Balasubramanian Poonkuzhali

Pharmacokinetics of oral busulphan in children with beta thalassaemia major undergoing allogeneic bone marrow transplantation

The pharmacokinetics of busulphan were studied in 23 thalassaemic children undergoing BMT. Patients received busulphan at a dose of either 16 mg/kg with cyclophosphamide and ATG (Group A) or 600 mg/m2 (with cyclophosphamide alone) (Group B) in 16 divided doses every 6 h over 4 days. Busulphan levels were analyzed by a modified GC-MS method. The dose of busulphan/kg for patients in group B was 64% (range 56–71%) higher than that for patients in group A. The mean AUC, Css, Cmax and MRV were significantly higher in group B as compared with group A for both doses 1 and 13. There was no significant difference in Vd/F, T1/2 and Kel between the two groups. A significant decrease in AUC and Css was found between 1st and 13th doses in group B, but not in group A. The Cl/F values in group A were significantly higher than those in group B after dose 1, but not after dose 13. No increase in toxicity due to the higher dose of busulphan was noted. We conclude that busulphan at 600 mg/m2 results in much higher systemic exposure to the drug as compared to 16 mg/kg, without increase in toxicity in children with beta thalassaemia major.

Quantification of busulfan in plasma by gas chromatography-mass spectrometry following derivatization with tetrafluorothiophenol

A specific and highly sensitive method has been developed for the determination of busulfan in plasma by gas chromatography-mass spectrometry using a deuterium-labeled busulfan (busulfan-d8) as internal standard. Plasma containing busulfan and busulfan-d8 were extracted with ethyl acetate and derivatized with 2,3,5,6-tetrafluorothiophenol prior to the monitoring of specific ions. The limit of quantification of the assay was 20 ng/ml and the calibration curve was linear over the range of 10 to 2000 ng/ml of derivatized busulfan. This method was in good agreement with the GC-MS assay using derivatization with sodium iodide and measuring diiodobutane. In addition, a pharmacokinetic study of busulfan was conducted in six children. The apparent oral clearance was 5.7+/-1.9 ml/kg/min and the volume of distribution was 1.0+/-0.4 l/kg and were similar to those previously reported in pediatric patients.

High-performance liquid chromatographic method for quantification of busulfan in plasma after derivatization by tetrafluorothiophenol

A high-performance liquid chromatographic (HPLC) method was developed and validated for the determination of busulfan in plasma. Busulfan was extracted in toluene, derivatized by 2,3,5,6-tetrafluorothiophenol to obtain di-TFTP-butane, the derivatization product was then re-extracted in toluene and injected into the HPLC system with ultraviolet detection (wavelength: 275 nm). Recovery from extraction was 80%, the limit of quantification was 50 ng/ml and linearity ranged from 50 to 2000 ng/ml. In addition, forty-two samples obtained from pediatric patients treated with busulfan were analyzed by the HPLC and GC-MS assays based on the same derivatization procedure. The correlation between the di-TFTP-butane concentrations was highly significant (p<0.0001), demonstrating that the two methods were in good agreement.

Rapid Detection of β-Globin Gene Mutations and Polymorphisms by Temporal Temperature Gradient Gel Electrophoresis

Background: Inherited hemoglobin disorders represent the most common Mendelian disease worldwide. Prevention programs based on molecular diagnosis of heterozygous carriers and/or patients require the use of reliable mutation scanning methods in at-risk populations. Methods: We developed a rapid and highly specific mutation-screening test based on temporal temperature gradient gel electrophoresis (TTGE). We analyzed 889 β-thalassemia genes from homozygous β-thalassemia patients and unrelated individuals with heterozygous β-thalassemia. Previously reported common mutations were screened by reverse dot blots using allele-specific probes. The rare mutations were analyzed by TTGE. Results: We found common mutations in 753 β-thalassemia genes. TTGE analysis in the rest of the genes showed the presence of mutations in different regions of the β-globin gene in 134 of them, and these mutations were characterized by DNA sequencing. In the two genes in which mutations were not identified, large deletions spanning β-globin gene were suspected. Conclusions: Compared with other approaches for comprehensive mutation screening, the reported method is rapid, highly sensitive, cost-effective, and suitable for high-throughput screening of a large number of samples.

Association between CYP1A2 gene single nucleotide polymorphisms and clinical responses to clozapine in patients with treatment-resistant schizophrenia.

Objectives Despite clozapines superior clinical efficacy in treatment-resistant schizophrenia (TRS), its adverse effects, need for periodic leukocyte monitoring, cost and variable clinical outcomes mandate a clinical need to predict its treatment response. Although cytochrome P450 1A2 (CYP1A2) is the principal determinant of metabolism of clozapine, the role of CYP1A2 gene in the clinical response to clozapine is uncertain. Hence, we investigated its association with treatment responses and adverse events of clozapine in TRS. Methods We evaluated four single nucleotide polymorphisms (SNP) in the CYP1A2 gene, clinical responses and serum clozapine levels in 101 consecutive patients with TRS on stable doses of clozapine. We defined clozapine response a priori and investigated allelic and genotypic associations. We assessed the socio-demographic and clinical profiles, premorbid adjustment, traumatic life events, cognition and disability of the participants, using standard assessment schedules for appropriate multivariate analyses. Results Our results revealed that CYP1A2 gene SNP (*1C, *1D, *1E and *1F) were not associated with clozapine treatment response, adverse effects, serum clozapine levels or with disability (p values > 0.10). Conclusion As CYP1A2 gene SNP do not help to predict the clinical response to clozapine, routine screening for them prior to start clozapine is currently unwarranted. We suggest future longitudinal genome-wide association studies investigating clinical and pharmacogenetic variables together.

Cytochrome P4501A1 and Glutathione S Transferase Gene Polymorphisms in Patients with Aplastic Anemia in India

The etiology of acquired aplastic anemia (AA) in most patients remains unclear. It is believed that patients with a reduced ability to detoxify environmental toxins are at increased risk of developing AA. Cytochrome P450 (CYP450) and glutathione S transferase (GST) are the major phase I and phase II xenobiotic-metabolizing enzymes. We analyzed the impact of the polymorphisms in CYP4501A1 and GSTM1 and GSTT1 genes on the susceptibility and disease severity in 200 patients with AA and compared the frequency with the normal population. There was a significantly increased frequency of the CYP1A1m4 allele in AA patients compared with normal controls (odds ratio = 3.01; 95% confidence interval 1.76–5.17; p = 0.00001). None of the other CYP1A1 genotypes or the GST genotypes were significantly different between AA patients and controls. Altered metabolism of benzo(a)pyrene due to the polymorphism in the CYP1A1 gene might be an etiologic factor in the increased incidence of AA in these patients. The CYP1A1m4 allele may play a role in determining the risk of AA in India.

The t(8;14)(q24.1;q32) and its variant translocations: A study of 34 cases

BACKGROUND The t(8;14)(q24.1;q32) and its variants - the t(2;8)(p12;q24.1) and t(8;22)(q24.1;q11.2) are associated with B-cell neoplasia and result in MYC/immunoglobulin (IG) gene rearrangement. PATIENTS AND METHODS We correlated the cytogenetic, molecular and clinico-pathological findings of patients with 8q24 translocations seen in the Department of Haematology, Christian Medical College, Vellore, from January 2003 to December 2015. RESULTS There were 34 patients with 8q24 translocations (31, ALL and three myeloma). The t(8;14) was seen in 25 patients, t(8;22) in seven and t(2;8) in two. The salient findings were as follows: 85% males; 79% adults, median age 37 years; L3 morphology in 61%; mature B immunophenotype in 77%; extra-medullary disease in 41%; additional abnormalities in 28 (85%), notably, structural abnormalities of chromosome 1q (41%) and 13q (9%) and monosomy 13 (15%); complex karyotypes in 68%. There were two double-hit lymphoma/leukemia, one with a t(14;18)(q32;q21) and the other with a t(3;14)(q27;q11.2), associated with nodal high grade B cell lymphoma and dermal leukemic infiltrates respectively. Only 13 samples were processed for DNA PCR and all these samples were positive for MYC-IgH (c-gamma type) rearrangement. Only in one patient, in addition to c-gamma, c-alpha rearrangement was also detected. CONCLUSION The frequency (1.7%) and distribution of these translocations in our series and the association with 1q and 13q abnormalities is similar to the literature. Trisomies 7 and 12 were seen in less than 10% of our patients.