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Safety, Tolerability, and Pharmacodynamics of the ADAMTS-5 Nanobody M6495: Two Phase 1, Single-Center, Double-Blind, Randomized, Placebo-Controlled Studies in Healthy Subjects and Patients With Osteoarthritis.
No full textTo assess the safety, pharmacokinetics (PK), and pharmacodynamics (PD) of single and multiple injections of M6495, a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS-5) nanobody, in healthy volunteers and patients with osteoarthritis.Two randomized, placebo-controlled, double-blind studies were performed. Study 1 enrolled 54 healthy male volunteers who received one subcutaneous (s.c.) injection of M6495 (1-300 mg) or placebo (ratio 2:1), evaluating safety, PK, and PD as changes in the serum aggrecan fragment alanine-arginine-glycine-serine (ARGS). Study 2 enrolled 32 patients with osteoarthritis with Kellgren-Lawrence grades 2 to 4 and pain greater than or equal to 40 on the Western Ontario and McMaster Universities Arthritis Index pain subscale at screening and evaluated the safety, PK, and PD of three doses every two weeks (75-300 mg per dose) or six once-weekly M6495 s.c. doses (300 mg) or placebo (ratio 3:1) over 106 days' follow-up.M6495 in single and multiple doses of less than or equal to 300 mg s.c. weekly was well tolerated with no clinically significant changes in any safety parameter. Adverse events more frequently reported in the M6495 groups were mostly mild cases of injection site reactions, myalgia, and nausea, which resolved after treatment cessation. The elimination half-life of single s.c. doses of M6495 ranged from 79 to 267 hours. M6495 administration substantially reduced serum ARGS levels, indicative of target engagement and indicating disease-modifying potential of M6495.Treatment with M6495 in single and multiple doses up to and including 300 mg s.c. was found to be well tolerated and adequately safe for further clinical evaluation of potential disease-modifying effects
Aptamer Proteomics for Biomarker Discovery in Heart Failure With Preserved Ejection Fraction: The PARAGON‐HF Proteomic Substudy
No full textPrognostic markers and biological pathways linked to detrimental clinical outcomes in heart failure with preserved ejection fraction (HFpEF) remain incompletely defined.We measured serum levels of 4123 unique proteins in 1117 patients with HFpEF enrolled in the PARAGON-HF (Efficacy and Safety of LCZ696 Compared to Valsartan, on Morbidity and Mortality in Heart Failure Patients With Preserved Ejection Fraction) trial using a modified aptamer proteomic assay. Baseline circulating protein concentrations significantly associated with the primary end point and the timing and occurrence of total heart failure hospitalization and cardiovascular death were identified by recurrent events regression, accounting for multiple testing, adjusted for age, sex, treatment, and anticoagulant use, and compared with published analyses in 2515 patients with heart failure with reduced ejection fraction from the PARADIGM-HF (Prospective Comparison of ARNI With ACEI to Determine Impact on Global Mortality and Morbidity in Heart Failure) and ATMOSPHERE (Efficacy and Safety of Aliskiren and Aliskiren/Enalapril Combination on Morbidity-Mortality in Patients With Chronic Heart Failure) clinical trials. We identified 288 proteins that were robustly associated with the risk of heart failure hospitalization and cardiovascular death in patients with HFpEF. The baseline proteins most strongly related to outcomes included B2M (β-2 microglobulin), TIMP1 (tissue inhibitor of matrix metalloproteinase 1), SERPINA4 (serpin family A member 4), and SVEP1 (sushi, von Willebrand factor type A, EGF, and pentraxin domain containing 1). Overall, the protein-outcome associations in patients with HFpEF did not markedly differ as compared with patients with heart failure with reduced ejection fraction. A proteomic risk score derived in patients with HFpEF was not superior to a previous proteomic score derived in heart failure with reduced ejection fraction nor to clinical risk factors, NT-proBNP (N-terminal pro-B-type natriuretic peptide), or high-sensitivity cardiac troponin.Numerous serum proteins linked to metabolic, coagulation, and extracellular matrix regulatory pathways were associated with worse HFpEF prognosis in the PARAGON-HF proteomic substudy. Our results demonstrate substantial similarities among serum proteomic risk markers for heart failure hospitalization and cardiovascular death when comparing clinical trial participants with heart failure across the ejection fraction spectrum.URL: https://www.clinicaltrials.gov; Unique Identifiers: NCT01920711, NCT01035255, NCT00853658
iPS-derived human macrophages as an infection model for Leishmania donovani
No full textThe parasite Leishmania donovani is one of the species causing visceral leishmaniasis in humans, a deadly infection claiming up to 40 000 lives each year. Despite its huge burden, the current drugs available all have severe drawbacks and there is an urgent need to find new antileishmanial compounds. However, the search for drug candidates is complicated by the intracellular lifecycle of Leishmania. Here, we investigated the use of human induced pluripotent stem cell (iPS) derived macrophages (iMAC) as host cells for L. donovani. iMAC obtained through embryoid body differentiation were infected with L. donovani promastigotes and high content imaging techniques were used to optimize the iMAC seeding density and multiplicity of infection, allowing us to reach infection rates up to 70% five days after infection. IC50 values calculated for miltefosine and amphotericin B using the infected iMAC or mouse peritoneal macrophages as host cells in an assay are comparable to each other and to what has been reported in literature, showing its potential as a new drug screening assay
Liquid Chromatography ICP-MS to Assess the Stability of 175Lu- and natGa-Based Tumor-Targeting Agents towards the Development of 177Lu- and 68Ga-Labeled Radiopharmaceuticals.
No full textIn recent years, nuclear medicine has gained great interest, partly due to the success story of [177Lu]Lu-PSMA-617 (PluvictoTM). Still, in-depth preclinical characterization of radiopharmaceuticals mainly happens at centers that allow working with radioactive material. To support the development of novel radiopharmaceuticals, alternative non-radioactive characterization assays are highly desirable. The aim of this study was to demonstrate that inductively coupled plasma mass spectrometry (ICP-MS) associated with a chromatographic system can serve as a surrogate for the classical high-performance liquid chromatography (HPLC)-radiodetector combination for preclinical in vitro characterization of non-radioactive metal-labeled analogs of radiopharmaceuticals. In this proof-of-concept study, we demonstrate the applicability of HPLC-ICP-MS by assessing the stability of 175Lu- and natGa-labeled prostate-specific membrane antigen (PSMA)-targeting peptidomimetics, single domain antibody (sdAb) conjugates, and monoclonal antibody (mAb) conjugates. 175Lu-labeled DOTAGA-conjugated and natGa-labeled NODAGA-conjugated sdAbs and mAbs showed the highest stability with >90% still intact after 24 h. The peptidomime-tics [175Lu]Lu-PSMA-617 and [natGa]Ga-PSMA-11 showed identical in vitro serum stability as it was reported for their corresponding radioligands with >99% intact species after 24 h incubation in mouse serum, demonstrating the reliability of the method. Hence, the established HPLC-ICP-MS methods can support the development of novel radiopharmaceuticals in a classical pharmaceutical setting
De-risking ASO-induced histopathology in spinal cord of monkeys by molecular localization investigations
No full textThe safety of a 2′-O-methoxyethyl antisense oligonucleotide (2'-MOE ASO) was investigated in cynomolgus monkeys in a chronic toxicity study after repeated intrathecal (IT) administration. Histopathological examination revealed formation of lymphoid follicles in the spinal cord (SC) at the injection site and presence of granular material in motor neurons of the SC in high dose animals. The granular material was seen in all the segments of the SC, but mainly in the lumbar segment and persisted at recovery. Findings associated with repeated intrathecal administration of 2' MOE ASOs have been characterized in non-human primate toxicity studies, especially in the brain but findings in the SC are poorly documented. We reported a high incidence of findings in the SC compared to brain, especially in the lumbar segment in proximity with IT injection sites. An extensive panel of immunohistochemistry (IHC) markers showed that the lymphoid follicles had a cellular composition and organization consistent with a tertiary lymphoid structure (TLS) and were not associated with axonal damage in the adjacent nervous tissue. IHC markers of neuronal degeneration and function, in additional to ISH with a miRNA probe complementary to the ASO revealed that the granular material was composed of a dose proportional ASO accumulation in the cytoplasm of neurons without inducing cell death or apoptosis but perturbing the lysosomal trafficking. Glial and ependymal cells in the SC also showed dose dependent ASO accumulation in the absence of granular material noted by H&E. Based on these molecular localization data, the presence of lymphoid follicles in SC suggests a chronic local immune activation due to repetitive IT injections of ASO and the granular material in SC neurons were caused by ASO accumulation. Based on the lack of cellular dysfunction or axonal damage, these findings were considered as non-adverse
A phase 1b/2 trial of capmatinib plus spartalizumab or spartalizumab alone in patients with pretreated hepatocellular carcinoma
No full textBackground & Aims: This phase 1b/2 trial evaluated the safety and efficacy of capmatinib in combination with spartalizumab or spartalizumab alone in patients with advanced hepatocellular carcinoma (HCC).
Methods: Eligible patients who had progressed or were intolerant to sorafenib received escalating doses of capmatinib 200 mg, 300 mg, and 400 mg bid plus spartalizumab 300 mg q3w in phase 1b. Once the recommended phase 2 dose (RP2D) was declared, phase 2 commenced with randomization 1:1 to treatment with either capmatinib + spartalizumab (n = 32) or spartalizumab alone (n = 30). Primary endpoints were safety and tolerability (phase 1b); and investigator-assessed ORR per RECIST v1.1 for combination versus single-agent arms using a Bayesian logistic regression model (phase 2).
Results: In phase 1b, the RP2D for capmatinib in combination with spartalizumab was determined as 400 mg bid. Dose-limiting toxicity of grade 3 diarrhea was reported in 1 patient in the capmatinib 400 mg bid + spartalizumab 300 mg q3w dose level. The primary endpoint in phase 2 was not met. The observed ORR in the capmatinib + spartalizumab arm was 9.4% versus 10% in the spartalizumab arm. The most common any-grade treatment-related adverse events (TRAEs ≥20%) were nausea (37.5%), asthenia and vomiting (28.1% each), diarrhea, pyrexia, and decreased appetite (25.0% each) in the combination arm; TRAEs ≥10% were pruritus (23.3%) and rash (10.0%) in the spartalizumab-alone arm.
Conclusion: Capmatinib at 400 mg bid plus spartalizumab 300 mg q3w was established as the RP2D, with manageable toxicities and no significant safety signals, but the combination did not show superior clinical activity compared to spartalizumab single-agent treatment in patients with advanced HCC who had been previously treated with sorafenib
Asymmetric Synthesis of α-Chloroamides via Photoenzymatic Hydroalkylation of Olefins.
No full textPhotoenzymatic intermolecular hydroalkylations of olefins are highly enantioselective for chiral centers formed during radical termination but poorly selective for centers set in the C-C bond-forming event. Here, we report the evolution of a flavin-dependent "ene"-reductase to catalyze the coupling of α,α-dichloroamides with alkenes to afford α-chloroamides in good yield with excellent chemo- and stereoselectivity. These products can serve as linchpins in the synthesis of pharmaceutically valuable motifs. Mechanistic studies indicate that radical formation occurs by exciting a charge-transfer complex templated by the protein. Precise control over the orientation of molecules within the charge-transfer complex potentially accounts for the observed stereoselectivity. The work expands the types of motifs that can be prepared using photoenzymatic catalysis
Understanding an impact of patient enrollment pattern on predictability of central randomization in a multi-center clinical trial
No full textIn a multi-center randomized controlled trial (RCT) with competitive recruitment, eligible patients are enrolled sequentially by different study centers and are randomized to treatment groups using the chosen randomization method. Given the stochastic nature of the recruitment process, some centers may enroll more patients than others, and in some instances, a center may enroll multiple patients in a row, e.g., on a given day. If the study is open label, the investigators might be able to make intelligent guesses on upcoming treatment assignments in the randomization sequence, even if the trial is centrally randomized and not stratified by center. In this
paper, we use the enrollment data from a real multi-center RCT to quantify the susceptibility of two restricted randomization procedures, the permuted block design and the big stick design, to selection bias under the convergence strategy of Blackwell and Hodges (1957) applied at the center level. We provide simulation evidence that the expected proportion of correct guesses may be greater than 50% (i.e., increased risk of the selection bias) and depends on the chosen randomization method and the number of study patients recruited by a given center that takes consecutive positions on the central allocation schedule. We propose some strategies for ensuring stronger encryption of the randomization sequence to mitigate the risk of selection bias
¬The impact of earth-abundant metals as a replacement for Pd in cross coupling reactions
No full textSubstitution of one metal catalyst for another is not straightforward as simply justifying this change based on the availability and/or cost of the metals. A life cycle-like assessment was performed leading to the conclusion that the commonly held view that use of earth-abundant metals (and in this case study, Ni) are replacements for palladium most notably in cross coupling reactions, and Suzuki-Miyaura couplings, in particular, is an incomplete analysis of the entire picture. This notion can be misleading, and unfortunately derives nowadays mainly from the standpoint of cost, and to some degree, the relative natural abundance and mining impact of each metal. However, a more realistic appreciation emerges when several additional reaction parameters involved in the couplings are considered. Such an analysis unequivocally points to the major impact on climate change brought about by use of organic solvents, while the metals actually play subordinate roles in terms of CO2-release into the environment. Clearly, this study reveals that several factors contribute in various ways to the overall carbon footprint. Hence, a far more detailed analysis is required than that typically being utilized
Characterizing Glycosylation of Adeno-Associated Virus Serotype 9 Capsid Proteins Generated from HEK293 Cells through Glycopeptide Mapping and Released Glycan Analysis
No full textRecombinant adeno-associated viral (AAV) vectors have emerged as prominent gene delivery vehicles for gene therapy. AAV capsid proteins determine tissue specificity and immunogenicity and play important roles in receptor binding, the escape of the virus from the endosome, and the transport of the viral DNA to the nuclei of target cells. Therefore, the comprehensive characterization of AAV capsid proteins is necessary for a better understanding of the vector assembly, stability, and transduction efficiency of AAV gene therapies. Glycosylation is one of the most common post-translational modifications (PTMs) and may affect the tissue tropism of AAV gene therapy. However, there are few studies on the characterization of the N- and O-glycosylation of AAV capsid proteins. In this study, we identified the N- and O-glycosylation sites and forms of AAV9 capsid proteins generated from HEK293 cells using liquid chromatography–tandem mass spectrometry (LC-MS)-based glycopeptide mapping and identified free N-glycans released from AAV9 capsid proteins by PNGase F using hydrophilic interaction (HILIC) LC-MS and HILIC LC-fluorescence detection (FLD) methods. This study demonstrates that AAV9 capsids are sprinkled with sugars, including N- and O-glycans, albeit at low levels. It may provide valuable information for a better understanding of AAV capsids in supporting AAV-based gene therapy development