Glutathione S-transferase activity influences busulfan pharmacokinetics in patients with beta thalassemia major undergoing bone marrow transplantation

Busulfan, at a dose of 16 mg/kg, is widely used in combination with cyclophosphamide as a conditioning regimen for patients undergoing bone marrow transplantation. Wide interindividual variation in busulfan kinetics and rapid clearance of the drug have been reported, especially in children. Some of the factors contributing to interpatient variability have been identified. They include circadian rhythms, age, disease, drug interaction, changes in hepatic function, and busulfan bioavailability. In this study, we demonstrate that hepatic glutathione S-transferase (GST) activity correlates negatively with busulfan maximum and minimum concentrations (Pearson's correlation r = -0.74 and -0.77, respectively) and positively with busulfan clearance (Pearson's correlation r = 0.728) in children with thalassemia major in the age range of 2 to 15 years. We also found that plasma alpha GST levels were 5 to 10 times higher in patients with thalassemia than in normal controls and age-matched leukemic patients, either reflecting extensive liver damage, elevated expression of the enzyme, or both in thalassemic patients. Plasma alpha GST concentrations showed a similar correlation with busulfan kinetic parameters to that observed for hepatic GST. The status of hepatic GST activity accounts, at least in part, for the observed interindividual variation in busulfan kinetics, while the observed association with plasma alpha GST is difficult to explain at present

Arsenic trioxide in the treatment of newly diagnosed acute promyelocytic leukemia: a single center experience

Arsenic trioxide (As2O3) has been found effective in the treatment in the treatment of acute promyelocytic leukemia (APML). Most studies with As2O3 involve patients with APML who have relapsed following standard therapy. Between January 1998 and July 2000, 14 patients were recruited for an ongoing trial of As2O3 in the treatment of newly diagnosed APML. Arsenic trioxide was administered at a dose of 10 mg/day until complete remission (CR) was achieved. Afterward, a consolidation course and a maintenance schedule consisting of As2O3 as a single agent were administered over 6 months. There were 3 early deaths related to intra-cerebral hemorrhage: two on day 3 and one on day 4. Of the 11 evaluable patients, one died on day 21 secondary to uncontrolled sepsis, while the remaining 10 (91%) have attained CR. The average time to CR was 52.3 days (range: 34-70 days). One patient developed an isolated central nervous system (CNS) relapse and subsequently went into a second CR following therapy with triple intrathecal chemotherapy, cranial irradiation, and an additional 4-week course of systemic As2O3. This patient, as well as the remaining nine, has continued to remain in CR at a median follow up of 15 months (range: 2-33 months). Eight out of 10 patients achieved molecular remission at variable periods during their consolidation and maintenance schedules. One patient developed an ATRA syndrome and was administered daunorubicin (40 mg/day) for 2 days. The side effects with this therapy were minimal and did not require cessation of therapy in any patient. There was no significant hepatic toxicity. In our experience, arsenic trioxide is effective in inducing and maintaining remission in patients with APML with minimal side effects. The optimal regimen and total dose required need to be defined

Glutathione S-transferase M1 polymorphism: a risk factor for hepatic venoocclusive disease in bone marrow transplantation

Hepatic venoocclusive disease (HVOD) in bone marrow transplantation (BMT) is attributed to toxicity of cytoreductive agents, especially busulfan and cyclophosphamide, in the conditioning therapy. Busulfan, as well as the metabolites of cyclophosphamide, are conjugated with glutathione (GSH), catalyzed by enzymes of the glutathione S-transferase (GST) family. To assess the impact of polymorphisms of the GST genes, GSTM1 and GSTT1, on the risk of HVOD, we evaluated 114 consecutive patients with -thalassemia major undergoing BMT. There was a significantly increased incidence of HVOD in patients with the GSTM1-null genotype compared with those with the GSTM1-positive genotype (46.5% vs 18.3%; P = .001). Pharmacokinetic analysis in these patients showed that the clearance of busulfan was higher and first-dose steady-state concentration was lower among those with HVOD (0.403 ± 0.06 vs 0.33 ± 0.071 L/h/kg, Student t test P value = .000 01; and 508 ± 125 vs 656 ± 255 ng/mL, t test P value = .001, respectively). We conclude that the GSTM1-null genotype predisposes to HVOD, and the sinusoidal endothelial cells and hepatocyte damage may be mediated by metabolites of busulfan through depletion of the cellular GSH pool

Single-agent arsenic trioxide in the treatment of newly diagnosed acute promyelocytic leukemia: durable remissions with minimal toxicity

Arsenic trioxide, as a single agent, has proven efficacy in inducing molecular remission in patients with acute promyelocytic leukemia (APL). There is limited long-term outcome data with single-agent As2O3 in the management of newly diagnosed cases of APL. Between January 1998 to December 2004, 72 newly diagnosed cases of APL were treated with a regimen of single-agent As2O3 at our center. Complete hematologic remission was achieved in 86.1%. At a median follow-up of 25 months (range: 8-92 months), the 3-year Kaplan-Meier estimate of EFS, DFS, and OS was 74.87% ± 5.6%, 87.21% ± 4.93%, and 86.11% ± 4.08%, respectively. Patients presenting with a white blood cell (WBC) count lower than 5 × 109/L and a platelet count higher than 20 × 109/L at diagnosis (n = 22 [30.6%]) have an excellent prognosis with this regimen (EFS, OS, and DFS of 100%). The toxicity profile, in the majority, was mild and reversible. After remission induction, this regimen was administered on an outpatient basis. Single-agent As2O3, as used in this series, in the management of newly diagnosed cases of APL, is associated with responses comparable with conventional chemotherapy regimens. Additionally, this regimen has minimal toxicity and can be administered on an outpatient basis after remission induction

Efficient deletion of microRNAs using CRISPR/Cas9 with dual guide RNAs

MicroRNAs (miRNAs) are short non-coding RNAs that play crucial roles in gene regulation, exerting post-transcriptional silencing, thereby influencing cellular function, development, and disease. Traditional loss-of-function methods for studying miRNA functions, such as miRNA inhibitors and sponges, present limitations in terms of specificity, transient effects, and off-target effects. Similarly, CRISPR/Cas9-based editing of miRNAs using single guide RNAs (sgRNAs) also has limitations in terms of design space for generating effective gRNAs. In this study, we introduce a novel approach that utilizes CRISPR/Cas9 with dual guide RNAs (dgRNAs) for the rapid and efficient generation of short deletions within miRNA genomic regions. Through the expression of dgRNAs through single-copy lentiviral integration, this approach achieves over a 90% downregulation of targeted miRNAs within a week. We conducted a comprehensive analysis of various parameters influencing efficient deletion formation. In addition, we employed doxycycline (Dox)-inducible expression of Cas9 from the AAVS1 locus, enabling homogeneous, temporal, and stage-specific editing during cellular differentiation. Compared to miRNA inhibitory methods, the dgRNA-based approach offers higher specificity, allowing for the deletion of individual miRNAs with similar seed sequences, without affecting other miRNAs. Due to the increased design space, the dgRNA-based approach provides greater flexibility in gRNA design compared to the sgRNA-based approach. We successfully applied this approach in two human cell lines, demonstrating its applicability for studying the mechanisms of human erythropoiesis and pluripotent stem cell (iPSC) biology and differentiation. Efficient deletion of miR-451 and miR-144 resulted in blockage of erythroid differentiation, and the deletion of miR-23a and miR-27a significantly affected iPSC survival. We have validated the highly efficient deletion of genomic regions by editing protein-coding genes, resulting in a significant impact on protein expression. This protocol has the potential to be extended to delete multiple miRNAs within miRNA clusters, allowing for future investigations into the cooperative effects of the cluster members on cellular functions. The protocol utilizing dgRNAs for miRNA deletion can be employed to generate efficient pooled libraries for high-throughput comprehensive analysis of miRNAs involved in different biological processes

Allogeneic stem cell transplantation for thalassemia major in India

Allogeneic stem cell transplantation (allo-SCT) is the only currently available curative treatment for thalassemia major. Since it was first done in 1981, several thousand patients have benefited from it and it is now possible to offer this treatment in different parts of the world with good results. With better risk stratification and supportive care, the results of allo-SCT are now very good even in high risk patients who have significant iron overload related organ dysfunction. The improvements have mainly been in the conditioning strategies with less toxic myeloablation and management of the complications of SCT. However, several challenges remain. Transplant related complications still cause significant morbidity and mortality. There is data to show that the results of transplantation as best if done in well transfused and chelated patients <7 years of age. As only a third of the patients will have a matched related donor, there is need for investigating SCT with alternative donors. Experience with SCT for thalassemia major from matched unrelated donors or haplo-identical donors is still limited but needs further exploration. Adequate management needs to be provided post-SCT for all pre-existing complications particularly iron chelation to prevent further organ dysfunction. Systematic follow-up is needed to measure long term outcomes. The biggest challenges in India are the cost of this treatment and access to centres capable of providing this treatment. With greater support from the government, health insurance and philanthropic programs, there has been a rapid increase in the number of SCTs for thalassemia major in India. The number centres providing this treatment are also increasing making this curative treatment more widely available in India

Arsenic Trioxide in the Treatment of Newly Diagnosed Acute Promyelocytic Leukemia: A Single Center Experience

Arsenic trioxide (As 2 O 3 ) has been found effective in the treatment in the treatment of acute promyelocytic leukemia (APML). Most studies with As 2 O 3 involve patients with APML who have relapsed following standard therapy. Between January 1998 and July 2000, 14 patients were recruited for an ongoing trial of As 2 O 3 in the treatment of newly diagnosed APML. Arsenic trioxide was administered at a dose of 10 mg/day until complete remission (CR) was achieved. Afterward, a consolidation course and a maintenance schedule consisting of As 2 O 3 as a single agent were administered over 6 months. There were 3 early deaths related to intra-cerebral hemorrhage: two on day 3 and one on day 4. Of the 11 evaluable patients, one died on day 21 secondary to uncontrolled sepsis, while the remaining 10 (91%) have attained CR. The average time to CR was 52.3 days (range: 34-70 days). One patient developed an isolated central nervous system (CNS) relapse and subsequently went into a second CR following therapy with triple intrathecal chemotherapy, cranial irradiation, and an additional 4-week course of systemic As 2 O 3 . This patient, as well as the remaining nine, has continued to remain in CR at a median follow up of 15 months (range: 2−33 months). Eight out of 10 patients achieved molecular remission at variable periods during their consolidation and maintenance schedules. One patient developed an ATRA syndrome and was administered daunorubicin (40 mg/day) for 2 days. The side effects with this therapy were minimal and did not require cessation of therapy in any patient. There was no significant hepatic toxicity. In our experience, arsenic trioxide is effective in inducing and maintaining remission in patients with APML with minimal side effects. The optimal regimen and total dose required need to be defined. Am

Effect of cytosine arabinoside metabolizing enzyme expression on drug toxicity in acute myeloid leukemia

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Central nervous system relapse in a patient with acute promyelocytic leukaemia treated with arsenic tri-oxide

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