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Green Chemistry Articles of Interest to the Pharmaceutical Industry (24)
No full textThis is a compilation of literature articles - Edition 2
Protein formulations containing polysorbates: are metal chelators needed at all?
No full textProteins are prone to post-translational modifications at specific sites, which can affect their physicochemical properties, and consequently also their safety and efficacy. Sources of post-translational modifications include oxygen and reactive oxygen species. Also, catalytic amounts of Fe(II) or Cu(I) can promote increased activities of reactive oxygen species, and thus catalyse the production of particularly reactive hydroxyl radicals. When oxidative post-translational modifications are detected in the biopharmaceutical industry, common practice is to add chelators to the formulation. However, the resultant complexes with metals can be even more damaging. Indeed, this is supported here using an ascorbate redox system assay and peptide mapping. EDTA addition strongly accelerated formation of hydroxyl radicals in an iron-ascorbate system, while DTPA addition did not. When Fe(III) was substituted with Cu(II), EDTA addition almost stopped hydroxyl radical production, whereas DTPA addition showed continued production, but at a reduced rate. Further, EDTA accelerated metal-catalysed oxidation of proteins, and thus did not protect them from Fe-mediated oxidative damage. As every formulation is unique, justification for EDTA or DTPA addition should be based on experimental data and not common practice
Microplate Selection and Recommended Practices in High-throughput Screening and Quantitative Biology
No full textHigh-throughput screening (HTS) can efficiently efficiently assay multiple discrete biological reactions using multi-well microplates. The choice of microplate is a crucial yet often overlooked technical decision in HTS and quantitative biology. This This chapter reviews key criteria for microplate selection, including: well number, well volume and shape, microplate color, surface treatments/coatings, and considerations for specialized applications such as high-content screening. This This chapter then provides important technical advice for microplate handling including mixing, dispensing, incubation, and centrifugation. Special topics are discussed, including microplate surface properties, plate washing, well-to-well contamination, microplate positional effects, effects,effects, well-to-well and inter-lot variability, and troubleshooting. This This information and best practices derived from academic, government, and industry screening centers, as well as microplate vendors, should accelerate assay development and enhance assay quality
Tg.rasH2 Mouse Model for Assessing Carcinogenic Potential of Pharmaceuticals: Industry Survey of Current Practices
No full textThe Tg.rasH2 mouse was developed as an alternative model to the traditional 2-year mouse bioassay for pharmaceutical carcinogenicity testing. This model has found extensive use in support of pharmaceutical drug development over the last few decades. It has the potential to improve quality and timeliness, reduce animal usage, and in some instances allow expedient decision-making regarding the human carcinogenicity potential of a drug candidate. Despite the increased use of the Tg.rasH2 model, there has been no systematic survey of current practices in the design, interpretation of results from the bioassay, and global health authority perspectives. Therefore, the aim of this work was to poll the pharmaceutical industry on study design practices used in the dose range finding and definitive 6-month studies and on results relative to the ongoing negotiations to revise The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use S1 Guidance. Twenty-two member companies of International Consortium for Innovation and Quality in Pharmaceutical Development DruSafe Leadership Group participated in the survey, sharing experiences from studies conducted with 55 test compounds between 2010 and 2018. The survey results provide very useful insights into study design and interpretation. Importantly, the results identified several key opportunities for reducing animal use and increasing the value of testing for potential human carcinogenicity using this model. Recommended changes to study designs that would reduce animal usage include eliminating the requirement to include positive control groups in every study, use of nontransgenic wild-type littermates in the dose range finding study, and use of microsampling to reduce or eliminate satellite groups for toxicokinetics
TFEB Transcriptional Responses Reveal Negative Feedback by BHLHE40 and BHLHE41
No full textTranscription factor EB (TFEB) activates lysosomal biogenesis genes in response to environmental cues. Given implications of impaired TFEB signaling and lysosomal dysfunction in metabolic, neurological, and infectious diseases, we aim to systematically identify TFEB-directed circuits by examining transcriptional responses to TFEB subcellular localization and stimulation. We reveal that steady-state nuclear TFEB is sufficient to activate transcription of lysosomal, autophagy, and innate immunity genes, whereas other targets require higher thresholds of stimulation. Furthermore, we identify shared and distinct transcriptional signatures between mTOR inhibition and bacterial autophagy. Using a genome-wide CRISPR library, we find TFEB targets that protect cells from or sensitize cells to lysosomal cell death. BHLHE40 and BHLHE41, genes responsive to high, sustained levels of nuclear TFEB, act in opposition to TFEB upon lysosomal cell death induction. Further investigation identifies genes counter-regulated by TFEB and BHLHE40/41, adding this negative feedback to the current understanding of TFEB regulatory mechanisms.
Employing RNA sequencing, genome-wide CRISPR screening, and high-content subcellular imaging, Carey et al. systematically unravel localization- and stimulation-specific transcriptional responses to TFEB, including target gene activation at steady state. The authors further uncover a negative feedback loop by BHLHE40 and BHLHE41 that counteracts a TFEB transcriptional signature induced by lysosomal stress
Comparison of pharmacokinetics, safety and tolerability of secukinumab administered subcutaneously using different 2 mL delivery systems in healthy volunteers and in psoriasis patients
No full textAims
The aim of the study was to compare the pharmacokinetics, safety and tolerability of secukinumab with different devices for subcutaneous administration of 2 mL (i.e. 300 mg) secukinumab.
Methods
A phase 1 study in healthy subjects with six devices to administer 2 mL injection volumes was conducted to evaluate the serum pharmacokinetics (PK), safety and tolerability of secukinumab following s.c. injection of 300 mg in the abdomen or in the thigh at either the left or right side of the body. Primary PK endpoints were maximum observed concentration (Cmax), and areas under the curve (AUCinf and AUClast). The impact of site and side of injection on serum exposure was evaluated. In a phase 3 study in psoriasis patients, PK of secukinumab was evaluated following multiple s.c. injections of 300 mg by either 2 x 1 mL prefilled syringe or 1 x 2 mL prefilled syringe.
Results
Mean serum concentration-time profiles for administrations as 2 x 1mL injections or as 1 x 2 mL injections were similar. With an injection volume of 2 mL, perceived injection pain was not different from 2 x 1 mL injections. A non-clinically significant difference in PK endpoints was observed between thigh and abdomen. Results with a 2 mL prefilled syringe in a one year phase 3 study in patients confirmed PK results observed in the phase 1 study.
Conclusions
Collective evidence from a phase 1 study and phase 3 study demonstrated that 2 mL injections of secukinumab into the abdomen or thigh using different devices resulted in comparable PK characerisitics and were all well tolerated without noticable pain experiences
Increased lysosomal biomass is responsible for the resistance of triple-negative breast cancers to CDK4/6 inhibition
No full textBreast cancer is the most common type of cancer in women and the second leading cause of cancer death among women. While significant progress has been made in the treatment of hormone receptor (HR) positive, HER2 negative and HER2-amplified breast cancer in the last decades, Triple negative breast cancer (TNBC) is lacking behind. Still today, chemotherapy is the only treatment option, but response rates are low and only a small fraction of patients can successfully be treated. Here we show, that CDK4/6 inhibitors which are FDA-approved for the treatment of HR+/HER2negative breast cancer and significantly improved treatment outcome in this type of breast cancer, also represent a promising therapy option for TNBC in contrast to the current view. We show, that a subset of TNBCs despite being resistant to chemical inhibition of CDK4/6 strictly depend on CDK4/6 kinases for proliferation. We identified lysosomal sequestration of the drug and therefore limited bioavailability at its target site responsible for the discrepancy. We show that increased number of lysosomes correlates with resistance and inhibition of the lysosome renders these cells fully sensitive to CDK4/6 inhibitors. We provide strategies how to overcome resistance by means of FDA-approved lysosomotropic agents that raise lysosomal pH, co-inhibition of CDK2 or the use of structurally altered CDK4/6 inhibitors with decreased basic characteristics. We also provide a biomarker to stratify patients for successful CDK4/6 inhibitor therapy. Moreover, we show that this mechanism of resistance also underlies cases of acquired resistance in HR+/HER2negative breast cancer and importantly we provide evidence that increased lysosomal sequestration operates in patients presenting resistant to treatment with the CDK4/6 inhibitor palbociclib. Our study therefore suggests that CDK4/6 inhibitor therapy could be a treatment option for a subset of TNBCs where new targeted therapeutic intervention is so urgently needed and furthermore, offers strategies how to overcome resistance in HR+/HER2-negative breast cancer where CDK4/6 inhibition is already successfully applied. In summary, our study provides ways on how to improve therapeutic use of this promising new class of anti-cancer agents
Protective anti‐prion antibodies in human immunoglobulin repertoires
No full textPrion immunotherapy may hold great potential, but antibodies against certain PrP epitopes can be neurotoxic. Here, we identified > 6,000 PrP‐binding antibodies in a synthetic human Fab phage display library, 49 of which we characterized in detail. Antibodies directed against the flexible tail of PrP conferred neuroprotection against infectious prions. We then mined published repertoires of circulating B cells from healthy humans and found antibodies similar to the protective phage‐derived antibodies. When expressed recombinantly, these antibodies exhibited anti‐PrP reactivity. Furthermore, we surveyed 48,718 samples from 37,894 hospital patients for the presence of anti‐PrP IgGs and found 21 high‐titer individuals. The clinical files of these individuals did not reveal any enrichment of specific pathologies, suggesting that anti‐PrP autoimmunity is innocuous. The existence of anti‐prion antibodies in unbiased human immunological repertoires suggests that they might clear nascent prions early in life. Combined with the reported lack of such antibodies in carriers of disease‐associated PRNP mutations, this suggests a link to the low incidence of spontaneous prion diseases in human populations
Discovery and Characterization of Clinical Candidate LXE408 as a Kinetoplastid-Selective Proteasome Inhibitor for the Treatment of Leishmaniases
No full textVisceral leishmaniasis is responsible for up to 30,000 deaths every year. Current treatments have shortcomings that include toxicity and variable efficacy across endemic regions. Previously, we reported the discovery of GNF6702, a selective inhibitor of the kinetoplastid proteasome, which cleared parasites in murine models of leishmaniasis, Chagas disease and human African trypamomiasis. Here, we describe the discovery and characterization of LXE408, a structurally related kinetoplastid-selective proteasome inhibitor currently in Phase 1 human clinical trials. In addition, we present high-resolution cryo-EM structures of the Leishmania tarentolae proteasome in complex with LXE408, which provides an explanation for the non-competitive mode of binding of this novel class of inhibitors to the kinetoplastid proteasome
EFMC, medicinal chemistry and chemical biology in Europe
No full textA reflection on the development of medicinal chemistry and chemical biology in Europe, and how the European Federation for Medicinal Chemistry (EFMC) helps the scientific community address its changing scope in a proactive manner. Networking, knowledge exchange and representation, as well as the acceptance and integration of novel scientific developments are keys to a healthy and dynamic scientific community. This article also reviews some of the forces that shape the medicinal chemistry and chemical biology continuum, including cross-fertilization, digitalization, and the outsourcing of synthetic and characterization work to focus on productivity and scientific excellence