IL-15 enhances the antitumor effect of human antigen-specific CD8+ T cells by cellular senescence delay.

Optimal expansion protocols for adoptive human T-cell therapy often include interleukin (IL)-15; however, the mechanism by which IL-15 improves the in vivo antitumor effect of T cells remains to be elucidated. Using human T cells generated from HLA-A2+ donors against novel T-cell epitopes derived from the human U266 myeloma cell line Ig light chain V-region (idiotype) as a model, we found that T cells cultured with IL-15 provided superior resistance to tumor growth in vivo, compared with IL-2, after adoptive transfer into immunodeficient hosts. This effect of IL-15 was associated with delayed/reversed senescence in tumor antigen-specific memory CD8+ T cells mediated through downregulation of P21WAF1, P16INK4a, and P53 expression. Compared to IL-2, IL-15 stimulation dramatically activated JAK3-STAT5 signaling and inhibited the expression of DNA damage genes. Thus, our study elucidates a new mechanism for IL-15 in the regulation of STAT signaling pathways and CD8+ T-cell senescence.

Nilotinib combined with multiagent chemotherapy for newly diagnosed Philadelphia-positive acute lymphoblastic leukemia.

We investigated the effects of nilotinib plus multiagent chemotherapy, followed by consolidation/maintenance or allogeneic hematopoietic cell transplantation (allo-HCT) for adult patients with newly diagnosed Philadelphia-positive (Ph-pos) acute lymphoblastic leukemia (ALL). Study subjects received induction treatment that comprised concurrent vincristine, daunorubicin, prednisolone, and nilotinib. After achieving complete hematologic remission (HCR), subjects received either 5 courses of consolidation, followed by 2-year maintenance with nilotinib, or allo-HCT. Minimal residual disease (MRD) was assessed at HCR, and every 3 months thereafter. The molecular responses (MRs) were defined as MR3 for BCR-ABL1/G6PDH ratios ≤10(-3) and MR5 for ratios <10(-5). Ninety evaluable subjects, ages 17 to 71 years, were enrolled in 17 centers. The HCR rate was 91%; 57 subjects received allo-HCT. The cumulative MR5 rate was 94%; the 2-year hematologic relapse-free survival (HRFS) rate was 72% for 82 subjects that achieved HCR, and the 2-year overall survival rate was 72%. Subjects that failed to achieve MR3 or MR5 were 9.1 times (P = .004) or 6.3 times (P = .001) more prone to hematologic relapse, respectively, than those that achieved MR3 or MR5. MRD statuses just before allo-HCT and at 3 months after allo-HCT were predictive of 2-year HRFS. Adverse events occurred mainly during induction, and most were reversible with dose reduction or transient interruption of nilotinib. The combination of nilotinib with high-dose cytotoxic drugs was feasible, and it effectively achieved high cumulative complete molecular remission and HRFS rates. The MRD status at early postremission time was predictive of the HRFS. This trial was registered at www.clinicaltrials.gov as #NCT00844298.

Dynamics of a wellness program: a conservation of resources perspective.

We leverage conservation of resources theory to explain possible dynamics through which a holistic wellness program results in positive longer-term outcomes. Specifically, we hypothesize that wellness self-efficacy at the end of a wellness program will create a positive resource gain spiral, increasing psychological availability (a sense of having cognitive, physical, and emotional resources to engage oneself) 6 months later, and career satisfaction, 1 year later. To test these hypotheses, using a time-lagged with control group design, we gathered questionnaire data from 160 Episcopal priests who participated in a 10-day off-site wellness program. We developed a scale measuring self-efficacy in the 4 wellness areas the program was designed to improve: physical, spiritual, financial, and vocational. Our findings provide evidence from a field setting of a relatively untested tenet of conservation of resources theory, resource gain spirals. The wellness program that we studied served as an opportunity for participants to gain new resources in the form of wellness self-efficacy, which in turn helped participants experience positive outcomes over time. We discuss theoretical and practical implications of the findings.

Up-front allogeneic hematopoietic cell transplantation in acute myeloid leukemia arising from the myelodysplastic syndrome.

In patients with secondary acute myeloid leukemia (s-AML) arising from the myelodysplastic syndrome (MDS), treatment outcome is unsatisfactory. We compared up-front allogeneic hematopoietic cell transplantation (HCT) to induction chemotherapy (IC) as an initial treatment in patients with s-AML arising from MDS. This retrospective study included 85 patients who were diagnosed with s-AML arising from MDS; 11 patients proceeded to up-front HCT without IC (HCT group) and 74 received IC (IC group) as an initial treatment for s-AML, 28 of whom subsequently underwent HCT. In the IC group, 41.9% achieved complete remission (CR) compared to 81.8% in the HCT group (p = 0.013). The HCT group showed a significantly longer event-free survival (EFS) than the IC group (median 29.2 vs. 5.2 months, p = 0.042). Overall survival of the HCT group was higher than that of the IC group, but the difference was not statistically significant (median 34.6 vs. 7.6 months, p = 0.149). After adjustment for other clinical factors, outcome in the HCT group was significantly better than in the IC group in terms of CR rate (hazard ratio, HR, 11.195; p = 0.007) and EFS (HR, 0.384; p = 0.029). Up-front HCT is a viable option in s-AML arising from MDS if an appropriate donor is available.

Clinical outcome after failure of hypomethylating therapy for myelodysplastic syndrome.

Around half of patients with myelodysplastic syndrome (MDS) fail to respond to hypomethylating therapy (HMT) and most responders progress within 2 yr. Retrospective studies report poor outcomes after HMT failure. Here, we analyzed the outcomes of patients suffering HMT failure. Of 149 patients with MDS treated with either azacitidine or decitabine, 91 who experienced HMT failure were included in the study. Median overall survival (OS) was 12.1 months: 16.2 months for lower-risk MDS and 9.3 months for higher-risk MDS. Disease status and progression to acute myeloid leukemia (AML) at the time of HMT failure were independent prognostic factors for OS. Fifty-four patients received one or more treatment modalities, and 23 received allogeneic hematopoietic cell transplantation (HCT). The objective response to non-transplant treatments was poor (11-17%), whereas 17 transplanted patients showed a complete response. OS probability at 2 yr post-HCT was 60.9%: 78.6% for patients receiving HCT during MDS and 33.3% for those receiving HCT after developing AML. In conclusion, the clinical outcome of patients after HMT failure was poor. Long-term disease-free survival was observed in approximately 50% of patients who received allogeneic HCT. Therefore, allogeneic HCT should be performed early in appropriate patients, and particularly before progression to AML.

Allogeneic hematopoietic cell transplantation in adult patients with myelodysplastic/myeloproliferative neoplasms.

BACKGROUND: In adults, the 2 main types of myelodysplastic/myeloproliferative neoplasms (MDS/MPN) are chronic myelomonocytic leukemia (CMML) and atypical chronic myeloid leukemia (aCML). Both are associated with a poor prognosis. Allogeneic hematopoietic cell transplantation (HCT) is the only known curative treatment modality for these diseases, but data on outcomes following such treatment are limited. We analyzed the outcomes of patients with MDS/MPN after allogeneic HCT. METHODS: This retrospective study included 10 patients with MDS/MPN who received allogeneic HCT at Asan Medical Center from 2002 to 2010. Of these 10 patients, 7 had CMML, 2 had aCML, and 1 had unclassifiable MDS/MPN. Five patients received a myeloablative conditioning (MAC) regimen (busulfan-cyclophosphamide), and 5 received reduced-intensity conditioning (RIC) regimen. RESULTS: Neutrophil engraftment was achieved in all patients. After a median follow-up of 47.5 months among surviving patients, 4 had relapsed and 5 had died. There was only 1 treatment-related death. The 5-year rates of overall, relapse-free, and event-free survival were 42.2%, 51.9%, and 46.7%, respectively. Relapse was the leading cause of treatment failure, and all relapses were observed in patients who had received RIC and who did not develop chronic graft-versus-host disease. CONCLUSION: Allogeneic HCT can induce durable remission in patients with MDS/MPN, but RIC cannot replace MAC in patients eligible for myeloablative treatments.

Prognostic implications of CD14 positivity in acute myeloid leukemia arising from myelodysplastic syndrome.

Secondary acute myeloid leukemia (s-AML) arising from myelodysplastic syndrome (MDS) shows different clinical features from de novo AML. We assessed the prognostic significance of immunophenotypic markers in patients with s-AML arising from MDS. Sixty-five adults diagnosed with AML arising from MDS between 1996 and 2010 were retrospectively analyzed. Immunophenotyping was performed for markers including CD3, CD7, CD10, CD13, CD14, CD19, CD33, CD34, CD41, CD45, CD56, CD65, CD117, HLA-DR, and TdT. Of these immunophenotypic markers, only CD14 positivity was significantly associated with lower complete remission rate (P = 0.034) and significantly shorter overall survival (OS, P < 0.001) and event-free survival (EFS, P < 0.001) on univariate analysis. On multivariate analysis, these differences remained significant in terms of OS [hazard ratio (HR) 4.49; P < 0.001] and EFS (HR 4.06; P < 0.001). Other significant prognostic variables included age ≥60 years [shorter OS (P = 0.003) and EFS (P = 0.020)], higher WBC count (>60,000/µL) [shorter OS (P < 0.001) and EFS (P = 0.001)], and poor cytogenetic risk group [shorter OS (P = 0.005)]. CD14 expression on leukemic blasts is an independent prognostic factor for survival outcomes in patients with AML arising from MDS.

Comparison of 7-day azacitidine and 5-day decitabine for treating myelodysplastic syndrome.

Two DNA methyltransferase inhibitors, azacitidine and decitabine, are currently approved for the treatment of myelodysplastic syndrome (MDS). Choosing between these drugs is an important practical issue. In this retrospective study, patients receiving AZA-7d (azacitidine 75 mg/m2 subcutaneously × 7 days, n = 75) or DEC-5d (decitabine 20 mg/m2 intravenously × 5 days, n = 74) were compared. The rates of hematologic response (complete response [CR]/partial response [PR]/marrow CR) were 12.0 % (AZA-7d) vs. 29.7 % (DEC-5d) (P = 0.008), and the overall response rates (CR/PR/marrow CR/hematologic improvement) were 52.0 % (AZA-7d) vs. 63.5 % (DEC-5d) (P = 0.155). Grade 3 or higher neutropenia occurred more frequently with DEC-5d (79.6 %) than with AZA-7d (72.2 %) (P = 0.040). Overall survival probabilities at 2 years were 42.1 % (AZA-7d) vs. 42.2 % (DEC-5d) (P = 0.944). Subgroup analysis revealed that AZA-7d associated with higher survival rates than DEC-5d in patients whose MDS duration exceeded 1 year or who had poor performance status. In conclusion, both AZA-7d and DEC-5d regimens were effective in treating patients with MDS. However, the two regimens differed in terms of clinical responses and toxicities. One hypomethylating regimen may be superior to the other regimen in particular subgroups.

Randomized trial of myeloablative conditioning regimens: busulfan plus cyclophosphamide versus busulfan plus fludarabine.

PURPOSE: We conducted a phase III randomized clinical trial to compare two myeloablative conditioning regimens for allogeneic hematopoietic cell transplantation (HCT) in patients with leukemia and myelodysplastic syndrome. PATIENTS AND METHODS: After randomization, 64 patients received busulfan (3.2 mg/kg per day × 4 days) plus cyclophosphamide (60 mg/kg per day × 2 days; BuCy), and 62 patients received busulfan (same dose and schedule) plus fludarabine (30 mg/m(2) per day × 5 days; BuFlu). RESULTS: The median age was 41 years (range, 17 to 59 years). Five patients in the BuFlu arm experienced graft failure (primary, n = 1; secondary, n = 4). At 4 weeks after HCT, the median percentage of recipient hematopoietic chimerism was significantly greater in the BuFlu arm (0% v 5.5%; P < .001), and complete donor chimerism was greater in the BuCy arm (97.2% v 44.4%; P < .001). Severe (grade 3 or higher) infection and gastrointestinal adverse events were significantly more common in the BuCy arm, but the frequencies of hepatic adverse events were similar in the two arms. Nonrelapse mortality was similar in the two arms, but the BuCy arm had better overall survival (OS), relapse-free survival (RFS), and event-free survival (EFS; OS at 2 years, 67.4% v 41.4%, P = .014; RFS, 74.7% v 54.9%, P = .027; EFS, 60.7% v 36.0%, P = .014). CONCLUSION: Our results indicate that the BuFlu regimen is not a suitable replacement for the BuCy regimen in young adults who are eligible for myeloablative conditioning therapy for allogeneic HCT.

Two cycles of the PS-341/bortezomib, adriamycin, and dexamethasone combination followed by autologous hematopoietic cell transplantation in newly diagnosed multiple myeloma patients.

INTRODUCTION: Treatment with 3-6 cycles of PS-341/bortezomib, adriamycin, and dexamethasone (PAD) has been explored in terms of induction therapy prior to autologous stem cell transplantation (ASCT) in patients with multiple myeloma (MM). We evaluated the effects of two cycles of PAD given before ASCT. PATIENTS AND METHODS: Patients received two 21-d cycles of PAD (bortezomib 1.3 mg/m(2) × 4 d, adriamycin 9 mg/m(2) × 4 d, and dexamethasone 40 mg × 4 d × 2). Starting on day 12 of cycle 2, patients were given subcutaneous granulocyte-colony stimulating factor to mobilize peripheral blood stem cells (PBSCs). Following PBSC harvesting, ASCT was performed using high-dose melphalan, followed by thalidomide. RESULTS: A total of 32 patients were enrolled. Of 31 who completed two cycles of PAD, 25 (81%) achieved a partial response (PR) or better. Major adverse events were cytopenia, with grade I/II neurotoxicity evident during 4.8% of PAD cycles. Two patients were withdrawn from the study prior to PBSC collection. Thirty patients showed successful mobilization of PBSCs and underwent ASCT, with all 30 showing adequate neutrophil and platelet recovery. Following ASCT, 14 patients (47%) achieved a complete response (CR), 8 (27%) a very good partial response (VGPR), and 6 (20%) PR. Thalidomide was given to 25 patients after ASCT, as maintenance therapy. Twelve patients showed better responses after administration of thalidomide, and a total of 21 patients (70%) achieved CR. The 5-yr probabilities of overall and progression-free survival were 71.1% and 23.5%, respectively. CONCLUSION: A short course of PAD was effective as an induction treatment before ASCT in patients newly diagnosed with MM. Prospective comparisons with longer courses of such treatment are needed.

Single nucleotide polymorphism of Wilms' tumor 1 gene rs16754 in Korean patients with cytogenetically normal acute myeloid leukemia.

A recent study from Germany showed that WT1 single nucleotide polymorphism (SNP) rs16754 was an independent prognostic factor in cytogenetically normal acute myeloid leukemia (CN-AML). We analyzed clinical impact of the WT1 rs16754 genotype on disease characteristics and outcomes in Korean patients with CN-AML. A total of 73 patients with CN-AML were included in the study. All patients received standard induction chemotherapy and their bone marrow or peripheral blood samples were cryopreserved at the time of diagnosis. WT1 exons 7 and 9 were amplified using polymerase chain reaction and directly sequenced. The genotype frequency for WT1 rs16754 was 6.8% for AA, 39.7% for GA, and 53.4% for GG. G was a minor allele in German population, whereas it was a major allele in Korean (13.7% vs. 73.3%, P < 0.001). Complete remission was induced in 85.3% of patients with GA/AA and 84.6% of those with GG (P = 0.936). Survival rates were also similar between patients with GG and those with GA/AA. Asian and Western populations exhibited significant differences in allele and genotype frequencies of WT1 rs16754. In Korean patients with CN-AML, WT1 SNP rs16754 had no significant impact on clinical outcomes and further investigations are needed to define prognostic implication of WT1 SNP rs16754 in CN-AML.

Clinical features of adult patients with secondary hemophagocytic lymphohistiocytosis from causes other than lymphoma: an analysis of treatment outcome and prognostic factors.

Although hemophagocytic syndrome (HS) featuring secondary hemophagocytic lymphohistiocytosis (HLH) has a grave prognosis, little is known about the natural course of the disease. Patients who showed the clinical features of HLH as well as tissue-proven hemophagocytosis when seen at Asan Medical Center between 1999 and 2010 were included in this analysis. Patients with proven lymphoma were excluded. The median age of our 23 study patients was 49 years. Epstein-Barr virus was suspected to have caused HS in 16 (70%) patients and hepatitis A virus in one patient. Twenty-two patients were treated, 13 according to the HLH protocol and nine using immunosuppressive agents such as corticosteroid and/or cyclosporine. Five patients undertook allogeneic hematopoietic cell transplantation (HCT) during their treatment-dependent relapse (n = 4) or responsive status (n = 1). After the median follow-up of 180 days, 17 (74%) died and six (26%) were alive. The median time from initial presentation until death was 41 days among those patients who died. The serum fibrinogen level ≥166 mg/dL determined at the initial visit was significantly associated with the survival time according to univariate analysis. The low histiocyte proportion in bone marrow and early initiation of treatment tended to correlate with a favorable outcome. On multivariate analysis, serum fibrinogen ≥166 mg/dL (hazard ratio, 0.175, P = 0.018) was an independent clinical factor for determining the patient survival time. Despite appropriate patient management, the outcome of HS featuring HLH was grave. The serum fibrinogen level at the initial presentation was significant, and selected patients obtained some benefit from allogeneic HCT.

Pre-engraftment graft-versus-host disease after allogeneic hematopoietic cell transplantation for acute leukemia.

OBJECTIVES: Acute graft-versus-host disease (GVHD) usually occurs with neutrophil engraftment following allogeneic hematopoietic cell transplantation (HCT), but it can also occur before engraftment. We intended to analyze the effects of timing of acute GVHD on leukemia relapse and mortality. METHODS: The outcomes of pre- and postengraftment GVHD were investigated in 384 patients who underwent allogeneic HCT for acute leukemia. RESULTS: Acute GVHD occurred in 100 patients, pre-engraftment in 22 and postengraftment in 78. Compared with postengraftment GVHD, pre-engraftment GVHD was more severe, as assessed by overall grade, with more frequent and more severe skin involvement and higher incidences of non-infectious fever, diarrhea, hepatic dysfunction, renal insufficiency, and weight gain. Compared with patients without acute GVHD, those with postengraftment GVHD had lower cumulative incidence of relapse [CIR; hazard ratio (HR), 0.470; P=0.006] and higher cumulative incidence of non-relapse mortality (CINRM; HR, 2.568; P<0.001), while those with pre-engraftment GVHD had similar CIR (HR, 0.815; P=0.059) and higher CINRM (HR, 2.872; P=0.036). Overall survival of patients with pre-engraftment GVHD was lower than that of those without acute GVHD (HR, 1.976; P=0.017), which was similar to that of those with postengraftment GVHD (HR, 0.969; P=0.878). Separate analyses of the effects of timing of acute GVHD on post-transplant outcomes in patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) showed similar trends. CONCLUSION: Pre-engraftment GVHD might be a 'cytokine storm' type syndrome rather than 'real' GVHD, indicating the need for separate analyses of pre- and postengraftment GVHD in future trials.

A prediction model for complete remission upon reinduction for patients with acute myeloid leukemia after failure of anthracycline and cytarabine standard chemotherapy.

The aim of this study was to investigate clinical parameters that might predict complete remission (CR) following reinduction therapy in patients with acute myeloid leukemia (AML). We retrospectively analyzed outcomes in 142 patients who failed to achieve CR with standard anthracycline-plus-cytarabine induction chemotherapy (IND1) and who received the same regimen as a reduction therapy (IND2). The CR rate after reinduction was 54%. Multivariate analysis showed that the presence of peripheral blood (PB) blasts on the day of commencement of IND2 and ≥20% blasts in an interim BM sample taken during IND1 (C1-INT-BM) were independent risk factors for failure of remission. Our scoring system for prediction of CR after reinduction (CRAR score) included a favorable chromosome risk group, absence of PB blasts on the day of commencement of IND2, and ≤21% blasts in the C1-INT-BM. This scoring system predicted that the rates of CR in the four subgroups would be 92.1%, 68.9%, 29.5%, and 7.3%, respectively, in good agreement with observed CR rates. The CRAR scoring model might be associated with the possibility of achieving CR after reinduction, and may be helpful in choosing alternative strategy for AML patients refractory to standard induction chemotherapy, although further prospective validation is required.

Influence of GST gene polymorphisms on the clearance of intravenous busulfan in adult patients undergoing hematopoietic cell transplantation.

Intravenous (i.v.) busulfan can produce a more consistent pharmacokinetic profile than oral formulations can, but nonetheless, significant interpatient variability is evident. We investigated the influence of polymorphisms of 3 GST isozyme genes (GSTA1, GSTM1, and GSTT1) on i.v. busulfan clearance. Fifty-eight adult patients who received 3.2 mg/kg/day of busulfan as conditioning for hematopoietic cell transplantation were included in this study. Stepwise multiple linear regression demonstrated that GSTA1 variant GSTA1∗B (P = .004), GSTM1/GSTT1 double-null genotype (P = .039), and actual body weight (P = .001) were significantly associated with lower clearance of i.v. busulfan. A trend test analyzing the overall effect of GST genotype on busulfan pharmacokinetics, combining GSTA1 gene polymorphism and the number of GSTM1- and GSTT-null genotypes, showed a significant correlation between GST genotype and busulfan clearance (P = .001). The clearance of i.v. busulfan was similar between patients with GSTA1∗A/∗A and GSTM1/GSTT1 double-null genotypes and those with GSTA1∗A/∗B and GSTM1/GSTT1 double-positive genotypes. In conclusion, a pharmacogenetic approach using GST gene polymorphisms may be valuable in optimizing the i.v. busulfan dosage scheme. Our results also highlight the importance of including polygenic analyses and addressing interactions among isozyme genes in pharmacogenetic studies.

Clinical evaluation of efficacy and tolerability of HMC05 in healthy subjects with normal and high-normal blood pressure: a pilot study.

HMC05, a formulation containing eight different herbal extracts, has been used widely for several thousand years in China, Japan, and Korea as a remedy for hypertension and headache. Although its anti-inflammatory effects in mouse monocytic cell lines and anti-atherosclerotic effects in apoE-knockout mice have been reported, the pharmacodynamic effects of HMC05 in human subjects have not yet been investigated. We evaluated the efficacy and tolerability of this drug in 14 healthy male Korean subjects with normal or high-normal blood pressure (BP) in a randomized, single-blind, crossover study with a 2-week washout period. Four 500-mg tablets of HMC05 or placebo were orally administered three times daily to nine subjects with normal BP and five subjects with high-normal BP for 4 weeks. To assess the pharmacodynamic effects of HMC05, levels of high-sensitivity C-reactive protein and homocysteine, BP, and flow-mediated vasodilation were measured before and after the 4-week medication period with evaluation of tolerability. All 14 subjects completed the study, and HMC05 was well tolerated with no significant adverse events. HMC05 did not exhibit a significant BP-lowering effect in either BP group, and there were no significant differences in other pharmacodynamic values after HMC05 or placebo administration in the two groups. Further study is needed to evaluate the efficacy and tolerability of HMC05 in an adequate number of patients with hypertension.

Significance of fibrinogen, D-dimer, and LDH levels in predicting the risk of bleeding in patients with acute promyelocytic leukemia.

This study was performed to determine whether any clinical parameters measured at diagnosis or during treatment are associated with bleeding in patients with acute promyelocytic leukemia (APL). Ninety patients with APL who were treated with all-transretinoic acid (ATRA) and anthracycline-based chemotherapy were analyzed. There were 24 significant bleeding events, classified by onset as 'hyperacute' (n = 5), 'acute' (n = 11) and 'late' (n = 8). Fifteen patients (17%) died because of bleeding. A poor fibrinogen (FBG) response during ATRA and chemotherapy was associated with an increased risk of bleeding (p = 0.003). Increased LDH and decreased FBG levels were associated with an increased incidence of bleeding, and low PLT count was correlated with death from bleeding. Our findings suggest that LDH/FBG levels and FBG response may be associated with morbidity and mortality from bleeding in patients with APL.

Decreased incidence of febrile episodes with antibiotic prophylaxis in the treatment of decitabine for myelodysplastic syndrome.

We analyzed the role of antibiotic prophylaxis during decitabine treatment for MDS. The primary endpoint was the incidence of febrile episodes. The total number of decitabine cycles given to 28 patients was 131, and febrile episodes occurred in 15 cycles (11.5%). Antibiotic prophylaxis was orally administered in 95 cycles (72.5%). Febrile episodes were significantly less frequent among patients who received antibiotic prophylaxis (7.4%) than in those without prophylaxis (22.2%) (P=0.017). In conclusion, antibiotic prophylaxis reduced the incidence of febrile episodes in patients who received decitabine treatment for MDS, especially at earlier cycles and in the presence of severe cytopenia.

Rapid determination of finasteride in human plasma by UPLC-MS/MS and its application to clinical pharmacokinetic study.

A rapid, specific, and sensitive method utilizing reversed-phase ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was developed and validated to determine finasteride levels in human plasma. The plasma samples were prepared by liquid-liquid extraction with ethyl acetate, evaporation, and reconstitution. MS/MS analyses were performed on a triple-quadrupole tandem mass spectrometer by monitoring protonated parent-->daughter ion pairs at m/z 373-->305 for finasteride and m/z 237-->194 for carbamazepine (internal standard, IS). The method was validated with respect to linearity, recovery, specificity, accuracy, precision, and stability. The method exhibited a linear response from 0.1 to 30 ng/mL (r(2)>0.998). The limit of quantitation for finasteride in plasma was 0.1 ng/mL. The relative standard deviation (RSD) of intra- and inter-day measurements was less than 15% and the method was accurate within -6.0% to 2.31% at all quality-control levels. The mean extraction recovery was higher than 83% for finasteride and 84% for the IS. Plasma samples containing finasteride were stable under the three sets of conditions tested and the processed samples were stable up to 29 h in an autosampler at 5 degrees C. Detection and quantitation of both analytes within 3 min make this method suitable for high-throughput analyses. The method was successfully applied to a pharmacokinetic study of finasteride in healthy volunteers following oral administration.

Bioequivalence and tolerability of two clopidogrel salt preparations, besylate and bisulfate: a randomized, open-label, crossover study in healthy Korean male subjects.

BACKGROUND: Clopidogrel, a potent antiplatelet agent, reduces the risk for thrombotic events in patients with atherothrombotic diseases. Clopidogrel is marketed primarily as a bisulfate salt. A different salt preparation of clopidogrel, clopidogrel besylate, has been developed and might provide an additional treatment option for patients. OBJECTIVE: The aim of this study was to compare the pharmacokinetic, pharmacodynamic, and tolerability profiles of clopidogrel besylate with those of clopidogrel bisulfate to determine bioequivalence for the purposes of marketing approval. METHODS: A randomized, open-label, 2-period, single- and multiple-dose, comparative crossover study was conducted in healthy Korean male subjects. The subjects received either clopidogrel bisulfate or clopidogrel besylate as a single 300-mg oral loading dose (day 1) followed by a 75-mg/d (once daily) maintenance dose on days 2 to 6. After a 15-day washout period, subjects were administered the alternative salt preparation according to the same protocol. The plasma concentrations of clopidogrel and its primary metabolite (SR26334) were assessed using high-performance liquid chromatography/tandem mass spectrometry after administration of the loading dose. The platelet aggregation response to 10-mumol/L adenosine diphosphate was measured using turbidometric aggregometry during the single- and multiple-dosing periods and at steady state (day 6). Tolerability was monitored using physical examination, including vital sign measurements, and laboratory analysis. RESULTS: Forty-four subjects were enrolled and completed the study (mean [SD] age, 24.3 [2.7] years; weight, 70.0 [8.2] kg). The mean values for C(max), T(max), and AUC(0-t) with clopidogrel (parent drug) of clopidogrel besylate (5.2 ng/mL, 0.9 hour, and 10.1 ng/mL/h, respectively) were similar to those with clopidogrel bisulfate (5.4 ng/mL, 0.9 hour, and 10.3 ng/mL/h). The mean values for Cmax, AUC(0-t), and AUC(0-infinity) with the SR26334 of clopidogrel besylate (10.9 microg/mL, 38.8 microg/mL/h, and 43.0 microg/mL/h, respectively) were not significantly different from those with the SR26334 of clopidogrel bisulfate (11.9 microg/mL, 40.6 microg/mL/h, and 43.8 microg/mL/h). The mean values for maximal antiplatelet effect (Emax) and area under the time-effect curve (AUEC) with the 2 clopidogrel salt preparations were as follows: clopidogrel besylate, 58.8 h . % and 4299.1 h . % inhibition, respectively; and clopidogrel bisulfate, 61.7 h . % and 4406.9 h . % inhibition; these differences were not statistically significant. The 90% CIs for the ratios of the log-transformed C(max), AUC, E(max), and AUEC values were within the predetermined bioequivalence range of 80% to 125%. Three adverse events (6.8%) were reported during the study and included abdominal discomfort (1 subject [2.3%] in the group that received clopidogrel bisulfate), easy fatigability (1 subject [2.3%] immediately before administration of loading dose of clopidogrel besylate), and thrombocytopenia (1 subject [2.3%] in the group receiving the clopidogrel bisulfate). All adverse events were transient and mild. CONCLUSIONS: In these healthy Korean male subjects, the differences in the pharmacokinetic and pharmacodynamic properties between the 2 clopidogrel salt preparations did not reach statistical significance and met the regulatory requirements for bioequivalence. Both preparations were well tolerated.