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Background In 2018, the World Health Organisation (WHO) released interim guidelines, advising a change of regimens to dolutegravir-based first- and second-line antiretroviral therapy (ART), based on which, in 2021, the National Aids Control Organisation (NACO) updated its guidelines to include the tenofovir + lamivudine + dolutegravir (TLD) regimen as a first line of therapy for all people living with HIV (PLHIV) and second- and third-line regimens to dolutegravir-based regimens. Considering this change of regimen, the adverse drug reaction (ADR) profiling and longitudinal prescription pattern of antiretroviral and concomitant medications in adult patients at the ART centre of a tertiary care hospital were assessed in this study. Methods Ninety-seven PLHIV out of all the patients who attended the ART centre from September 2021 to July 2022 were enrolled and followed up for six months. The ADRs that occurred during this period were collected along with details of prescription patterns and analyzed by descriptive statistics. Causality assessment for ADR was done using the World Health Organisation-Uppsala Monitoring Centre (WHO-UMC) scale. Results Seventy-eight percent (n=76 out of 97) of patients experienced at least one ADR, and 128 ADRs were seen in 97 patients. The most common ADRs were increased alkaline phosphatase (39.0%, n=128), dyslipidaemia (12.5%, n=128), and nephrotoxicity (10.1%, n=128). The drug most suspected of causing ADRs was dolutegravir (27.5%, n=342). The most common therapeutic regimen was TLD (71.2%, n=97). The most prescribed drug was lamivudine (30.6%, n=1183). The most prescribed concomitant medication was cotrimoxazole (15%, n=312). Conclusions Dolutegravir-based regimens have been implemented for PLHIV in a phased-out manner from previous non-dolutegravir-based ART regimens, which is in line with the recent NACO guidelines. However, it has also led to an increase in dolutegravir-associated ADRs like increased alkaline phosphatase, dyslipidaemia, and nephrotoxicity. Continuous monitoring of prescriptions and ADRs can add to our knowledge regarding their use and ADRs.
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Introduction: In 2019, the Central Drugs Standard Control Organization (CDSCO) introduced the New Drugs and Clinical Trials Rules 2019 (NDCTR), which separated the research guidelines for "Clinical Trials" and "Biomedical and Health Research." As a result, guidelines issued by Indian Council of Medical Research were stated to apply to academic clinical trials (ACTs). This change is important because academic studies are crucial for scientific advancement and repurposing of approved drugs in health-care industry. However, conducting an ACT can pose challenges. We assessed the level of awareness, knowledge, and challenges faced by investigators. Our aim is to overcome some of these challenges and encourage more academic studies for the betterment of healthcare and scientific knowledge in India. Methodology: The study was conducted in two phases after obtaining approval from the Institutional Ethics Committee (EC) of three tertiary care hospitals in Mumbai. In the first phase, the number of ACTs was assessed from the clinical trial registry India website, while the number of registered and re-registered ECs were assessed from the CDSCO website. The second phase involved assessing investigator awareness and knowledge about ACTs using a prevalidated questionnaire with a content validity index score of 0.93. Results: In 2020, the highest numbers of studies were registered, with the highest numbers of registered and re-registered ECs from Maharashtra. All participants completed the questionnaire and were aware of the need to follow guidelines for clinical trials. Sixty-seven percent of participants knew that the guidelines for ACTs differed from those of sponsored clinical trials, but only 58% were aware of the exact definition of an ACT as per NDCTR, 2019. Eighty-five percent of participants knew who could initiate an ACT, but only 27% knew about the applicability of results of an ACT and 33% had in-depth knowledge about the required approvals, while only 10% knew the archival period. Although 71% of participants had knowledge about serious adverse event reporting, few answered in-depth questions correctly. Only 31 participants reported facing varied challenges. Conclusion: To conduct ACTs effectively and contribute to healthcare and scientific advancement, it is crucial to enhance investigators' existing knowledge about ACTs.
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Introduction Obstructive Airway Diseases (OADs) are the leading cause of death among chronic respiratory diseases worldwide, and novel therapies are direly needed. Fluticasone furoate/vilanterol (FF/Vi) (100/25 µg) is the first once-daily ICS/uLABA marketed in India for COPD since 2021. Considering its limited real-world experience in OAD patients in Indian clinical settings, a large drug utilization study (DUS) was planned. Methodology We conducted a cross-sectional, observational DUS at 1900 outpatient clinics in India from October 2021 to March 2022. Prescription data and medical history of patients who were prescribed the FF/Vi combination were collected. Results It was observed that FF/Vi was prescribed in an almost equal number of patients with COPD (44.2%) and asthma (42.9%). The majority of the patients (74%) were switched from previous ICS/LABA to this ICS/uLABA, while 26% of patients were treatment naïve. The average CAT score was 19.5±7.8 (43.2% GOLD Group C and 32.2% GOLD Group B) in COPD patients, while the average ACQ-5 score was 2.6±1.3 (33.1% GINA Step 3, 29.5% GINA Step 2) in asthmatic patients. Most of the patients (63.9%) had raised biomarkers (Blood eosinophil count >300 cells/µl). Prior history of exacerbation was present in 65% of patients with annual exacerbation rates of 1.2 in COPD, 1.1 in asthma, and 1.2 in asthma-COPD overlap syndrome (ACOS). Leukotriene inhibitors (42%) and LAMAs (30.8%) were common add-on medications. Conclusion We observed a trend towards a shift to once-daily ICS/uLABA (FF/Vi) by physicians, especially in symptomatic and exacerbating OAD patients with underlying comorbidities.
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Context: Number of trials in India shows an increasing trend. As these trials will shape clinical practice, their quality is of utmost importance. Among many tools to assess the quality of randomized control trials (RCTs), risk of bias (RoB) is most robust. Aims: To understand the quality of trials being carried out in India in terms of RoB. Settings and Design: We aimed to assess the RoB in a set of RCTs published in Indian pharmacology of randomized trials from journals pertaining to pharmacology. Subjects and Methods: We used published journal articles as source of information for randomized clinical trials and evaluated them using Cochrane RoB tool 2.0. Statistical Analysis Used: Descriptive statistics were used. Results: 158 trials published in seven journals were evaluated in six different domains. Overall evaluation for 97% (153) trials was "high risk," while 3% (5) were in "some concerns" category, with no trials categorized as "low risk. 74% articles showed a high risk of bias in the domain of 'selection of reported results. Nearly half articles scored "low risk" in domains of "missing data" and "deviations in assignment to intervention." The study results showed a slowly increasing trend of average RoB over the last 10 years. Conclusions: The study shows concerning rise in RoB in various domains RCTs published in Pharmacology journals in India.
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A large hexanucleotide (G4C2) repeat expansion in the first intronic region of C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Several mechanisms have been proposed to explain how the repeat expansion drives disease, and we hypothesize that a variant-selective approach, in which transcripts affected by the repeat expansion are preferentially decreased, has the potential to address most of them. We report a stereopure antisense oligonucleotide, WVE-004, that executes this variant-selective mechanism of action. WVE-004 dose-dependently and selectively reduces repeat-containing transcripts in patient-derived motor neurons carrying a C9orf72-repeat expansion, as well as in the spinal cord and cortex of C9 BAC transgenic mice. In mice, selective transcript knockdown was accompanied by substantial decreases in dipeptide-repeat proteins, which are pathological biomarkers associated with the repeat expansion, and by preservation of healthy C9orf72 protein expression. These in vivo effects were durable, persisting for at least 6 months. These data support the advancement of WVE-004 as an investigational stereopure antisense oligonucleotide targeting C9orf72 for the treatment of C9orf72-associated ALS or FTD.
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OBJECTIVE: A GGGGCC repeat expansion in the C9orf72 gene is the most common cause of genetic frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). As potential therapies targeting the repeat expansion are now entering clinical trials, sensitive biomarker assays of target engagement are urgently required. Our objective was to develop such an assay. METHODS: We used the single molecule array (Simoa) platform to develop an immunoassay for measuring poly(GP) dipeptide repeat proteins (DPRs) generated by the C9orf72 repeat expansion in cerebrospinal fluid (CSF) of people with C9orf72-associated FTD/ALS. RESULTS AND CONCLUSIONS: We show the assay to be highly sensitive and robust, passing extensive qualification criteria including low intraplate and interplate variability, a high precision and accuracy in measuring both calibrators and samples, dilutional parallelism, tolerance to sample and standard freeze-thaw and no haemoglobin interference. We used this assay to measure poly(GP) in CSF samples collected through the Genetic FTD Initiative (N=40 C9orf72 and 15 controls). We found it had 100% specificity and 100% sensitivity and a large window for detecting target engagement, as the C9orf72 CSF sample with the lowest poly(GP) signal had eightfold higher signal than controls and on average values from C9orf72 samples were 38-fold higher than controls, which all fell below the lower limit of quantification of the assay. These data indicate that a Simoa-based poly(GP) DPR assay is suitable for use in clinical trials to determine target engagement of therapeutics aimed at reducing C9orf72 repeat-containing transcripts.
Subject(s)
Amyotrophic Lateral Sclerosis , Frontotemporal Dementia , Amyotrophic Lateral Sclerosis/cerebrospinal fluid , Amyotrophic Lateral Sclerosis/diagnosis , Amyotrophic Lateral Sclerosis/genetics , Biomarkers/cerebrospinal fluid , C9orf72 Protein/genetics , DNA Repeat Expansion/genetics , Frontotemporal Dementia/diagnosis , Frontotemporal Dementia/genetics , Frontotemporal Dementia/metabolism , HumansABSTRACT
Attaining sufficient tissue exposure at the site of action to achieve the desired pharmacodynamic effect on a target is an important determinant for any drug discovery program, and this can be particularly challenging for oligonucleotides in deep tissues of the CNS. Herein, we report the synthesis and impact of stereopure phosphoryl guanidine-containing backbone linkages (PN linkages) to oligonucleotides acting through an RNase H-mediated mechanism, using Malat1 and C9orf72 as benchmarks. We found that the incorporation of various types of PN linkages to a stereopure oligonucleotide backbone can increase potency of silencing in cultured neurons under free-uptake conditions 10-fold compared with similarly modified stereopure phosphorothioate (PS) and phosphodiester (PO)-based molecules. One of these backbone types, called PN-1, also yielded profound silencing benefits throughout the mouse brain and spinal cord at low doses, improving both the potency and durability of response, especially in difficult to reach brain tissues. Given these benefits in preclinical models, the incorporation of PN linkages into stereopure oligonucleotides with chimeric backbone modifications has the potential to render regions of the brain beyond the spinal cord more accessible to oligonucleotides and, consequently, may also expand the scope of neurological indications amenable to oligonucleotide therapeutics.
In this study, the authors explore the impact of nitrogen-containing (PN) backbones on oligonucleotides that promote RNase H-mediated degradation of a transcript in the central nervous system (CNS). Using Malat1, a ubiquitously expressed non-coding RNA that is predominately localized in the nucleus, and C9orf72, a challenging RNA target requiring a more nuanced targeting strategy, as benchmarks, they show that chimeric oligonucleotides containing stereopure PS and one of the more promising PN backbones (PN-1) have more potent and durable activity throughout the CNS compared with more traditional PS-modified molecules in mouse models. They demonstrate that potency and durability benefits in vivo derive at least in part from increased tissue exposure, especially in more difficult to reach regions of the brain. Ultimately, these benefits enabled the authors to demonstrate pharmacodynamic effects on Malat1 and C9orf72 RNAs in multiple brain regions with relatively low doses.
Subject(s)
Oligonucleotides, Antisense , Animals , Cells, Cultured , Central Nervous System , Guanidine/chemistry , Mice , Neurons/drug effects , Oligonucleotides, Antisense/chemistry , Oligonucleotides, Antisense/pharmacology , Phosphorothioate Oligonucleotides , Ribonuclease H/metabolismABSTRACT
Objective: The aim of the study is to evaluate the effect of deferred androgen deprivation therapy on biochemical recurrence (BCR) and other survival parameters in node-positive prostate cancer patients after robot-assisted radical prostatectomy with bilateral extended pelvic lymph node dissection (RARP + EPLND). Materials and methods: Of the 453 consecutive RARP procedures performed from 2011 to 2018, 100 patients with no prior use of androgen deprivation therapy were found to be lymph node (LN) positive and were observed, with initiation of salvage treatment at the time of BCR only. Patients were divided into 1 or 2 LNs (67)-and more than 2 LNs (33)-positive groups to assess survival outcomes. Results: At a median follow-up of 21 months (1-70 months), the LN group (p < 0.000), preoperative prostate-specific antigen (PSA, p = 0.013), tumor volume (TV, p = 0.031), and LND (p = 0.004) were significantly associated with BCR. In multivariate analysis, only the LN group (p = 0.035) and PSA level (p = 0.026) were statistically significant. The estimated BCR-free survival rates in the 1/2 LN group were 37.6% (27%-52.2%), 26.5% (16.8%-41.7%), and 19.9% (9.6%-41.0%) at 1, 3, and 5 years, respectively, with a hazard of developing BCR of 0.462 (0.225-0.948) compared with the more than 2 LN-positive group. Estimated 5-year overall survival, cancer-specific, metastasis-free, and local recurrence-free survival rates were 88.4% (73.1%-100%), 89.5% (74%-100%), 65.1% (46.0%-92.1%), and 94.8% (87.2%-100.0%), respectively, for which none of the factors were significant. Based on cutoff values for PSA, TV, and LND of 30 ng/mL, 30%, and 10%, respectively, the 1/2 LN group was substratified, wherein the median BCR-free survival for the low- and intermediate-risk groups was 40 and 12 months, respectively. Conclusions: Nearly one fourth and one fifth of 1/2 node-positive patients were BCR-free at 3 and 5 years after RARP + EPLND. Further substratification using PSA, TV, and LN density may help in providing individualized care regarding the initiation of adjuvant therapy.
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Robotic assistance is being increasingly utilised for nephron-sparing surgery for complex renal masses. We evaluated the outcomes of robot-assisted partial nephrectomy (RAPN) for cT1a versus cT1b + renal masses by a comparative analysis of trifecta outcomes between these two groups of patients. We utilised our prospectively maintained database to identify patients undergoing RAPN for cT1a (group 1, n = 41) and cT1b + (group 2, n = 37) renal masses from April 2016 to March 2020. The oncological and trifecta outcomes were analysed using appropriate statistical methods. Out of 78 patients, trifecta was achieved in 30 (38.4%) patients. There was no statistically significant difference in trifecta between cT1a and cT1b + tumours (p = 0.152). We found a statistically significant difference between the two groups in terms of RENAL scores (p = 0.0005), PADUA score (p = 0.0002), and robotic console time (133.8 ± 42.8 Vs 170 ± 54.8 min for cT1a versus cT1b + , respectively) (p = 0.002). On multivariate analysis, warm ischemia time (p = 0.069), blood loss (p = 0.345), UCS repair (p = 0.691) and GFR reduction (p = 0.152) were not statistically different. There was no statistically significant difference in intraoperative and post-operative complications (p = 0.9317) or length of hospital stay (p = 0.112). Although recurrences were observed in two patients (5.4%) of the cT1b group, there was no statistical difference in the recurrence-free survival at 12 and 24 months. Our study shows that RAPN can be safely done for cT1b + renal tumours. These findings reinforce the view that RAPN should be considered a viable option for cT1b + lesions whenever technically feasible.
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Prostate-specific antigen (PSA) screening for prostate cancer (PCa) is limited by the lack of specificity but is further complicated in the benign prostatic hyperplasia (BPH) population which also exhibit elevated PSA, representing a clear unmet need to distinguish BPH from PCa. Herein, we evaluated the utility of FLNA IP-MRM, age, and prostate volume to stratify men with BPH from those with PCa. Diagnostic performance of the biomarker panel was better than PSA alone in discriminating patients with negative biopsy from those with PCa, as well as those who have had multiple prior biopsies (AUC 0.75 and 0.87 compared to AUC of PSA alone 0.55 and 0.57 for patients who have had single compared to multiple negative biopsies, respectively). Of interest, in patients with PCa, the panel demonstrated improved performance than PSA alone in those with Gleason scores of 5-7 (AUC 0.76 vs. 0.56) and Gleason scores of 8-10 (AUC 0.74 vs. 0.47). With Gleason scores (8-10), the negative predictive value of the panel is 0.97, indicating potential to limit false negatives in aggressive cancers. Together, these data demonstrate the ability of the biomarker panel to perform better than PSA alone in men with BPH, thus preventing unnecessary biopsies.
Subject(s)
Biomarkers, Tumor/blood , Diagnosis, Differential , Prostatic Hyperplasia/diagnosis , Prostatic Neoplasms/diagnosis , Aged , Humans , Male , Middle Aged , Neoplasm Grading , Prostate/metabolism , Prostate-Specific Antigen/blood , Prostatic Hyperplasia/blood , Prostatic Hyperplasia/pathology , Prostatic Neoplasms/blood , Prostatic Neoplasms/pathologyABSTRACT
BACKGROUND: B-cell maturation antigen (BCMA) is expressed on malignant plasma cells from patients with multiple myeloma (MM). These patients have higher levels of serum (s)BCMA than healthy subjects, and levels correlate with disease status. The half-life of sBCMA is only 24-36 h, and levels are independent of renal function. OBJECTIVE: We determined whether baseline sBCMA values, a ≥ 25% increase, and a ≥ 50% decrease during treatment predicted progression-free survival (PFS) and overall survival (OS) among 81 patients with relapsed/refractory MM (RRMM) starting new treatments. METHODS: Serum was obtained on day 22 of each patient's 28-day cycle of new therapy. Kaplan-Meier survival analysis and log-rank comparison tests were used to determine the effect of baseline sBCMA. The effect of percentage change in sBCMA was investigated using time-dependent Cox proportional hazard models. RESULTS: Patients with baseline sBCMA levels above the median had a shorter PFS (p = 0.0077), and those in the highest quartile had a shorter PFS (p = 0.0012) and OS (p = 0.0022). A ≥ 25% increase at week 4, week 8, and anytime through week 12 predicted a shorter PFS (p = 0.0011, p = 0.0005, and p < 0.0001, respectively). A ≥ 50% decrease at week 4, week 8, and anytime through week 12 predicted a longer PFS (p = 0.0045, p = 0.029, p = 0.0055, respectively). A ≥ 25% increase in sBCMA occurred before progression according to International Myeloma Working Group criteria in 67.5% of patients. CONCLUSIONS: Our results indicate the potential for the use of sBCMA as a new biomarker for monitoring patients with RRMM.
Subject(s)
B-Cell Maturation Antigen/blood , Multiple Myeloma/blood , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Multiple Myeloma/mortality , Multiple Myeloma/physiopathology , Survival AnalysisABSTRACT
The serum B-cell maturation antigen (sBCMA) has been identified as a novel serum biomarker for patients with multiple myeloma. However, no study has yet established a reference range for sBCMA levels. Its levels were determined in 196 healthy subjects and showed a right-tailed distribution with a median value of 37·51 ng/ml with a standard deviation of 22·54 ng/ml (range 18·78-180·39 ng/ml). Partitioning of subgroup reference ranges was considered but determined to be irrelevant. A non-parametric method using the median ± 2 standard deviations suggests using a universal reference interval of <82·59 ng/ml.
Subject(s)
B-Cell Maturation Antigen/blood , Biomarkers, Tumor/blood , Multiple Myeloma/blood , Neoplasm Proteins/blood , Adult , Aged , Female , Humans , Male , Middle AgedABSTRACT
Serum B-cell maturation antigen (sBCMA) is a novel biomarker for B-cell malignancies. A normal reference range (<82·59 ng/ml) has been recently established but the impact of achieving normal levels to outcomes for patients receiving treatment for B-cell malignancies has not been studied. We first found that among multiple myeloma (MM) patients starting a new treatment, those who begin treatment within normal sBCMA limits (<82·59 ng/ml) have improved progression-free survival (PFS; P = 0·0398) and overall survival (OS; P = 0·0217) than those who do not. Furthermore, among patients who begin treatment with elevated (≥82·59 ng/ml) sBCMA levels, we assessed the relationship of a decrease in sBCMA to the normal range to OS and found that those who normalize sBCMA demonstrated improved OS (P = 0·0078). Normalizing patients also experienced a markedly improved overall response rate (P < 0·0001). Moreover, all patients who achieved complete remission (CR) showed normalization of sBCMA, and time to normalization (median 0·9 months) was faster than time to CR (5·0 months; P = 0·0036) for these patients. These results suggest that normalization of sBCMA may be an accurate predictor of OS for MM patients during treatment and predict for a higher likelihood of response.
Subject(s)
B-Cell Maturation Antigen/blood , Multiple Myeloma/blood , Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Multiple Myeloma/diagnosis , Multiple Myeloma/therapy , Prognosis , Survival Analysis , Treatment OutcomeABSTRACT
INTRODUCTION AND OBJECTIVE: Carcinoma prostate is considered highly aggressive in Asian countries such as India. This raises an argument whether active surveillance (AS) gives a false sense of security as opposed to upfront radical prostatectomy (RP) in Indian males with low-risk prostate cancer (PCa). We analyzed our prospectively maintained robot-assisted RP (RARP) database to address this question. MATERIALS AND METHODS: Five hundred and sixty-seven men underwent RARP by a single surgical team from September 2013 to September 2019. Of these, 46 (8.1%) were low risk considering the National Comprehensive Cancer Network criteria. Gleason grade group and stage were compared before and after surgery to ascertain the incidence of upgrading and upstaging. Preoperative clinical and pathological characteristics were analyzed for association with the probability of upstaging and upgrading. RESULTS: The mean age was 60.8 ± 6.8 years. Average prostate-specific antigen level was 6.7 ± 2.0 ng/mL. 40 (86.9%) patients had a T1 stage disease and 6 (13%) patients were clinically in T2a stage. A total of 25 (54.3%) cases were either upstaged or upgraded, 19 (41.3%) showed no change, and the remaining 2 (4.3%) had no malignancy on the final RP specimen. Upstaging occurred in 8 (17.4%) cases: 5 (10.9%) to pT3a and 3 (6.5%) to pT3b. Upgrading occurred in 23 (50%) cases: 19 (41.3%) to Grade 2; 3 (6.5%) to Grade 3; and 1 (2.2%) to Grade 4. CONCLUSIONS: There is a 50% likelihood of upstaging or upgrading in Indian males with low-risk PCa eligible for AS. Decision to proceed with AS should be taken carefully.
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INTRODUCTION: The objective of this study was to evaluate the perioperative outcomes of patients undergoing robot-assisted radical cystectomy (RARC) with intracorporeal ileal conduit (IIC) urinary diversion treated in line with the enhanced recovery after surgery (ERAS) protocol. METHODS: After approval from the institutional ethics committee, we conducted an analysis of a prospectively maintained database of patients undergoing RARC + IIC using ERAS protocol by a single surgical team with the da Vinci Xi® system from March 2016 till December 2018. To minimize the effect of the learning curve of this complex procedure, we excluded the first thirty patients from analysis. RESULTS: Thirty-five consecutive patients (33 males and 2 females) with a median age of 69 years (range: 50-82) were evaluated. The median total console time and console time for diversion were 253 min (range: 191-370) and 80 min (range: 65-90), respectively. The median estimated blood loss was 300 cc (range: 50-500). The median length of stay was 8 days (range: 4-30). Per-urethral pelvic drain was removed at a median of 2 days (range: 1-17). Overall, complications occurred in 16/35 (45.7%) patients, of which major complications (≥Grade 3) were seen in 5/35 (14.3%) patients, without any 90-day mortality. The median follow-up for the cohort was 14 months (1-34). CONCLUSIONS: While the initial outcomes of this combined treatment strategy appear promising in terms of complication rates and perioperative parameters, greater insight is required from multi-institutional data sets and prospective comparative studies to establish the true value of RARC + IIC and ERAS protocol for bladder cancer.
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A 12-year-old male child, during evaluation of chronic constipation, was incidentally diagnosed to have a fronded bladder growth. Transurethral en bloc excision of the tumor was achieved using holmium laser. Histopathology confirmed it to be an inflammatory pseudotumor. This case is reported for its unusual presentation and management by holmium laser.
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Ubiquitin plays an essential role in modulating protein function, and deregulation of the ubiquitin system leads to the development of a variety of human diseases. E3 Ubiquitin ligases that mediate ubiquitination and degradation of caspases prevent apoptosis, and as such belong to the family of inhibitors of apoptosis proteins (IAPs). Diablo is a substrate of IAPs but also a negative regulator of IAPs in apoptotic pathway as it blocks the interaction between IAPs and caspases. In efforts to identify IAP inhibitors, we developed sandwich immunoassays in conjunction with an electrochemical luminescence (ECL) platform for quantitation of total Diablo, ubiquitinated Diablo, and ubiquitinated Diablo with K48-specific linkage. The assay panel detects Diablo ubiquitination level changes in the presence of IAP inhibitor or proteasome inhibitor, demonstrating its potential as a cost-efficient high-throughput method for drug discovery involving IAP ubiquitination cascade. The ECL based sandwich assay panel performance was subsequently evaluated for precision, linearity, and limit of quantification.
Subject(s)
Apoptosis Regulatory Proteins/isolation & purification , Drug Discovery/methods , High-Throughput Screening Assays/methods , Mitochondrial Proteins/isolation & purification , X-Linked Inhibitor of Apoptosis Protein/antagonists & inhibitors , Apoptosis Regulatory Proteins/metabolism , Cell Line, Tumor , Humans , Immunoassay/methods , Luminescent Measurements/methods , Mitochondrial Proteins/metabolism , Proteasome Inhibitors/pharmacology , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Ubiquitination/drug effects , X-Linked Inhibitor of Apoptosis Protein/metabolismABSTRACT
B-cell maturation antigen (BCMA), a tumor necrosis factor receptor (TNFR) family member, is selectively expressed on terminally differentiated B-lymphocytes including multiple myeloma (MM) tumor cells. We sought to determine whether circulating (c)BCMA in MM serum interferes with antiBCMA antibody binding to MM cells. An enzyme-linked immunosorbent assay (ELISA) was used to determine serum (s) BCMA levels among 379 samples from patients with relapsed/refractory MM (RRMM). Furthermore, flow cytometric and immunofluorescent studies were used to examine if concentrations of BCMA in patients' serum were high enough to interfere with the binding of anti-BCMA antibody to MM tumor cells. We have shown that BCMA is elevated in the serum from MM patients and that the median concentration of sBCMA from RRMM patients was 176 ng/mL (n = 379). Additionally, there was a consistent decrease in the binding of anti-BCMA antibody to MM tumor cells with sBCMA level ≥156 ng/mL. Together, these results demonstrate that circulating BCMA levels in most RRMM patients are high enough to interfere with anti-BCMA antibody binding to MM tumor cells and may interfere with BCMA-targeted immune-based therapies.