Review of DBS methods as a quantitative tool for anticancer drugs.

An overview of published dried blood spot (DBS) methods for the quantitation of various classes of anticancer drugs from clinical and preclinical studies is presented. The increased reporting of DBS methods in the literature for quantitation of various classes of drugs is a testimony to their utility in bioanalytical applications. While DBS offers several advantages as compared with conventional wet sampling techniques, there remain a number of nuances that may impede the assay adaptability of DBS method in routine quantitative bioanalysis. This review covers several case studies of DBS application in the quantitation of anticancer drugs. Some perspectives are provided on the optimization of the DBS method with respect to the selection of DBS card, spot volume, hematocrit effect and other regular validation parameters, which are essential in quantitative bioanalysis. Some thoughts are provided on the existing gaps in the DBS method and possible remedial measure(s) to address such gaps. Although DBS methods have great potential, there is the need for a global consensus including regulatory support on the type of validation experiments to be performed to support quantitative data.

Validated HPLC method for quantification of copanlisib in mice plasma: application to a pharmacokinetic study.

Copanlisib is a pan phosphatidylinositol 3-kinase (PI3K) inhibitor approved for follicular lymphoma. In this paper, we present the data of development and validation of a high-performance liquid chromatography (HPLC) method for the quantitation of copanlisib in mice plasma as per the FDA regulatory guideline. The method involves the extraction of copanlisib along with internal standard (IS, enasidenib) from mice plasma (100 µL) using ethyl acetate as an extraction solvent. The chromatographic resolution of copanlisib and the IS was achieved on a Hypersil Gold C18 column maintained at 40 °C using a binary gradient mobile phase [10 mM ammonium formate (pH 4.0) and acetonitrile]. The flow-rate was 0.8 mL/min. For the detection of copanlisib and the IS, the photo-diode array detector was set at λmax 310 nm. Copanlisib and the IS eluted at 6.60 and 7.80 min, respectively with a total run time of 10 min. The calibration curve was linear over a concentration range of 50 to 5000 ng/mL for copanlisib (r2≥ 0.998). The results of intra- and inter-day accuracy and precision studies were within the acceptable limits. Copanlisib was stable on bench-top, in auto-sampler, up to three freeze/thaw cycle and long-term storage at -80 °C. The application of the validated method was shown in a mice pharmacokinetic study.

Terminalia tomentosa Bark Ameliorates Inflammation and Arthritis in Carrageenan Induced Inflammatory Model and Freund's Adjuvant-Induced Arthritis Model in Rats.

Terminalia tomentosa bark belongs to the family Combretaceae. The plant bark is astringent and useful in the treatment of ulcers, vata, fractures, hemorrhages, bronchitis, and diarrhea. Phytochemical investigation of T. tomentosa bark confirms the presence of flavonoids, polyphenols, and tannins. The plant has not been investigated for its anti-inflammatory and antiarthritic activity. The present study was undertaken to explore its possible anti-inflammatory and antiarthritic activity. Anti-inflammatory activity of alcoholic and aqueous extracts of the bark was assessed by in vivo methods. In vivo antiarthritic potential of the extracts was evaluated by Complete Freund's Adjuvant (CFA) induced arthritis in Wistar rats. Our findings showed that the alcoholic and aqueous extracts exhibited anti-inflammatory activity at 500 mg/kg oral dose in carrageenan-induced hind paw edema and carrageenan-induced air pouch inflammation models. We also found alcoholic as well as aqueous extracts of the bark restored the altered blood and serum parameters caused by the Complete Freund's Adjuvant-induced arthritis in Wistar rats. This study shows that the T. tomentosa bark extracts possess anti-inflammatory activity and have pronounced effects on adjuvant arthritis also. Future studies are necessary to provide deeper insight into the exact mechanism of the action of anti-inflammatory and antiarthritic activity of T. tomentosa.

Validation of an LC-MS/MS method for simultaneous quantitation of enzalutamide, N-desmethylenzalutamide, apalutamide, darolutamide and ORM-15341 in mice plasma and its application to a mice pharmacokinetic study.

A sensitive and rapid LC-MS/MS method was developed and validated for the simultaneous quantitation of enzalutamide, N-desmethylenzalutamide (active metabolite of enzalutamide), apalutamide, darolutamide and ORM-15341 (active metabolite of darolutamide) in mice plasma as per regulatory guidelines. The analytes and the internal standard (I.S.: apalutamide-d3) were extracted from 50 µL mice plasma by simple protein precipitation using acetonitrile, followed by chromatographic separation using an Atlantis C18 column with an isocratic mobile (0.2% formic acid:acetonitrile; 30:70, v/v) at a flow rate of 0.8 mL/min within 2.5 min. Detection and quantitation was done by multiple reaction monitoring on a triple quadrupole mass spectrometer following the transitions: m/z 465 → 209, 451 → 195, 478 → 450, 399 → 178, 397 → 194 and 481 → 453 for enzalutamide, N-desmethylenzalutamide, apalutamide, darolutamide, ORM-15341 and the I.S. respectively. The calibration curves were linear from 1.07 to 2000 ng/mL with r2 ≥0.99 for all the analytes. The intra- and inter-batch accuracy and precision (% CV) across quality controls varied from 88.5-111% and 1.13-13.1, 85.4-106% and 3.15-14.3, respectively for all the analytes. All the analytes were found to be stable under different conditions. The method was applied to an intravenous pharmacokinetic study in mice.

Validation of an LC-ESI-MS/MS method for the determination of apalutamide, a novel non-steroidal anti-androgen in mice plasma and its application to a pharmacokinetic study in mice.

A sensitive, specific, selective and rapid LC-ESI-MS/MS method has been developed and validated for the quantification of apalutamide in mice plasma using apalutamide-d3 as an internal standard (I.S.). Sample preparation was accomplished through a simple protein precipitation process. Chromatography of apalutamide and the I.S. was achieved on an Atlantis dC18 column using an isocratic mobile phase comprising 0.2% formic acid in water and acetonitrile (20:80, v/v) delivered at a flow rate of 0.8 mL/min. LC-MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique in positive ion mode and the transitions of m/z 478 → 450 and m/z 481 → 453 were used to measure the derivative of apalutamide and the I.S, respectively. The total chromatographic run time was 2.5 min and the elution of apalutamide and I.S. occurred at 1.10 and 1.09 min, respectively. Method validation was performed as per regulatory guidelines and the results met the acceptance criteria. Linearity was established in the concentration range of 1.02-2030 ng/mL (r > 0.995) for apalutamide. The intra- and inter-day accuracy and precision for apalutamide in mice plasma were in the range of 2.11-8.44 and 2.51-6.09%, respectively. Apalutamide was found to be stable under various stability conditions. This novel method has been applied to a pharmacokinetic study in mice.

Biological activity of a small molecule indole analog, 1-[(1H-indol-3-yl)methylene]-2-phenylhydrazine (HMPH), in chronic inflammation.

A synthetic small molecule, 1-[(1H-indol-3-yl)methylene]-2-phenylhydrazine (HMPH) was conveniently synthesised by a one-step reaction, purified and characterised by chromatographic and spectroscopic methods. HMPH scavenged free radicals and inhibited lipopolysaccharide (LPS)-induced ROS generation and NO release in RAW-264.7 cells without signs of any detectable cytotoxicity. HMPH inhibited lipid peroxidation (LPO) with IC50 of 135 ± 9 as against 58 ± 8 µM for α-tocopherol. Further, HMPH (>50 µM) significantly reduced the LPS-induced TNF-α release in mouse peritoneal macrophages and in human peripheral blood mononuclear cells (PBMCs). HMPH did not show any visible signs of toxicity in rats up to 400 mg/kg/intraperitoneal and 2000 mg/kg/oral. HMPH at 25 and 50 mg/kg attenuated neutrophil infiltration in air-pouch lavage and bronchoalveolar lavage (BAL) in rat models. HMPH also reduced myeloperoxidase (MPO), nitrite and TNF-α in air-pouch lavage in addition to MPO in plasma. HMPH reduced acute paw-inflammation in carrageenan-induced paw-edema. HMPH consistently decreased both ipsilateral and contralateral paw inflammation, minimised the clinical scores of arthritis, prevented body weight (B.wt.) loss, attenuated serum C-reactive protein (C-RP) and rheumatoid factors (RF) in rat model of adjuvant-induced arthritis. Histopathology and radio-graphical reports show that HMPH reduced bone erosion in both ipsilateral and contralateral paw joints. Failure to inhibit COX suggests that effectiveness of HMPH in both acute and chronic inflammation is mediated by a multimodal mechanism involving modulation of immunity, attenuating TNF-α, protecting bone attrition and reducing oxidative stress.