Toxicity of clopidogrel and ticlopidine on human myeloid progenitor cells: importance of metabolites

Ticlopidine and clopidogrel are thienopyridine derivatives used for inhibition of platelet aggregation. Not only hepatotoxicity, but also bone marrow toxicity may limit their use. Aims of the study were to find out whether non-metabolized drug and/or metabolites are responsible for myelotoxicity and whether the inactive clopidogrel metabolite clopidogrel carboxylate contributes to myelotoxicity. We used myeloid progenitor cells isolated from human umbilical cord blood in a colony-forming unit assay to assess cytotoxicity. Degradation of clopidogrel, clopidogrel carboxylate or ticlopidine (studied at 10 and 100 μM) was monitored using LC/MS. Clopidogrel and ticlopidine were both dose-dependently cytotoxic starting at 10 μM. This was not the case for the major clopidogrel metabolite clopidogrel carboxylate. Pre-incubation with recombinant human CYP3A4 not only caused degradation of clopidogrel and ticlopidine, but also increased cytotoxicity. In contrast, clopidogrel carboxylate was not metabolized by recombinant human CYP3A4. Pre-incubation with freshly isolated human granulocytes was not only associated with a myeloperoxidase-dependent degradation of clopidogrel, clopidogrel carboxylate and ticlopidine, but also with dose-dependent cytotoxicity of these compounds starting at 10 μM. In conclusion, both non-metabolized clopidogrel and ticlopidine as well as metabolites of these compounds are toxic towards myeloid progenitor cells. Taking exposure data in humans into account, the myelotoxic element of clopidogrel therapy is likely to be secondary to the formation of metabolites from clopidogrel carboxylate by myeloperoxidase. Concerning ticlopidine, both the parent compound and metabolites formed by myeloperoxidase may be myelotoxic in vivo. The molecular mechanisms of cytotoxicity have to be investigated in further studies.     

Social facial touch in rats

We know much about how rats use their whiskers to discriminate simple tactile properties, but little about how they are used in natural settings. Here we studied whisker motion during social interactions between rats in order to gain a better understanding of natural whisker use in this model system for sensorimotor integration. In the first set of experiments, an intruder was placed in a second rat's home cage. Anogenital sniffing immediately ensued; later in the trial, facial interactions occurred at least as frequently. Whereas much previous work has focused on the importance of anogenital sniffing during social interactions, these facial interactions were accompanied by some of the most intense whisker behaviors described to date. Whisker trimming increased biting but reduced boxing. In addition, whiskers were more protracted and whisking amplitude was larger in aggressive than in nonaggressive interactions. In a second set of experiments, rats interacted facially across a gap. As rats approached each other, whisking amplitude decreased and whiskers were more protracted. Whisker trimming disrupted facial alignment and reduced the frequency of interactions, indicating that whisker use, and possibly whisker protraction, is important for rats to orient themselves with respect to one another. We also found that females whisked with smaller amplitude when interacting with males than with females, and that they held their whiskers less protracted than males. The natural whisker use described here should further our understanding of this important somatosensory system during social interactions.     

Severity of heroin use in Israel: comparisons between native Israelis and former Soviet Union immigrants

AIMS: This study examined drug use patterns and severity among native-Israeli and former Soviet Union (FSU) immigrants in Israel who reported heroin use. DESIGN, SETTING AND PARTICIPANTS: a total of 272 native Israelis and 300 FSU heroin users were interviewed from 2002 to 2006 as part of a large drug use surveillance study in Israel. Individuals were sampled at an intake centre, a methadone clinic and a day-treatment facility in the Negev region of Israel. Participants were assessed using the Addiction Severity Index, fifth edition. Native Israeli and FSU users were compared within two groups: those interviewed at intake and those interviewed in treatment. FINDINGS: Overall, ASI composite scores suggested generally comparable levels of addiction severity between the two ethnic groups. Native-born Israelis reported more years of heroin use; however, the FSU immigrants reported longer use of other opiates. The FSU reported significantly more heroin use by injection, and a significantly higher rate of hepatitis C and other chronic medical problems. Comparisons by gender within each group revealed higher drug severity scores for females (native-born Israeli and FSU combined). Females in the intake group had significantly higher severity scores in the areas of employment and psychiatric status when compared to individuals who had been in treatment for some time. CONCLUSIONS: Except for higher levels of alcohol use, the FSU did not have more severe drug problems than the native Israelis as measured by ASI severity scores. Injection use among FSU, however, is a critical public health problem, especially given the well-established link between injection drug use, hepatitis C and HIV/AIDS.     

Screening of amide analogues of Trichostatin A in cultures of primary rat hepatocytes: search for potent and safe HDAC inhibitors

Summary  The vast majority of preclinical studies of HDAC inhibitors (HDAC-I) focus on the drug–target (cancer) cell interaction, whereas little attention is paid to the effects on non-target healthy cells, which could provide decisive information to eliminate potential cytotoxic compounds at a very early stage during drug development. In the current study we used cultures of primary rat hepatocytes as a read out system to select for the most potent HDAC-I in the group of structural analogues of an archetypal HDAC-I, namely Trichostatin A. This kind of approach allowed selecting compounds with high biological activity and with no apparent toxicity towards cultured hepatocytes. Joanna Fraczek and Sarah Deleu contributed equally to this article. T. Vanhaecke is a postdoctoral research fellow of the Fund for Scientific Research Flanders (FWO-Vlaanderen, Belgium)     

Synthesis of C5-dicarboxylic acids from C2-units involving crotonyl-CoA carboxylase/reductase: the ethylmalonyl-CoA pathway

Fifty years ago, Kornberg and Krebs established the glyoxylate cycle as the pathway for the synthesis of cell constituents from C2-units. However, since then, many bacteria have been described that do not contain isocitrate lyase, the key enzyme of this pathway. Here, a pathway termed the ethylmalonyl-CoA pathway operating in such organisms is described. Isotopically labeled acetate and bicarbonate were transformed to ethylmalonyl-CoA by cell extracts of acetate-grown, isocitrate lyase-negative Rhodobacter sphaeroides as determined by NMR spectroscopy. Crotonyl-CoA carboxylase/reductase, catalyzing crotonyl-CoA + CO2 + NADPH --> ethylmalonyl-CoA- + NADP+ was identified as the key enzyme of the ethylmalonyl-CoA pathway. The reductive carboxylation of an enoyl-thioester is a unique biochemical reaction, unprecedented in biology. The enzyme from R. sphaeroides was heterologously produced in Escherichia coli and characterized. Crotonyl-CoA carboxylase/reductase (or its gene) can be used as a marker for the presence of the ethylmalonyl-CoA pathway, which functions not only in acetyl-CoA assimilation. In Streptomyces sp., it may also supply precursors (ethylmalonyl-CoA) for antibiotic biosynthesis. For methylotrophic bacteria such as Methylobacterium extorquens, extension of the serine cycle with reactions of the ethylmalonyl-CoA pathway leads to a simplified scheme for isocitrate lyase-independent C1 assimilation.     

Pancreatoblastoma: a report from the European cooperative study group for paediatric rare tumours (EXPeRT)

Background

Pancreatoblastoma is a very rare malignant tumour typically occurring in the early years of life. Due to its rarity, standardised diagnostic and therapeutic guidelines are not available for pancreatoblastoma.

Methods

The newborn cooperative group denominated EXPeRT – European cooperative study group for paediatric rare tumours – combined in a joint analysis of all cases registered between 2000 and 2009 by the national groups of Italy, France, United Kingdom, Poland and Germany.

Results

Twenty patients <18 years old (median age 4 years) were analysed: nine had distant metastases at diagnosis. Seventeen patients had tumour resection, at initial or delayed surgery. Eighteen received chemotherapy (response rate 73%), seven received radiotherapy. For the whole series, 5-year event-free survival and overall survival were 58.8% and 79.4%, respectively. Outcome did not correlate with tumour site and size, but was strongly influenced by the feasibility of tumour complete resection.

Conclusions

This international study confirms the rarity of the disease, the critical role of surgical resection both as therapy and as a prognostic variable, and the potential efficacy of chemotherapy. The adoption of an intensive multidisciplinary approach is required, as well as the referral to highly experienced centres. Further international cooperation is needed to collect larger series and stimulate biological studies to improve our understanding of the biology and the natural history of PBL.     

Prognosis and Determinants of Outcome Following Locoregional or Distant Recurrence in Patients with Cutaneous Melanoma

Objective Information on prognosis for patients with cutaneous melanoma after locoregional or distant recurrence is sparse and controversial. The aim of this study was to analyze factors influencing outcome after the development of a first relapse. Methods Information was extracted from the Sydney Melanoma Unit database for 873 melanoma patients with American Joint Committee on Cancer (AJCC) Stage I and II disease treated between 1960 and 2002 who relapsed following treatment of their primary melanoma. Clinical and pathologic factors predicting survival were analyzed using the Cox proportional hazards regression model. Results Initial presentation of recurrence was local: 95 patients (10.9%), in transit: 86 patients (9.9%), regional lymph node: 300 patients (34.4%), and distant: 392 patients (44.9%). Independent prognostic factors for survival of the 481 patients with only locoregional recurrence were type of recurrence, primary tumor ulceration, and patient age. Predictors for longer survival in the 392 patients with distant metastasis at the time of first presentation with recurrence were lung vs other sites and diagnosis of relapse after 1990 compared with diagnosis before 1980. Conclusions The type of recurrence is the most important prognostic factor in melanoma patients who relapse. Primary tumor ulceration is the most important pathologic predictor. The results of this study suggest that management of distant metastases may have improved over the last 25 years, but many confounders and improved staging techniques make assessment of this unreliable.     

A keratinocyte's course of life

An adequate permeability barrier function of the mammalian epidermis is guaranteed by the characteristic architecture of the stratum corneum. This uppermost layer consists of a highly organized extracellular lipid compartment which is tightly joined to the corneocytes. The generation of the extracellular lipid compartment and the transformation of the keratinocytes into corneocytes are the main features of epidermal differentiation. However, equally important is the continuous renewal of the stratum corneum, which is insured by a careful balance between the replenishment of new keratinocytes from the proliferating basal layer, and the well-orchestrated loss of the most superficial cells after the so-called 'epidermal programmed cell death'. In this overview, the complete life of keratinocytes is described, from the proliferative organization to the process of desquamation.     

Detection of First Relapse in Cutaneous Melanoma Patients: Implications for the Formulation of Evidence-Based Follow-up Guidelines

Background The value of follow-up surveillance for patients with cutaneous melanoma remains uncertain. In this prospective study the frequency of detection of first melanoma recurrence (FMR) by patient or doctor was analyzed to assist in the future design of evidence-based follow-up guidelines. Methods Patients who had a recurrence of a previously treated American Joint Committee on Cancer (AJCC) stage I–III primary melanoma (PM) were interviewed to ascertain how their PM and FMR were detected. Factors predicting the detection of PM and FMR were analyzed. Results The study group comprised 211 patients. In 168 patients, information on detection of their PM was available; 102 PMs (61%) were detected by the patient and 18 (11%) by their partner. Higher AJCC stage, visible location for the patient, and female sex were independent predictive factors for patient-detected PM (P = .03, .002, and .02 respectively). The FMR type was local in 28 (13%), in transit in 35 (17%), in regional lymph nodes in 97 (46%), and distant in 51 (24%). Seventy-three percent of all FMRs were detected by the patient. The presence of a symptom was the only independent predictor of a patient-detected FMR (P < .0001). There was no statistically significant survival difference between the patient-detected and doctor-detected FMRs. Conclusions Three-quarters of FMRs were detected by patients or their partners, and it should be possible to improve this rate even further by better education. More frequent follow-up visits are thus unlikely to be valuable. Reductions in follow-up frequency may therefore be safe and economically responsible. Presented at the American Society of Clinical Oncology annual meeting June 5–8 2004, New Orleans, Louisiana.