Development and multicenter validation of an LC-MS-based bioanalytical method for antisense therapeutics.

Background: Many bioanalytical methods for antisense oligonucleotides (ASOs) using LC-MS have been reported. However, no data have been available on the reproducibility and robustness of a single bioanalytical method for ASOs. As such, in the current study, we evaluated the reproducibility and robustness of LC-MS-based bioanalytical methods for ASOs in multiple laboratories. Methods/Results: Seven independent laboratories were included in this study. Mipomersen was measured by ion-pairing LC-MS (IP-LC-MS) as a model ASO using different LC-MS. The validation results of calibration curve, accuracy, precision and selectivity met the criteria of conventional bioanalytical method validation guidelines using LC/GC-MS for drugs in all laboratories. Meanwhile, carryover (>20%) was detected in three laboratories. Conclusion: We first demonstrated the multicenter-validated IP-LC-MS bioanalytical method for ASOs. Our data showed that the method was sensitive, robust and reproducible. However, the occurrence of carryover should be carefully monitored in its future application.

Bioanalysis of therapeutic monoclonal antibody by peptide adsorption-controlled LC-MS.

Aim: We aimed to develop an easy, low-cost and versatile mass spectrometric method for the bioanalysis of a therapeutic monoclonal antibody (mAb) in human serum that employs peptide adsorption-controlled (PAC)-LC/MS using selected reaction monitoring mode (LC-MS/MS-SRM). Materials & methods: Rituximab was used as a model mAb. To apply the method to human serum samples, a peptide of the complementarity-determining region was selected as the surrogate peptide. The usefulness of PAC-LC-MS/MS-SRM was evaluated by a collaborative study. Results: The calibration curve ranged from 0.5 (or 1.0) to 1000.0 µg/ml. The selectivity, linearity, accuracy and precision met the predefined acceptance criteria. Conclusion: Our method could be a useful bioanalytical method for the quantification of mAbs in clinical samples.

Development of a bioanalytical method for an antisense therapeutic using high-resolution mass spectrometry.

Background: Ion-pairing reverse-phase LC coupled with high-resolution mass spectrometry (IP-LC/HRMS) has gained attention in oligonucleotide therapeutic bioanalyses owing to its high sensitivity and selectivity. However, optimization and validation of IP-LC/HRMS-based methods are rare. The objective of this study is the development of a sensitive and reproducible IP-LC/HRMS-based bioanalytical method using clinically approved mipomersen as a model for antisense oligonucleotides. Materials & methods/results: Mipomersen was extracted from rat plasma using Clarity OTX SPE and quantified by IP-LC/HRMS. The calibration range was 0.5-250.0 ng/ml. The developed method met the general regulatory criteria for accuracy, precision, carry-over, selectivity, matrix effect and dilution integrity. Conclusion: A highly sensitive and reliable method for mipomersen measurement with potential antisense oligonucleotide bioanalysis applications has been developed.

Generic MS-based method for the bioanalysis of therapeutic monoclonal antibodies in nonclinical studies.

Aim: A generic bioanalytical method was developed to quantify therapeutic IgG1 monoclonal antibodies (mAbs) in mouse sera by combining an easy sample preparation method with LC/MS using selected reaction monitoring. Materials & methods: Rituximab and trastuzumab were used as model mAbs. A synthetic stable isotope-labeled peptide or a stable isotope-labeled mAb was used as an internal standard. The method feasibility was evaluated by a collaborative study involving six laboratories. Results: The calibration curve ranged from 1.0 to 1000.0 µg/ml (correlation coefficient >0.99). The validation parameters including selectivity, linearity of calibration curve, accuracy and precision met the predefined acceptance criteria. Conclusion: Our method is a useful bioanalytical method for the quantification of therapeutic IgG mAbs in nonclinical animal studies.

Metal-free α-CH amination of ethers with hypervalent sulfonylimino-λ3-bromane that acts as an active nitrenoid.

Hypervalent N-triflylimino-λ(3)-bromane undergoes direct and regioselective α-C-H amination of ethers at room temperature under transition metal-free conditions. Kinetic results, substituent and deuterium isotope effects suggest an asynchronous concerted organonitrenoid transition state with some hydride transfer character, analogous to that for alkane C-H insertions.

Inverse correlation between seasonal changes in home blood pressure and atmospheric temperature in treated-hypertensive patients.

We examined the relationships between home blood pressure (BP) and atmospheric temperature in 20 treated-hypertensive patients. A significant correlation between morning and evening BP and atmospheric temperature was found. For morning systolic blood pressure (SBP), the maximal seasonal difference was 13.2 mmHg with 25.5 degrees C temperature difference. Morning SBP increased by approximately 0.41 mmHg with a 1 degrees C decrease in atmospheric temperature. A similar but weaker correlation with temperature was observed for morning diastolic, evening systolic and diastolic blood pressure (DBP). The present study provides important information in respect to BP changes with atmospheric temperature that should be taken into account in the analysis and treatment of hypertension.

Design, synthesis, and structure-activity relationship of novel opioid kappa-agonists.

By focusing on 4,5-epoxymorphinan, a traditional opioid skeleton but a new structure in the opioid kappa-agonist research field, and by rationally applying the 'message-address concept' and 'accessory site hypothesis,' we discovered a new chemical class opioid kappa-agonist, TRK-820 (1). Its development as an antipruritus is now in the final stage. Here, the full scope of its design, synthesis, and structure-activity relationship are described.

Heart rate variability for evaluating surgical stress and development of postoperative complications.

Heart rate variability (HRV) has recently been used to detect autonomic nerve tone, which is affected by various stresses. To test out hypothesis that HRV can determine surgical stress, we examined perioperative HRV in 30 patients with surgical treatment. Relations between HRV and factors of surgical stresses, such as duration of the operation, amount of blood loss at the operation, and developments of complications, were evaluated. Mean heart rate (HR) increased and other HRV indices decreased postoperatively. Most indices correlated significantly to the duration of the operation and amount of blood loss at the operation on postoperative day 1. Only the standard deviation of normal to normal RR intervals (SDNN) and HRV triangular index showed significantly low values in complicated patients. HRV measurement in the perioperative period showed a significant relation to surgical stress. The present results indicated that HRV may provide useful information with respect to surgical stress.

Clindamycin in the treatment of Babesia gibsoni infections in dogs.

This report examines the effectiveness of clindamycin for the treatment of babesiosis in dogs (n=10) experimentally infected with Babesia gibsoni (B. gibsoni). Clindamycin (25 mg/kg body weight, per os, q 12 hours for 14 days) gradually reduced parasitemia levels and induced morphological changes that indicated degeneration of parasites (e.g., segmentation; size reduction; localization in the cell limbic and/or torn state of the nucleus; and swelling, decrease, or disappearance of the cytoplasm) in the majority of dogs. Clindamycin treatment reduced the clinical symptoms characteristic of Babesia infection, including anemia, anorexia, and listlessness. Clindamycin might be useful as a medicine for treatment of B. gibsoni infection.

Correlation of Lorenz scatterplots with frequency-domain heart rate variability.

Heart rate (HR) variability is important with respect to disease prognosis and the effects of drugs. Lorenz scatterplots provide a simple way to evaluate HR variability visually. The relation of Lorenz scatterplots to frequency-domain HR variability was examined in 75 Holter recordings and in simulated HR trends. The length of Lorenz scatterplots was double-exponentially correlated with total frequency and very-low frequency powers, with correlation coefficients (r) of 0.88. The width of Lorenz scatterplots was highly correlated with the high frequency power (r=0.98). The sum of the width and length of Lorenz scatterplots was highly correlated with the total frequency power (r=0.92). Identical results were obtained when simulated HR trends were used. In conclusion, Lorenz scatterplots provide a simple way to estimate the frequency-domain HR variability.