In vitro effect of r-verapamil on acute myelogenous leukemia blast cells: studies of cytokine secretion and cytokine-dependent blast proliferation

The in vitro effect of the dextroisomer r-verapamil on blast cells derived from patients with acute myelogenous leukemia (AML) was studied. R-verapamil caused a dose-dependent inhibition of AML blast proliferation in the presence of stem-cell factor, leukemia inhibitory factor, interleukin 4, interleukin 6, and interleukin 10 when these cytokines were tested both alone and in different combinations. R-verapamil also inhibited the growth of clonogenic AML blast cells. The antiproliferative effect was not specific for AML blast cells, because r-verapamil also inhibited cytokine-dependent proliferation of blast cells derived from patients with acute lymphoblastic leukemia. The inhibitory effects of r-verapamil and anti-IL1 serum were additive, suggesting that the antiproliferative effect of r-verapamil does not depend solely on inhibition of IL1-mediated effects. Although r-verapamil inhibited spontaneous AML blast proliferation, for a majority of patients it caused only minimal, if any, inhibition of spontaneous cytokine secretion (IL1, IL1, TNF, IL6) by AML blast cells. Thus, although inhibition of IL1 effects may contribute in certain patients to the antiproliferative effect of r-verapamil, mechanisms other than IL1 inhibition seem to be more important in mediating the effects of r-verapamil.Abbreviations ALL Acute lymphocytic leukemia - AML acute myelogenous leukemia - cpm counts per minute - ELISA enzyme-linked immunosorbent assay - G-CSF granulocyte colony-stimulating factor - GM-CSF granulocyte-macrophage colony-stimulating factor - IL interleukin - IF leukemia inhibitory factor - PBMC peripheral blood mononuclear cells - RR relative response - SCF stem cell factor - TNF tumor necrosis factor      

Abbreviated cyclosporine AUCs on Neoral – the search continues!

 Cyclosporine is a powerful immunosuppressant with a narrow therapeutic window and considerable inter- and intrapatient variability. The pre-dose trough concentration (C_min) is commonly used for therapeutic drug monitoring. With the new microemulsion (Neoral), intrapatient variability was reduced. However, the usefulness of Neoral C_min was questioned. Firstly, because of the improved and more-rapid absorption, accidental intake before blood sampling has a greater impact on C_min than with classic cyclosporine. Secondly, C_min may be low despite high drug exposure, due to rapid clearance in children. A full pharmacokinetic (PK) profile with determination of the area under the curve (AUC) is expensive and cumbersome, and therefore a search for an abbreviated AUC began. Here, we present a retrospective analysis of 84 PK profiles from 78 pediatric renal transplant recipients. By analysis of rejection episodes and toxicity, we estimated a target AUC above 5,000 ng×h/ml in the early post-transplant period and 3,900 ng×h/ml beyond 100 days. The abbreviated AUC using the 2- and 6-h concentrations (C2 and C6) and a simple estimate derived from the 3-h concentration (C3) were equally well correlated with the AUC. From our data, we recommend a target C3 at approximately 800 ng/ml early after transplantation and 450–550 ng/ml beyond 100 days. Received: 28 January 1998 / Revised: 10 June 1998 / Accepted: 15 June 1998     

Nonoperative Management of Primary Colorectal Cancer in Patients With Stage IV Disease

Background: Traditional teaching maintains that patients with primary colorectal adenocarcinoma require timely resection to prevent bleeding, perforation, or obstruction. The true benefits of primary tumor resection remain undocumented for patients presenting with metastatic disease, however. We postulated that resection of primary colorectal tumors could be avoided safely in a select population of asymptomatic colorectal cancer patients presenting with incurable stage IV disease.Methods: A retrospective review of the Vanderbilt University Hospital tumor registry was performed for the years 1985 to 1997. During this period, 955 patients presented for management of primary colorectal cancer. From this group, all patients with stage IV disease at the time of diagnosis were identified. Patients who initially underwent resection of their primary lesion were included in the resection group; those who underwent initial nonoperative primary tumor management were included in the nonresection group. Data were obtained regarding age, extent of disease, nonsurgical therapy, tumor-specific complications, and palliative surgical procedures. Surgery-free survival and overall survival were analyzed using the Kaplan-Meier method. For patients with liver metastases, hepatic tumor burden was defined as either H1 (<25% parenchymal replacement), H2 (25% to 50%), or H3 (>50%) disease.Results: Sixty-six patients were included in the resection group, and 23 patients with intact asymptomatic primary colorectal lesions were included in the nonresection group. Among patients with hepatic metastases, most of the patients in both groups had H1 disease. Ten patients in the resection group and 3 patients in the nonresection group presented with exclusively extrahepatic metastases. In the nonresection group, primary therapy included chemotherapy in 13 patients, external beam radiation therapy in 1 patient, and combination chemoradiation in 9 patients. The median survival in the nonresection group was 16.6 months. The 2-year actuarial survival was 18%, and the surgery-free survival was 91.3%. Only 2 of 23 patients (8.7%) managed without resection eventually developed obstruction at the primary tumor site requiring emergent diversion. There were no episodes of tumor-related hemorrhage or perforation. For the resection group, the operative morbidity was 30.3%, and the perioperative mortality rate was 4.6%. The median survival in the resection group was 14.5 months (P = 0.59, log-rank test vs. nonresection group).Conclusions: Selected patients with asymptomatic primary colorectal tumors who present with incurable metastatic disease may safely avoid resection of their primary lesions, with an anticipated low rate of hemorrhage, perforation, or obstruction before death from systemic disease. No survival advantage is gained by resection of an asymptomatic primary lesion in the setting of incurable stage IV colorectal cancer.     

Endothelin-induced inositol phosphate formation in rat kidney. Studies on receptor subtypes, G-proteins and regulation during ontogenesis

The aim of this study was to characterize the properties of endothelin (ET)-receptor subtypes mediating inositol phosphate (IP)-formation in rat kidney and their regulation during ontogenesis. In renal cortical slices of adult rats (12–16 weeks old) ET's concentration-dependently increased IP-formation with an order of potency ET -1 ET 3. While the non-selective ET receptor antagonist bosentan (10 M) completely suppressed ET-induced IP-formation, the ETA-receptor antagonist BQ-123 (10 M) inhibited it only by 70%, the ET_B-receptor antagonist IRL 1038 (1 M) by 25%; combined application of BQ-123 + IRL 1038 caused complete inhibition of ET-1-induced IP-formation. Pretreatment of isolated renal cells with pertussis toxin (PTX, 500 ng/ml) overnight did not attenuate but significantly increased ET-1-induced IP-formation. Ontogenetic studies in renal slices from neonatal, 1, 2, 3, 6, 12 and 24 weeks old rats revealed that ET-1-induced IP-formation maturation-dependently declined being highest in neonatal rats (increase: 169% over basal) and lowest in 24 weeks old rats (increase: 47% over basal). This decline in ET-induced IP-formation was accompanied by a decrease in renal ET receptor number and the amount of immunodetectable G_q/11 (assessed by Western-blotting using the QL-antiserum). Moreover, ET receptor subtypes changed during the maturation process: from neonates to 12 weeks old rats number and functional responsiveness of ET_A-receptors declined, while that of ET_B-receptors increased. We conclude that in adult rat renal cortex ET-induced IP-formation is mediated by activation of both ET_A- and ET_B-receptors and does not involve a PTX-sensitive G-protein. ET-induced IP-formation declines during the maturation process; this is associated with a decrease in ET-receptor number and the immunodetectable amount of G_q/11.     

Immunobiology of experimental leishmaniasis

Self-cure versus uncontrolled disease progression in experimental murine cutaneous leishmaniasis depends upon a delicate interplay among various activated cells of the host's immune system. Susceptibility or resistance to infection with Leishmania major is correlated with the ability of different inbred strains of mice to produce the characteristic spectra of lymphokines upon infection. Appropriate experimental interventions now allow the modulation of these reponses, providing the possibility to render genetically susceptible mice resistant to infection and, vice versa, to cause genotypically healer strains to express a non-healer phenotype. These experimental manipulations have proven to be powerful tools in the dissection of the underlying immune mechanisms and cellular parameters responsible for susceptibility and resistance, and will perhaps allow the identification of molecules of parasite origin that induce deleterious immune responses to infection with Leishmania, and thus to exclude them from future vaccines. More importantly, rational immune intervention could permit the diversion of established host-damaging immune responses to host-protective immunization.     

Screening for chlamydia trachomatis in military personnel by urine testing

Summary In order to determine the infection rate ofChlamydia trachomatis in young males in Austria an epidemiological study was performed on 335 male Austrian soldiers attending the military hospital for a health check-up procedure. Three hundred twenty-nine (98.2%) of the screened males were clinically asymptomatic. Chlamydial diagnosis was established by testing first catch urine (FCU). Urine sediment was tested by an enzyme immunosorbent assay (EIA) and by a direct immunofluorescent test (DIF test). Positive results in both tests were defined true positives. In 41 (12.2%) of all the 335 soldiers a genital chlamydial infection could be demonstrated by a positive result of the sediment of the FCU in both tests. 93% of the positive results in the DIF test could be confirmed by the EIA whereas only 77% of the positive EIA tests were also positive in the DIF test. Data on genital symptoms and the history of sexually transmitted diseases (STDs) as well as information about sexual relationships were available from all persons included in the study and did not differ between chlamydia positive and negative ones. The study demonstrates a high infection rate withC. trachomatis in mostly asymptomatic young males when using FCU for chlamydial diagnosis. Due to the discrepancy between the EIA and the DIF test, positive results of the sediment of FCU in the EIA test should be confirmed by the DIF test to eliminate false positive cases.

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Screening auf Chlamydia trachomatis im Urin bei Militärpersonal

Zusammenfassung Um bei vorwiegend asymptomatischen männlichen Jugendlichen die Infektionsrate mitChlamydia trachomatis zu bestimmen, wurde im Rahmen einer Routinekontrolle in einem Militärspital eine Chlamydienuntersuchung bei insgesamt 335 Soldaten durchgeführt. Neben der Diagnose vonC. trachomatis wurden Daten über klinische Beschwerden, die Zahl früherer genitaler Kontaktinfektionen sowie über das Sexualverhalten der untersuchten Personen erhoben. Die Chlamydiendiagnostik erfolgte durch die Antigenbestimmung des Harnsediments mittels Enzymimmunassay (EIA) und direkter Immunfluoreszenz (DIF). Der Nachweis vonC. trachomatis im Harnsediment war bei 41 (12,2%) Männern durch ein positives Resultat in beiden Testverfahren möglich. 93% der im DIF-Test positiven Ergebnisse stimmten mit jenen im EIA überein, während nur 77% der positiven Resultate des EIA im DIF-Test bestätigt werden konnten. 98,2% der untersuchten Männer waren klinisch asymptomatisch, chlamydien-positive Personen unterschieden sich weder im Sexualverhalten noch in der Zahl früherer Infektionen von jenen mit negativem Chlamydienergebnis. Die erhobenen Daten weisen auf eine hohe Zahl klinisch asymptomatischer Chlamydieninfektionen bei vorwiegend jungen Männern hin. Aufgrund der diskrepanten Ergebnisse positiver Resultate in beiden Testverfahren sollte ein Antigennachweis im EIA aus dem Harnsediment durch den DIF-Test bestätigt werden. Der Nachweis einer Chlamydieninfektion aus dem Harnsediment kann lediglich dann empfohlen werden, wenn die Gewinnung von Patientenmaterial aus dem Urethralsekret nicht möglich ist.

     

Cytochrome P450 3A4 is the major enzyme responsible for the metabolism of laquinimod, a novel immunomodulator.

In the present study, the involvement of cytochrome P450 enzyme(s) in the primary metabolism of laquinimod, a new orally active immunomodulator, has been investigated in human liver microsomes. Hydroxylated and dealkylated metabolites were formed. The metabolite formation exhibited single enzyme Michaelis-Menten kinetics with apparent KM in the range of 0.09 to 1.9 mM and Vmax from 22 to 120 pmol/mg/min. A strong correlation between the formation rate of metabolites and 6beta-hydroxylation of testosterone was obtained within a panel of liver microsomes from 15 individuals (r2 = 0.6 to 0.94). Moreover, ketoconazole and troleandomycin, specific inhibitors of CYP3A4 metabolism, demonstrated a significant inhibition of laquinimod metabolism. Furthermore, in incubations with recombinant CYP3A4, all the primary metabolites were formed. In vitro interaction studies with CYP3A4 substrates and possible concomitant medication demonstrated that laquinimod inhibits the metabolism of ethinyl estradiol with an IC50 value of about 150 microM, which is high above the plasma level of laquinimod after clinically relevant doses. Ketoconazole, troleandomycin, erythromycin, prednisolone, and ethinyl estradiol inhibited the metabolism of laquinimod, and IC50 values of 0.2, 11, 24, 87, and 235 microM, respectively, were calculated. In conclusion, the present study demonstrates that laquinimod is a low affinity substrate for CYP3A4 in human liver microsomes. The likelihood for in vivo effects of laquinimod on the metabolism of other CYP3A4 substrates is minor. However, inhibitory effects on the metabolism of laquinimod by potent and specific inhibitors of CYP3A4, such as ketoconazole, are anticipated and should be considered in the continued clinical program for laquinimod.     

Comparing Medications in a Therapeutic Area Using an NNT Model

OBJECTIVES: Clinicians are told to use the number needed to treat (NNT) to compare the benefits of therapeutic strategies, and researchers are asked to report results this way, generally without considering differences among the studies from which these were derived. METHODS: The crude NNT currently advocated is compared to the NNT standardized for a common outcome, follow-up time, study population and comparator. An NNT model for cardiovascular disease is described as an example that addresses differences among studies of secondary prevention of cardiovascular disease. Crude NNTs are compared to those obtained from the model. RESULTS: Follow-up in the 18 trials identified varied from 1.0 to 6.2 years; rates of cardiovascular events in the untreated subgroups ranged from 4.8% to 45.9%. The crude NNTs were more variable (9.1-163.7) than those obtained from the model (9.1-75.2). The effect of standardization was substantial in some cases, with proportional changes ranging from a 91% decrease to a 223% increase. CONCLUSION: Using an NNT model to account for differences in study design allows for more meaningful comparisons.     

Phylogenetic reconstruction of Mycobacterium tuberculosis within four settings of the Caribbean region: tree comparative analyse and first appraisal on their phylogeography.

In order to compare phylogenetic methods and to reconstruct the evolutionary history of the tubercle bacilli, a set of macro-array-based genotyping data of Mycobacterium tuberculosis clinical isolates (called spoligotyping for spacer oligonucleotide typing, which assays the variability of the Direct Repeat -DR- locus), was analyzed in four settings of the Caribbean region (Guadeloupe, Martinique, Cuba and Haiti). A set of 47 alleles, split into 26 shared and 21 unique alleles) representative of 321 individual M. tuberculosis clinical isolates from patients residing in the above regions was studied. The following methods (and software in brackets) were investigated: numerical taxonomy distance methods (TAXOTRON), maximum parsimony procedure (PAUP), median-joining networks (NETWORK), and nested clade analysis (GEODIS). Results using these methods were analyzed, compared and discussed. The latter method (GEODIS) was investigated in detail by introducing geographical data together with genetic variability results to detect a link between population structure and population history, and to test the null hypothesis of no association between geography and genotypes. Irrespective of the methods used, our findings demonstrate that a core structure of four families (or clades) of M. tuberculosis strains is highly prevalent within the islands studied, indirectly reflecting passed colonization history of these different settings. Specificity of M. tuberculosis genotypes in each of the islands is discussed in the light of their respective colonial and contemporary histories.     

Retrospective analysis of selective lymphadenectomy in apparent early-stage endometrial cancer.

PURPOSE: Selective lymphadenectomy is widely accepted in the management of endometrial cancer. Purported benefits are individualization of adjuvant therapy based on extent of disease and resection of occult metastases. Our goal was to assess effects of the extent of selective lymphadenectomy on outcomes in women with apparent stage I endometrial cancer at laparotomy. PATIENTS AND METHODS: Patients with endometrial cancer who received primary surgical treatment between 1973 and 2002 were identified through an institutional tumor registry. Inclusion criteria were clinical stage I/IIA disease and procedure including hysterectomy and selective lymphadenectomy (pelvic or pelvic + aortic). Exclusion criteria included presurgical radiation, grossly positive lymph nodes, or extrauterine metastases at laparotomy. Recurrence and survival were analyzed using Kaplan-Meier analysis and Cox proportional hazards model. RESULTS: Among 509 patients, the median number of lymph nodes removed was 15 (median pelvic, 11; median aortic, three). Pelvic and aortic node metastases were found in 24 (5%) of 509 patients and 11 (3%) of 373 patients, respectively. Patients with poorly differentiated cancers having more than 11 pelvic nodes removed had improved overall survival (hazard ratio [HR], 0.25; P < .0001) and progression-free survival (HR, 0.26; P < .0001) compared with patients having poorly differentiated cancers with 11 or fewer nodes removed. Number of nodes removed was not predictive of survival among patients with cancers of grade 1 to 2. Performance of aortic selective lymphadenectomy was not associated with survival. Three (27%) of 11 patients with microscopic aortic nodal metastasis are alive without recurrence. CONCLUSION: These data add to the literature documenting the possible therapeutic benefit of selective lymphadenectomy in management of patients with apparent early-stage endometrial cancer.