Development and validation of high-performance liquid chromatographic method for quantification of irinotecan and its active metabolite SN-38 in colon tumor bearing NOD/SCID mice plasma samples: Application to pharmacokinetic study

Irinotecan (IRT) is an antineoplastic agent widely used in the treatment of various cancers primarily in colorectal cancer. A new, simple and sensitive high-performance liquid chromatography (HPLC) method coupled with fluorescence detector was developed and validated to quantify IRT and its active metabolite SN38 in the plasma of non-obese diabetic/severe combined immune-deficient mice (NOD/SCID) mice bearing colon tumor. The plasma samples were extracted by precipitation method using acetonitrile with 0.1% formic acid. The chromatographic separation was achieved using mobile phase consisted of water and acetonitrile (57:43 v/v) pH 3 at the flow rate of 0.8 mL/min in C18 column (internal diameter, 250 × 4.6 mm; pore size, 5 μm). The method was validated according to the bioanalytical guidelines defined by Food and Drug Administration (FDA) and European Medicine Agency (EMA). A regression (R2 ) value of 0.999 and 0.997 for IRT and SN38 suggested the good linearity in the range of 0.1–10 μg/mL and 5–500 ng/mL, respectively. The calculated lower limit of quantification (LLOQ) and limit of detection (LOD) for IRT were 0.1 and 0.065 μg/mL, respectively. However, for SN38, LLOQ and LOD were 5 and 2 ng/mL, respectively. The intra-day and inter-day variations (coefficient of variance; % CV) observed during the validation were found to be within the set limit of 15%. Both accuracy and percentage recovery analyzed and calculated from the quality control samples were in the between the defined range of 85–115%. Plasma samples were found to be stable when stored at room temperature for 2 h, after 2 freeze–thaw cycles and at −80 °C for 2 months. The developed method was successfully applied to study the plasma elimination profile of IRT in NOD/SCID mice with tumor. The results from plasma concentration time profile and pharmacokinetic parameter analyzed suggested the rapid elimination of IRT and SN38 from the plasma of NOD/SCID mice

Raman spectroscopy for detection of imatinib in plasma: A proof of concept

Imatinib is the standard first line treatment for chronic myeloid leukemia (CML). Owing to dose-related toxicities of Imatinib such as neutropenia, there is scope for treatment optimization through therapeutic drug monitoring (TDM). Trough concentration of 1 μg/mL is considered the therapeutic threshhold. Existing methods for the detection of Imatinib in plasma are limited by long read out time and expensive instrumentation. Hence, Raman spectroscopy was explored as a rapid and objective tool for monitoring Imatinib concentration. Three approaches: conventional Raman spectroscopy (CRS), Drop coating deposition Raman (DCDR) spectroscopy and surface-enhanced Raman spectroscopy (SERS) were employed to detect the required trough concentration of 1 μg/mL and above. Detection of therapeutically relevant concentrations (1 μg/mL) using SERS and suitable nanoparticle substrates has been demonstrated. Prospectively, rigorous validation using clinical samples is necessary to confirm the utility of this approach in routine clinical usage

Off-label use of anti-cancer drugs in India: To be or not to be!

Background: Administering drugs outside the terms of their official labeling is called off-label use. In the West, oncologists often use drugs off-label, which offers several advantages especially while treating patients with multiple comorbidities or advanced cancer. The practice of off-label prescribing of anticancer agents in India is not well documented. Materials and Methods: A survey on prescribing practices of 10 important drugs used in cancer was conducted in March 2010. Ten centers representing all parts of India were identified. One oncologist from each center was contacted by phone or email and explained about the survey. The list of drugs was sent to them by email if they agreed to participate and they were requested to fill all the indications for which these drugs were used in their center. Labeling for each drug was obtained from the Food and Drug Administration (FDA) website. Off-label practice was categorized as those recommended by the National Comprehensive Cancer Network (NCCN) and those without. Results: Nine out of 10 centers agreed to participate in the survey. Four centers responded to the questionnaire. Six out of 10 drugs were used for off-label indications, cisplatin being the most commonly used. All drugs were either used as per FDA labeling or according to the recommendations of NCCN, except for gemcitabine which was used in one center for some indications based on phase II data. Conclusion: Very limited off-label prescribing was found in oncology practice in India. Since off-label use offers several advantages, judicious use of this practice should be encouraged among the oncologists

Novel NUDT15 germline variant in acute lymphoblastic leukaemia â Increase susceptibility to mercaptopurine toxicity responsible for relapse and severe life threatening sepsis: A case report

Acute lymphoblastic leukaemia (ALL) has become one of most curable malignancies among all childhood cancers. Such results are possible because of improved understanding of the biology and pathophysiology of the disease with discovery of novel mutations effecting the pharmacodynamics of the drugs. One of the important drug used in ALL treatment is mercaptopurine (6-MP). Implementation of TPMT polymorphisms in 6-MP dose regime in acute lymphoblastic leukaemia (ALL) is an epitome of pharmacogenetic evidence based clinical practice in oncology. Recent advances in genome wide associations studies (GWAS) have unearthed more germline mutations, notable among which is a polymorphism (rs116855232) in the NUDT15 gene. Several cohorts of Asian population have reported a higher frequency of this mutation as compared to the TPMT mutations, necessitating more research into the clinical significance of this particular genetic variation

Preclinical evaluation of engineered L-asparaginase variants to improve the treatment of Acute Lymphoblastic Leukemia

Introduction: Escherichia coli l-asparaginase (EcA), an integral part of multi-agent chemotherapy protocols of acute lymphoblastic leukemia (ALL), is constrained by safety concerns and the development of anti-asparaginase antibodies. Novel variants with better pharmacological properties are desirable. Methods: Thousands of novel EcA variants were constructed using protein engineering approach. After preliminary screening, two mutants, KHY-17 and KHYW-17 were selected for further development. The variants were characterized for asparaginase activity, glutaminase activity, cytotoxicity and antigenicity in vitro. Immunogenicity, pharmacokinetics, safety and efficacy were tested in vivo. Binding of the variants to pre-existing antibodies in primary and relapsed ALL patients’ samples was evaluated. Results: Both variants showed similar asparaginase activity but approximately 24-fold reduced glutaminase activity compared to wild-type EcA (WT). Cytotoxicity against Reh cells was significantly higher with the mutants, although not toxic to human PBMCs than WT. The mutants showed approximately 3-fold lower IgG and IgM production compared to WT. Pharmacokinetic study in BALB/c mice showed longer half-life of the mutants (KHY-17- 267.28±9.74; KHYW-17- 167.41±14.4) compared to WT (103.24±18). Single and repeat-doses showed no toxicity up to 2000 IU/kg and 1600 IU/kg respectively. Efficacy in ALL xenograft mouse model showed 80–90 % reduction of leukemic cells with mutants compared to 40 % with WT. Consequently, survival was 90 % in each mutant group compared to 10 % with WT. KHYW-17 showed over 2-fold lower binding to pre-existing anti-asparaginase antibodies from ALL patients treated with l-asparaginase. Conclusion: EcA variants demonstrated better pharmacological properties compared to WT that makes them good candidates for further development