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Identification of Monoclonal Antibody Variants Involved in Aggregate Formation - Part 1: Charge Variants
No full textBiopharmaceutical products contain conformational and chemical variants, that are typically well characterized regarding identity and activity. However, little is known about their self-interaction propensity and tendency to unfold, which are critical characteristics for drug stability and safety. This study aimed to separate and compare charge variants of a monoclonal antibody (mAb) and to identify aggregation prone species. We show a semi-preparative cation exchange method, that we developed to separate the individual
acidic and basic variants from the naive mAb. Additionally, we demonstrate, that the yield and purity of the fractionated charge species, extracted by that method, were suffient for subsequent analysis of aggregate content, conformation stability and selfinteraction. Our analysis revealed a differently behaving acidic variant and confirmed its increased aggregation propensity by molecular modeling. During a stability study, the potentially aggregation prone charge variant posed a limited risk to the DS. We are the first
to look at the stability of single charge variants of biopharmaceuticals, and thus present
manufacturers and regulatory authorities with a method to enhance drug safety
Discovery of LYS006, a Potent and Highly Selective Inhibitor of Leukotriene A4 Hydrolase
No full textABSTRACT: The cytosolic metalloenzyme Leukotriene A4 Hydrolase (LTA4H) is the final and rate-limiting enzyme in the biosynthesis of pro-inflammatory lipid mediator Leukotriene B4 (LTB4). Genetic deletion as well as pharmacological inhibi-tion of LTA4H in preclinical models have validated this enzyme as an attractive drug target in chronic inflammatory dis-eases. Despite several attempts by different pharmaceutical companies, no LTA4H inhibitor has yet reached the market. Herein, we disclose the discovery and preclinical profile of LYS006, a highly potent and selective LTA4H inhibitor. A fo-cused DSF screen for binders of LTA4H afforded fragments 1 and 2 that could be co-crystallized with LTA4H and inspired a fragment merging. Further optimization led to chiral amino acids and ultimately to LYS006/(S)-22, a picomolar LTA4H inhibitor with exquisite whole blood potency (IC90 = 143 nM) and long-lasting in vivo pharmacodynamic effects. Due to its high selectivity and its ability to suppress LTB4 generation entirely in vivo at low exposures, LYS006 has the potential for a best-in-class LTA4H inhibitor and is currently being studied in phase II clinical trials in inflammatory acne, hidradenitis sup-purativa, ulcerative colitis and NASH
Continuous Flow as an Enabling Technology: A Fast and Versatile Entry to Functionalized Glyoxal Derivatives
No full textOrganometallic chemistry is a remarkable opportunity for continuous processing and has been applied to demonstrated
effect in the industrial landscape. We herein report two complementary strategies employing organolithium chemistry for the synthesis of glyoxal derivatives. Micro-mixer technology allows for the generation of unstable organometallic intermediates and their instantaneous in-line quench with esters as electrophiles. Selective mono-addition was observed via putative stabilized tetrahedral intermediates. Advantages offered by flow chemistry technologies facilitate a direct and efficient access to masked 1,2-dicarbonyl compounds while mitigating undesired by-product formation. These two approaches enable the production of advanced and valuable synthetic building blocks for heterocyclic chemistry with throughputs of grams per minute
Letter to the Editor Regarding “Pathology Informatics Education Committee of the American College of Veterinary Pathologists (ACVP)”
No full textNo abstrac
In Vitro Kinetic Degradation and In Vivo Biocompatibility Evaluation of Polycaprolactone-Based Growth Factor Delivery Matrices in the Rotator Cuff
No full textThe recent years have seen a significant surge in the use of synthetic biodegradable polymers for growth factor delivery in the rotator cuff. While these polymers have been successfully applied in delivery of factors in other tissues, the anatomical complexity, hypovascularity, cellularity, and reduced clearance of degradation by-products in the rotator cuff, creates unique requirements in tailoring the physical dimensions, chemical constituents, drug release and degradation characteristics of biomaterials for implantation. In this study, we investigate poly-lactic acid co-epsilon-caprolactone (30:70 LA:CL ratio) at 35-45kDa range and varying polymeric films casting concentrations (5-20%) as potential growth factor delivery matrices in the rotator cuff. Matrices were fabricated of 300µm thickness and 3x3mm surface area to facilitate model protein encapsulation and controlled release, and smooth translation of the matrix under the bony acromion after implantation in the rotator cuff. The matrix with the highest casting concentration (20wt%) showed unique, highly regular, and controlled release of the protein payload compared to the lower- casting concentrations (15 and 10wt%) and - molecular weights (35kDa) matrices. All films were found to lose molecular weight rapidly during the first 4 weeks due to the preferential hydrolysis of lactide-rich regions within the polymer, and then maintain a relatively stable molecular weight between week 4 and 8 due to the emergence of highly-crystalline caprolactone-rich regions. Nevertheless, the cleaved lactide-chains were not small enough to exit through the polymeric matrix as was evident from the maintenance of bulk matrix weight, form, and polymer dispersity index. This resulted in recrystallization of the cleaved chains in the presence of water molecules increasing the crystallinity of the matrix as was evident from the H-NMR and thermal analysis. Kinetic analysis revealed an inverse-linear relationship between polymer casting concentration and polymer break down. The ‘context-dependent’ biocompatibility evaluation was carried in a clinically-relevant rat model of acute rotator cuff repair model to address the unique features of both the tissue and the biomaterial being investigated. The matrices were found to remodel locally without undergoing catastrophic breakdown or causing excessive inflammatory reaction at the tissue site during the study period and is anticipated to completely degrade within 6 months of implantation. Our study is significant as it provides a systematic assessment of polymer properties that can be modifies to engineer morphogen release, degradation rates and mechanisms for biologic delivery in the rotator cuff. It also provides a pilot assessment of in situ biocompatibility of the polymeric matrix in the complex rotator cuff tissue
Parkinson’s Disease–Associated LRRK2 Interferes with Astrocyte-Mediated Alpha-Synuclein Clearance
No full textParkinson’s disease (PD) is a neurodegenerative, progressive disease without a cure. To prevent PD onset or at least limit neurodegeneration, a better understanding of the underlying cellular and molecular disease mechanisms is crucial. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene represent one of the most common causes of familial PD. In addition, LRRK2 variants are risk factors for sporadic PD, making LRRK2 an attractive therapeutic target. Mutations in LRRK2 have been linked to impaired alpha-synuclein (α-syn) degradation in neurons. However, in which way pathogenic LRRK2 affects α-syn clearance by astrocytes, the major glial cell type of the brain, remains unclear. The impact of astrocytes on PD progression has received more attention and recent data indicate that astrocytes play a key role in α-syn-mediated pathology. In the present study, we aimed to compare the capacity of wild-type astrocytes and astrocytes carrying the PD-linked G2019S mutation in Lrrk2 to ingest and degrade fibrillary α-syn. For this purpose, we used two different astrocyte culture systems that were exposed to sonicated α-syn for 24 h and analyzed directly after the α-syn pulse or 6 days later. To elucidate the impact of LRRK2 on α-syn clearance, we performed various analyses, including complementary imaging, transmission electron microscopy, and proteomic approaches. Our results show that astrocytes carrying the G2019S mutation in Lrrk2 exhibit a decreased capacity to internalize and degrade fibrillar α-syn via the endo-lysosomal pathway. In addition, we demonstrate that the reduction of α-syn internalization in the Lrrk2 G2019S astrocytes is linked to annexin A2 (AnxA2) loss of function. Together, our findings reveal that astrocytic LRRK2 contributes to the clearance of extracellular α-syn aggregates through an AnxA2-dependent mechanism
Clinical design and analysis strategies for development of cell and gene therapies: quantitative drug development in the age of genetic medicine
No full textCell and gene therapies have shown enormous promise across a range of diseases in recent years. Numerous adoptive cell therapy modalities as well as systemic and direct to target tissue gene transfer administrations are currently in clinical development. The clinical trial design, development, analysis, and reporting of novel cell and gene therapies can differ significantly from established practices for small molecule drugs and biologics. Here we discuss important quantitative considerations and key competencies for drug developers in the preclinical, trial design, and lifecycle planning for gene therapies. We argue that the unique development path of gene therapies requires practicing quantitative drug developers—statisticians, pharmacometricians, pharamcokineticists, and medical and operational leads—to exercise active collaboration and cross-functional learning across development stages
Estimating drug potency in the competitive target mediated drug disposition (TMDD) system when the endogenous ligand is included.
No full textPredictions for target engagement are often used to guide drug development. In particular, when selecting the recommended phase 2 dose of a drug that is very safe, and where good biomarkers for response may not exist (e.g. in immuno-oncology), a receptor occupancy prediction could even be the main determinant in justifying the approved dose, as was the case for atezolizumab. The underlying assumption in these models is that when the drug binds its target, it disrupts the interaction between the target and its endogenous ligand, thereby disrupting downstream signaling. However, the interaction between the target and its endogenous binding partner is almost never included in the model. In this work, we take a deeper look at the in vivo system where a drug binds to its target and disrupts the target’s interaction with an endogenous ligand. We derive two simple steady state inhibition metrics (SSIMs) for the system, which provides intuition for when the competition between drug and endogenous ligand should be taken into account for guiding drug development
Bimagrumab to Improve Recovery after Hip Fracture in the Elderly: A Randomised, Double-Blind, Placebo-Controlled Phase 2a/b Study
No full textBackground: Elderly patients undergoing surgery for hip fracture repair frequently experience loss of muscle mass and strength due to immobility, which may delay functional recovery. This study investigated whether bimagrumab, a human monoclonal antibody targeting activin type II receptors (ActRII), can improve postsurgical recovery.
Methods: This was a randomised, double-blind, parallel-group, placebo-controlled Phase 2a/2b trial conducted at 50 centres in 18 countries. Patients aged ≥60 years who recently (past 6 weeks) underwent surgery for hip fracture were enrolled to receive intravenous treatment with either placebo or one of three doses of bimagrumab (70 mg, 210 mg, and 700 mg) every 4 weeks for 24 weeks. The primary endpoint was the change from baseline in total lean body mass (LBM) measured by dual-energy X-ray absorptiometry (DEXA) at Week 24. Key secondary endpoints included the respective changes in habitual gait speed (m/s) and the Short Physical Performance Battery (SPPB). Safety and tolerability were assessed by recording adverse events (AEs), physical examination, vital signs, laboratory assessments, and electrocardiography. (ClinicalTrials.gov, NCT02152761).
Findings: Of the 250 treated patients, 207 (83%) completed the treatment period. The absolute increase in LBM from baseline was dose dependent, with mean±standard deviation gains of 1·9±1·7 kg in the bimagrumab 210 mg group and 2·8±2·2 kg in the 700 mg group, compared with 0·3±2·0 kg in the placebo group (both p<0·001). Changes in the key secondary endpoints were similar across treatment groups suggesting no enhancement of physical recovery with bimagrumab over placebo. Bimagrumab was safe and well tolerated; the most frequently reported AEs were falls, muscle spasms, and arthralgia.
Interpretation: Twenty-four weeks of treatment with bimagrumab led to dose-dependent and clinically meaningful increases in LBM in elderly hip fracture patients. However, no functional benefit was seen in terms of the recovery of mobility or physical performance compared to placebo
First-in-Human, Phase I Dose-Escalation Study of CGM097, a HDM2 Inhibitor in Adult Patients With p53 Wild-type Advanced Solid Malignancies
No full textBackground: CGM097 inhibits p53-HDM2 interaction, thus activates p53 downstream effector pathways inducing cell cycle arrest and/or apoptosis. This phase I study aimed at assessing the safety, MTD, PK/PD, and preliminary antitumor activity of CGM097 in advanced solid tumors patients (NCT01760525).
Methods: Fifty-one patients received oral treatment with CGM097 10-400mg 3qw (n=31) and on an alternative regimen of 3qw 2 weeks on/1 week off (300-700mg; n=20). Choice of dose regimen was guided by PD biomarkers, PK/PD relationship, and modeling of drug-induced changes in platelet kinetics.
Results: No dose-limiting toxicities were reported in any regimens. Grade 3/4 AEs suspected to be drug-related were reported in 21 patients. Main reason for discontinuation was disease progression (n=41). CGM097 plasma concentrations increased in a dose proportional manner. Disease control rate was 39%, including one partial response (300mg, 3qw) and 19 patients in stable disease (10-700mg 3qw). Forty patients had a cumulative treatment duration of >16 weeks, with 8 patients on treatment for >32 weeks. The MTD was not determined.
Conclusions: Despite delayed-onset thrombocytopenia frequently observed, the tolerability of CGM097 appears manageable. Although CGM097 is not being further developed, this study led to important modeling-derived learnings to optimize dose scheduling of next generation HDM2 inhibitors