Amelioration of Cisplatin-induced Renal Inflammation by Recombinant Human Golimumab in Mice.

BACKGROUND: One of the most commonly used anti-cancer agents, Cisplatin (CDDP) often causes nephrotoxicity by eliciting inflammation and oxidative stress. Golimumab, an anti-TNF biologic, is prescribed for the management of numerous inflammatory ailments like psoriatic and rheumatoid arthritis, ulcerative colitis and ankylosing spondylitis. OBJECTIVE: Current study has explored the effects of anti-TNF biologics golimumab on mice due to cisplatin-induced nephrotoxicity. METHOD: Renal toxicity was caused by administration of single cisplatin injection at 22 mg/kg by intraperitoneal (i/p) route. Golimumab (24 mg/kg, s.c.) was administered consecutively for 7 days. The parameters such as renal functions, oxidative stress, inflammation, and renal damage were evaluated on the 7th day of experiments. RESULTS: Cisplatin administration caused nephrotoxicity as shown by a significant elevation of various parameters viz; serum creatinine, neutrophil gelatinase-associated lipocalin (NGAL), urea nitrogen (BUN), and cystatin C. There was a significant rise in urinary clusterin, kidney injury molecule 1 (KIM-1), and ß-N-acetylglucosaminidase (NAG) concentrations in the animals treated with cisplatin. The markers of oxidative stress (malondialdehyde, reduced glutathione, and catalase), inflammation (IL-6, TNF-α, IL-10, IL-1ß, MCP-1, ICAM-1, and TGF-ß1), and apoptosis (caspase-3) were also altered in serum and/or kidneys of cisplatin animals. Further, cisplatin-caused histopathological changes in proximal tubular cells as observed in the H&E staining of renal tissue. Golimumab treatment reduced all markers of kidney injury and attenuated cell death. Golimumab significantly reduced inflammatory cytokines TNFα, IL- 6, MCP-1, IL- 1ß, ICAM-1, and TGF-ß1 and increased anti-inflammatory cytokine IL-10 in cisplatin-intoxicated mice. CONCLUSION: The study's results suggest that golimumab prevented nephrotoxicity induced by cisplatin- through inhibition of oxidative stress, apoptotic cell death inflammatory response, thus improving renal function.

Protective effects of recombinant human golimumab and pentoxifylline in nephrotoxicity induced by cisplatin.

In cisplatin-induced nephrotoxicity, the significant role of activation of inflammatory pathways has been reported earlier. Studies indicate elevated expression and activity of tumor necrosis factor-α (TNF-α) in both, serum and kidneys of cisplatin-treated animals. Golimumab, an anti-TNF biologic, has been approved for the management of many inflammatory conditions. This experiment was planned and executed to evaluate the impact of golimumab on renal function, inflammation in cisplatin-induced nephrotoxicity in mice, and oxidative stress. Cisplatin (22 mg/kg) as a single intraperitoneal injection was used to induce nephrotoxicity in mice. Golimumab was administered at 24 mg/kg for 7 days by subcutaneous route. Pentoxifylline (PTX) was administered for 7 days (150 mg/kg) as a reference standard. Renal functions, oxidative stress, and inflammation were evaluated on the seventh day. Cisplatin administration in mice caused a significant rise in serum cystatin C, creatinine, blood urea nitrogen, and neutrophil gelatinase-associated lipocalin. A significant rise of urinary clusterin, kidney injury molecule-1, and ß-N-acetylglucosaminidase levels was also seen in cisplatin-treated animals. Furthermore, cisplatin-induced nephrotoxicity was associated with a significant increase in oxidative stress and inflammation in serum and kidneys. Golimumab treatment significantly prevented the cisplatin-induced alteration in markers of renal function and renal damage. There was a significant reduction in oxidative stress and inflammation in golimumab-treated animals. Furthermore, the histopathological evaluation also revealed inverted changes in the proximal tubules after golimumab treatment in kidneys after cisplatin toxicity. The standard drug, PTX, also prevented nephrotoxicity caused by cisplatin as indicated by the recovery in renal function, reduction in oxidative stress and inflammation. These results indicate that golimumab was effective in nephrotoxicity induced by cisplatin through inhibition of oxidative stress, and inflammatory response.

A selective and sensitive UPLC-ESI-MS/MS method for quantification of Pegylated Interferon Alfa-2b in human serum using signature peptide-based quantitation.

A sensitive method for determination of PEG-IFN-α-2b in human serum was developed using ultra performance liquid chromatography aligned with tandem mass spectrometric detection. A two-treatment, two-period, cross over study was conducted to establish bioequivalence between a test and reference formulation and the method was successfully applied to the quantification of PEG-IFN-α-2b in serum samples of this clinical study. The sample concentrations obtained from LC-MS/MS technique were compared with the concentrations obtained from ELISA technique. PEG-IFN-α-2b was isolated from serum using protein precipitation technique with isopropyl alcohol followed by overnight tryptic digestion. The signature peptide formed as result of tryptic digestion was separated on a chromatograph and detected using a mass detector. The mass transition ion-pair of m/z 741.3 → 1047.1 for PEG-IFN-α-2b and m/z 387.4 → 205.2 for internal standard were used for MS/MS detection. The sample extraction involves a simple protein precipitation method followed by tryptic digestion of the supernatant and further sample cleanup was not needed. The method has been validated over a linear range of 1.028-3200 ng/mL with a correlation coefficient ≥ 0.99. The precision (%RSD) was 5.52 to 7.90 and accuracy (%RE) was within -1.80 to 1.68. The total run time was 22.0 min. The sensitivity of LC-MS/MS method was 1.0 ng/ml which was found to be more sensitive than ELISA and resulted in improving the overall study data by being able to quantify all the samples without any below LOQ results helping to further improve the pharmacokinetic modeling. This improved method is a promising anti-body free LC-MS/MS based methodology for estimation of PEG-IFN-α-2b in human serum and may be applied for other such pegylated molecules.

Comparison of Strategies in Development and Manufacturing of Low Viscosity, Ultra-High Concentration Formulation for IgG1 Antibody.

Monoclonal antibodies requiring higher doses for exerting therapeutic effect but having lower stability, are administered as dilute infusions, or as two (low concentration) injections both resulting in reduced patient compliance. Present research summarizes impact of manufacturing conditions on ultra-high concentration (≥150 mg/mL) IgG1 formulation, which can be administered as one subcutaneous injection. IgG1 was concentrated to ~200 mg/mL using tangential flow filtration (TFF). Alternatively, spray dried (SPD) and spray freeze dried (SFD) IgG1, was reconstituted in 30%v/v propylene glycol to form ultra-high concentration (~200 mg/mL) injectable formulation. Reconstituted, SPD and SFD IgG1 formulations, increased viscosity beyond an acceptable range for subcutaneous injections (<20 cP). Formulations developed by reconstitution of SPD IgG1, demonstrated increase in high and low molecular weight impurities, at accelerated and stressed conditions. Whereas, the stability data suggested reconstituted SFD IgG1 was comparable to control IgG1 formulation concentrated by TFF. Also, formulation of IgG1 diafiltered with proline using TFF, reduce viscosity from ~21.9 cP to ~11 cP at 25 °C and had better stability. Thus, conventional TFF technique stands to be one of the preferred methods for manufacturing of ultra-high concentration IgG1 formulations. Additionally, SFD could be an alternative method for long term storage of IgG1 in a dry powder state.

Reconstruction of bone defects after osteomyelitis with nonvascularized fibular graft: a retrospective study in twenty-six children.

BACKGROUND: Persistent infection, soft-tissue fibrosis, and damage to periosteum compound the treatment of children with a bone defect following osteomyelitis. We report on a series of twenty-six patients treated with nonvascularized fibular graft and intramedullary fixation. METHODS: The series included eleven boys and fifteen girls (mean age, 6.8 years; range, three to twelve years) with gap nonunion after osteomyelitis. Initial treatment involved thorough debridement and sequestrectomy. When the infection was quiescent as indicated by inflammatory parameters, nonvascular fibular grafting with intramedullary Kirschner wire fixation (with or without additional external fixation) was performed. The time to union was noted, and a subgroup analysis was performed to correlate the size of the bone defect with the time to union. RESULTS: The mean duration of follow-up was 3.02 ± 0.74 years (range, 1.3 to 4.2 years), and the mean time to union was 38.76 ± 12.02 weeks (range, fifteen to sixty weeks). There was a weak positive correlation between the time to union and the preoperative bone defect size (Pearson correlation coefficient, 0.699). The mean time to union was 31.7 ± 11.5 weeks for a defect of <4 cm, 36.6 ± 9 weeks for a defect of 4 to 6 cm, and 51 ± 6.7 weeks for a defect of >6 cm. Delayed union was seen at one end of the fibular graft in four (15%) of the patients and was treated with plate fixation. One patient had recurrence of infection. Limb-length discrepancy (range, 2 to 5 cm) was seen in all patients in whom the lower limb was involved and was treated with a shoe lift. CONCLUSIONS: This series illustrates the potential benefits of staged sequestrectomy and nonvascular fibular grafting for the treatment of gap nonunion following osteomyelitis in children. The procedure is simple, does not require specialized training or equipment, and has a low complication rate.

Development and validation of RP-HPLC and RP-UPLC methods for quantification of parathyroid hormones (1-34) in medicinal product formulated with meta-cresol.

Rapid and sensitive reversed phase high performance liquid chromatography (RP-HPLC) and ultra performance liquid chromatography (RP-UPLC) method with UV detection has been developed and validated for quantification of parathyroid hormone (PTH) in presence of meta-cresol as a stabilizer in a pharmaceutical formulation. Chromatography was performed with mobile phase containing 0.1% Trifluoroacetic acid (TFA) in MilliQ water and 0.1% TFA in acetonitrile with gradient program and flow rate at 0.3 mL/min for HPLC and 0.4 mL/min for UPLC. Quantification was accomplished with internal reference standard (qualified against innovator product and National Institute for Biological Standards and Control (NIBSC) standard). The methods were validated for linearity (correlation coefficient=0.99), range, accuracy, precision and robustness. Robustness was confirmed by considering three factors; mobile phase composition, column temperature and flow rate/age of mobile phase. Intermediate precision was confirmed on different equipments, different columns and on different days. The relative standard deviation (RSD) (<2% for RP-HPLC and <1% for UPLC, n=30) indicated a good precision. Retention time was found about 17 min and 2 min by HPLC and UPLC methods, respectively. Both methods are simple, highly sensitive, precise and accurate and have the potential of being useful for routine quality control.

Development and validation of RP-HPLC and RP-UPLC methods for quantification of erythropoietin formulated with human serum albumin.

Rapid and sensitive reverse phase high performance liquid chromatography (RP-HPLC) and ultra performance liquid chromatography (UPLC) methods with UV detection for quantification of erythropoietin (EPO) in presence of human serum albumin (HSA) as a stabilizer in a pharmaceutical formulation of EPO have been developed and validated. Chromatography was performed with mobile phase containing 0.1% Trifluoroacetic acid (TFA) in MilliQ water and 0.1% TFA in acetonitrile with gradient program and a flow rate of 1.5 mL/min for HPLC and 0.35 mL/min for UPLC. Quantification was accomplished with internal reference standard (qualified using EP reference standard). The methods were validated for linearity (correlation coefficient=0.99), accuracy, precision and robustness. Robustness was confirmed by considering three factors; percentages of TFA in mobile phase, age of test sample and mobile phase and column temperature. Intermediate precision was confirmed by different analysts, different equipments and on different days. The relative standard deviation (RSD) value (<2%, n=30) indicated good precision of the developed method. The proposed RP-HPLC method had retention time less than 20 min while the developed UPLC method had retention time less than 4 min. Both the RP-HPLC and UPLC methods were simple, highly sensitive, precise and accurate, suggesting that the developed methods are useful for routine quality control.

Validation of a simple RP-HPLC method developed for the quantification of meta-cresol in parathyroid hormones formulation.

AIM: To develop and validate a rapid and sensitive reverse phase high performance liquid chromatography (RP-HPLC) method with UV detection for quantification of meta-cresol (m-cresol) in pharmaceutical preparation of parathyroid hormone (1-34) (PTH). MATERIALS AND METHODS: Chromatography was performed on a Jupiter RP C-18 (4.6 mm ID × 250 mm L, porosity 300 Å, particle size 5 µm) with a guard column (reversed-phase C18 column of 4.6 mm ID × 12.5 mm L, porosity 300 Å, particle size 5 µm) using a mobile phase containing 0.1% TFA in 60% methanol with isocratic program at 1.0 mL/min flow rate. Detection was carried out at 217 nm. The method was validated as per ICH guidelines for linearity (correlation coefficient = 0.99), range, accuracy, precision, and robustness (n = 9 during accuracy parameter whereas n =15 during linearity and range parameter and n = 6 during repeatability). Robustness was confirmed by considering two factors; age effect of the mobile phase and test sample and with different columns during method development. RESULTS: The method was linear over the concentration range of 75-120 µg/mL. The precision of the method in terms of relative standard deviation was evaluated from intra- and inter-day replicate injections of system suitability standards of m-cresol using different equipment and different columns. Components of within- and between-batch variances were found to be below 2% (n = 30) and 3%, respectively, which constituted an acceptable level of variation. Retention time was found to be about 5.2 min and 10.9 min for m-cresol and PTH, respectively. CONCLUSION: The developed method thus has the potential of being useful for routine quality control of m-cresol.

Physiochemical parameters optimization for enhanced nisin production by Lactococcus lactis (MTCC 440)

The influence of various physiochemical parameters on the growth of Lactococcus lactis sub sp. lactis MTCC 440 was studied at shake flask level for 20 h. Media optimization (MRS broth) was studied to achieve enhanced growth of the organism and also nisin production. Bioassay of nisin was done with agar diffusion method using Streptococcus agalactae NCIM 2401 as indicator strain. MRS broth (6 percent, w/v) with 0.15ìg/ml of nisin supplemented with 0.5 percent (v/v) skimmed milk was found to be the best for nisin production as well as for growth of L lactis. The production of nisin was strongly influenced by the presence of skimmed milk and nisin in MRS broth. The production of nisin was affected by the physical parameters and maximum nisin production was at 30(0)C while the optimal temperature for biomass production was 37(0)C.