) in 6 h, whereas Stat5b required nearly 48 h to attain the same inhibition, suggesting that the apparent turnover rate for Stat5a was 8-fold higher than that for Stat5b. Expression of the closely related Stat3 protein was unchanged after asODN treatment, however. Molecular ablation of Stat5a/b promoted apoptotic cell death in a significant population of primary PHA-activated T cells (72%) and lymphoid tumor cell line (e.g., YT; 74%) within 24 h, as assessed by 1) visualization of karyolytic nuclear degeneration and other generalized cytoarchitectural alterations, 2) enzymatic detection of TdT-positive DNA degradation, and 3) automated cytometric detection of annexin V translocation. Contrary to findings from Stat5a/b-null mice, cell cycle progression did not appear to be significantly affected. Interestingly, IL-2-insensitive and unprimed T cells and Jurkat cells remained mostly unaffected. Finally, evidence is provided that the cytotoxicity associated with Stat5a/b ablation may derive from activation of caspase-8, an initiator protease that contributes to apoptotic cell commitment. We propose that in lymphoid cells competent to activate Stat5a and Stat5b, both proteins preferentially mediate an antiapoptotic survival influence.