Functional Half-life Research Articles

Pharmacokinetics, Efficacy, and Safety of Upadacitinib in Pediatric Patients with Polyarticular-Course Juvenile Idiopathic Arthritis: An Interim Analysis of an Open-label, Phase 1 Trial.

This work aimed to evaluate the pharmacokinetics, efficacy, and safety of upadacitinib, an oral selective JAK inhibitor, in pediatric patients with polyarticular-course juvenile idiopathic arthritis (pcJIA). In an open-label, phase 1 study (SELECT-YOUTH), enrolled patients, aged 2 to <18 years with pcJIA, received bodyweight-based upadacitinib doses using a twice-daily (BID) oral solution or once-daily (QD) extended-release tablet based on their body weight and ability to swallow tablets. The study included a 7-day pharmacokinetic assessment, followed by a long-term efficacy and safety evaluation for up to 156 weeks, including an additional long-term safety cohort. This interim analysis included available pharmacokinetic and safety data and efficacy data collected through Week 48. A total of 57 patients received upadacitinib. The median time to maximum upadacitinib concentration was approximately 3 hours and 1 hour for the tablet and oral solution regimens, respectively; the harmonic mean functional half-life was approximately 5 hours and 2 hours, respectively. Juvenile idiopathic arthritis (JIA) American College of Rheumatology (ACR)30/50/70/90/100 responses at Week 12 were 91.8/89.8/69.4/49.0/32.7%, respectively. Efficacy was generally maintained through Week 48, and improvement in additional efficacy endpoints was also observed. At a median exposure duration of 412 days, 52 of 57 patients reported adverse events, of these 6 experienced serious adverse events. Adverse events were predominately mild to moderate in severity and consistent with the known safety profile of upadacitinib. This interim analysis demonstrates that the bodyweight-based dosing regimen of upadacitinib was well tolerated and efficacious in pediatric patients with pcJIA.

Pharmacokinetics, Safety, Tolerability, and Exploratory Efficacy of Upadacitinib in Children with Severe Atopic Dermatitis

PurposeThis study aims to characterize the pharmacokinetics, safety, tolerability, and exploratory efficacy of upadacitinib, an oral Janus kinase inhibitor approved for treating moderate to severe atopic dermatitis (AD) in adults and adolescents, in children with severe AD. MethodsIn an open-label, multiple-dose, Phase 1 study, pediatric patients with severe AD from two age groups (2 to <6 years and 6 to <12 years) received bodyweight-based dosing regimens of upadacitinib using either twice-daily immediate-release (IR) oral solution or once-daily extended-release (ER) tablets. A pharmacokinetic assessment was conducted on Day 7 of the study, which was followed by a long-term safety and exploratory efficacy evaluation for up to 108 weeks. The results reported here are based on an interim analysis when the study had completed enrollment and pharmacokinetic assessment. FindingsA total of 35 patients were enrolled and received upadacitinib. The maximum upadacitinib plasma concentration was attained within a median time of 0.5 to 2 hours and 2 to 2.5 hours for the IR oral solution and ER tablet formulations, respectively. Upadacitinib functional half-life was generally shorter with IR oral solution relative to ER tablets. Upadacitinib apparent oral clearance decreased with decreasing body weight in the pediatric patients enrolled in this study. Upadacitinib was generally safe and well tolerated. The most common (≥3 patients) adverse events were upper respiratory tract infection, COVID-19 infection, headache, abdominal discomfort, vomiting, asthma, and cough. No new safety risks were identified compared to the known safety profile for upadacitinib in adults and adolescents. In the 30 patients with available exploratory efficacy data at Week 12, 36.7% achieved validated Investigator's Global Assessment scale for AD score of 0 or 1 (Validated Investigator Global Assessment for AD 0/1), and 70.0% had Eczema Area and Severity Index (EASI) improvement of at least 75% (EASI 75). ImplicationsThe characterized pharmacokinetic profiles in this study, together with the observed safety and exploratory efficacy results, support further investigation of the current upadacitinib dosing regimen in future confirmatory Phase 3 clinical trials in children with AD.Clinical Trial Number: NCT03646604, registered 2018-08-23

Abstract 6319: Characterization of AB-1015 logic-gated CAR induction (ON kinetics), receptor turnover (OFF kinetics), and logic gate sensitivity to ALPG/P and MSLN

Abstract CAR T cell activity in solid tumors is limited, in part, by off-tumor toxicity. To address this obstacle, AB-1015 contains an “AND” logic gate, consisting of a priming receptor (PrimeR) and an inducible chimeric antigen receptor (CAR) that is upregulated by PrimeR activation. The AB-1015 logic gate targets ALPG/P and MSLN, which are coexpressed in ovarian tumors but not normal tissues. AB-1015 CAR induction within the tumor is expected to be a dynamic process influenced by multiple factors, including antigen density, target cell abundance, as well as tumor microenvironmental factors. To characterize CAR induction and receptor turnover, we developed an in vitro assay with plate-bound ALPG to induce CAR. CAR induction occurred quickly in response to ALPG priming, with most ICT cells expressing CAR within 24 hrs, and plateaued by 72 hrs. Once removed from ALPG, CAR expression returned to baseline within 96 hrs. Similar to conventional CAR T cells, the presence of the cytolytic target antigen (MSLN) was expected to increase the rate of CAR turnover on AB-1015. To assess this, we measured the CAR functional half-life of AB-1015 cells once removed from ALPG priming. The cytotoxic activity of AB-1015 cells rapidly decreased in the absence of ALPG, reaching baseline within 24 hrs. Of note, our in vitro assay did not result in 100% CAR induction from all PrimeR+ AB-1015 cells. To understand whether this reflected biology versus a limitation of our in vitro assay, we conducted a CAR induction, sorted the CAR- cells, then performed a second induction. ICT cells that did not express CAR after the first induction were shown to be able to induce CAR during the second induction assay, thereby demonstrating that all PrimeR+ ICT cells have the potential to induce CAR. To assess the sensitivity of AB-1015 for low density ALPG/P and MSLN, isogenic cell lines were engineered to express each antigen at variable levels. In co-culture, AB-1015 was able to prime and kill targets expressing ALPG and MSLN at or below the limit of detection using immunohistochemistry. In light of these observations, the specificity of AB-1015 was verified using a dual flank in vivo model where one tumor expressed both ALPG and MSLN, and the contralateral tumor expressed MSLN alone. While robust anti-tumor activity was observed against the ALPG+/MSLN+ tumor, no activity was detected against the MSLN+ tumor. In summary, AB-1015 CAR induction and receptor turnover kinetics is a dynamic process. A reductionist in vitro model system was developed, enabling analysis of CAR induction and receptor turnover. Importantly, we demonstrated that all PrimeR+ ICT cells have the potential to induce CAR. Finally, the in vivo specificity of AB-1015 for ALPG+/MSLN+ tumors was established using a dual flank specificity model. Together, these data support the continued development of AB-1015 in a phase I clinical trial (NCT05617755). Citation Format: Xinyan Tang, Marvin Chew, Rakesh Sudhakar, Hongruo Yun, Doreen Sakamoto, Vibhavari Sail, Amanda Fearon, Jasper Williams, Jun Feng, Stephen Santoro. Characterization of AB-1015 logic-gated CAR induction (ON kinetics), receptor turnover (OFF kinetics), and logic gate sensitivity to ALPG/P and MSLN [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6319.

Protein S antibody as an adjunct therapy for hemophilia B

AbstractHemophilia B (HB) is caused by an inherited deficiency of plasma coagulation factor IX (FIX). Approximately 60% of pediatric patients with HB possess a severe form of FIX deficiency (<1% FIX activity). Treatment typically requires replacement therapy through the administration of FIX. However, exogenous FIX has a limited functional half-life, and the natural anticoagulant protein S (PS) inhibits activated FIX (FIXa). PS ultimately limits thrombin formation, which limits plasma coagulation. This regulation of FIXa activity by PS led us to test whether inhibiting PS would extend the functional half-life of FIX and thereby prolong FIX-based HB therapy. We assayed clotting times and thrombin generation to measure the efficacy of a PS antibody for increasing FIX activity in commercially obtained plasma and plasma from pediatric patients with HB. We included 11 pediatric patients who lacked additional comorbidities and coagulopathies. In vivo, we assessed thrombus formation in HB mice in the presence of the FIXa ± PS antibody. We found an accelerated rate of clotting in the presence of PS antibody. Similarly, the peak thrombin formed was significantly greater in the presence of the PS antibody, even in plasma from patients with severe HB. Furthermore, HB mice injected with PS antibody and FIX had a 4.5-fold higher accumulation of fibrin at the thrombus induction site compared with mice injected with FIX alone. Our findings imply that a PS antibody would be a valuable adjunct to increase the effectiveness of FIX replacement therapy in pediatric patients who have mild, moderate, and severe HB.

Potential health risks of mRNA-based vaccine therapy: A hypothesis

Therapeutic applications of synthetic mRNA were proposed more than 30 years ago, and are currently the basis of one of the vaccine platforms used at a massive scale as part of the public health strategy to get COVID-19 under control. To date, there are no published studies on the biodistribution, cellular uptake, endosomal escape, translation rates, functional half-life and inactivation kinetics of synthetic mRNA, rates and duration of vaccine-induced antigen expression in different cell types. Furthermore, despite the assumption that there is no possibility of genomic integration of therapeutic synthetic mRNA, only one recent study has examined interactions between vaccine mRNA and the genome of transfected cells, and reported that an endogenous retrotransposon, LINE-1 is unsilenced following mRNA entry to the cell, leading to reverse transcription of full length vaccine mRNA sequences, and nuclear entry. This finding should be a major safety concern, given the possibility of synthetic mRNA-driven epigenetic and genomic modifications arising. We propose that in susceptible individuals, cytosolic clearance of nucleotide modified synthetic (nms-mRNAs) is impeded. Sustained presence of nms-mRNA in the cytoplasm deregulates and activates endogenous transposable elements (TEs), causing some of the mRNA copies to be reverse transcribed. The cytosolic accumulation of the nms-mRNA and the reverse transcribed cDNA molecules activates RNA and DNA sensory pathways. Their concurrent activation initiates a synchronized innate response against non-self nucleic acids, prompting type-I interferon and pro-inflammatory cytokine production which, if unregulated, leads to autoinflammatory and autoimmune conditions, while activated TEs increase the risk of insertional mutagenesis of the reverse transcribed molecules, which can disrupt coding regions, enhance the risk of mutations in tumour suppressor genes, and lead to sustained DNA damage. Susceptible individuals would then expectedly have an increased risk of DNA damage, chronic autoinflammation, autoimmunity and cancer. In light of the current mass administration of nms-mRNA vaccines, it is essential and urgent to fully understand the intracellular cascades initiated by cellular uptake of synthetic mRNA and the consequences of these molecular events.

Deep mutational scanning and massively parallel kinetics of plasminogen activator inhibitor-1 functional stability to probe its latency transition

Plasminogen activator inhibitor-1 (PAI-1), a member of the serine protease inhibitor superfamily of proteins, is unique among serine protease inhibitors for exhibiting a spontaneous conformational change to a latent or inactive state. The functional half-life for this transition at physiologic temperature and pH is ∼1 to 2h. To better understand the molecular mechanisms underlying this transition, we now report on the analysis of a comprehensive PAI-1 variant library expressed on filamentous phage and selected for functional stability after 48h at 37 °C. Of the 7201 possible single amino acid substitutions in PAI-1, we identified 439 that increased the functional stability of PAI-1 beyond that of the WT protein. We also found 1549 single amino acid substitutions that retained inhibitory activity toward the canonical target protease of PAI-1 (urokinase-like plasminogen activator), whereas exhibiting functional stability less than or equal to that of WT PAI-1. Missense mutations that increase PAI-1 functional stability are concentrated in highly flexible regions within the PAI-1 structure. Finally, we developed a method for simultaneously measuring the functional half-lives of hundreds of PAI-1 variants in a multiplexed, massively parallel manner, quantifying the functional half-lives for 697 single missense variants of PAI-1 by this approach. Overall, these findings provide novel insight into the mechanisms underlying the latency transition of PAI-1 and provide a database for interpreting human PAI-1 genetic variants.

Skin proteomic analysis of immune activation associated with regression of melanoma metastases induced by diphencyprone

To the Editor: Diphencyprone (DPCP), a hapten that causes delayed-type hypersensitivity reactions, has been used to treat warts, alopecia areata, and cutaneous metastases in melanoma patients.1Damian D.L. Saw R.P. Thompson J.F. Topical immunotherapy with diphencyprone for in transit and cutaneously metastatic melanoma.J Surg Oncol. 2014; 109: 308-313https://doi.org/10.1002/jso.23506Crossref PubMed Scopus (51) Google Scholar Previously, our group performed transcriptomic analysis of skin biopsies from melanoma metastases treated with topical DPCP, revealing significant increases in Th1-related genes but not in genes of other major T-cell subsets.2Gulati N. Garcet S. Fuentes-Duculan J. et al.Molecular profiling of immune activation associated with regression of melanoma metastases induced by diphencyprone.J Invest Dermatol. 2016; 136: 2101-2103https://doi.org/10.1016/j.jid.20 16.06.611Crossref PubMed Scopus (0) Google Scholar While gene expression profiling has long been used as a proxy for protein expression, correlation between messenger RNA (mRNA) and protein levels is inconsistent due to factors such as post-transcriptional regulation. Therefore, to more precisely ascertain the mechanisms involved in immune-mediated tumor regression induced by DPCP, we performed proteomic analysis of skin biopsies from 5 patients with cutaneous melanoma metastases treated with DPCP twice weekly for 7 to 14 weeks (Supplemental Material and Methods and Supplementary Table I, available via Mendeley at https://doi.org/10.17632/f5ttw4mygy.1). In this study, we assessed 96 proteins using the Olink immuno-oncology panel for the 5 patients who underwent molecular profiling. All patients had expected skin inflammation and at least partial regression of skin metastases, without systemic side effects. There were 10 proteins (interleukin 6 [IL-6], IL-12, IL-18, TIE2, HGF, PDGFB, TNFSF14, PD-L2, MMP7, MMP12) significantly upregulated (P < .05) in post-treatment versus pretreatment metastases (Fig 1), including biomarkers associated with Th1 response (IL-12), innate immunity (IL-6), and immune checkpoints (PD-L2) (P < .05). All of these proteins had progressively increased expression in cutaneous metastases with repeated DPCP applications, and similar increases were observed in nonlesional skin of these patients following a single DPCP application. Prior work has shown that administration of IL-18 in mice increases activated T cells and natural killer cells that contribute to effective antimelanoma immunity,3Zhou T. Damsky W. Weizman O.E. et al.IL-18BP is a secreted immune checkpoint and barrier to IL-18 immunotherapy.Nature. 2020; 583: 609-614https://doi.org/10.1038/s41586-020-2422-6Crossref PubMed Scopus (93) Google Scholar so the significant upregulation of this protein in our study may contribute to the successful skin metastasis regression observed in our patients. TNFSF14 also likely plays an active role in immune-mediated regression, as in vivo studies have demonstrated an association between the expression of this protein in melanoma metastasis lesions and proliferation of T cells.4Mortarini R. Scarito A. Nonaka D. et al.Constitutive expression and costimulatory function of LIGHT/TNFSF14 on human melanoma cells and melanoma-derived microvesicles.Cancer Res. 15 2005; 65: 3428-3436https://doi.org/10.1158/0008-5472.CAN-04-3239Crossref PubMed Scopus (35) Google Scholar Additionally, there were 40 proteins significantly upregulated in pretreatment metastases versus untreated nonlesional skin (P < .05) (Fig 1), suggesting baseline immune activation in melanoma metastases. Upon DPCP treatment, changes in nonlesional skin positively correlated with those in skin metastases (Supplementary Fig 1, available via Mendeley at https://doi.org/10.17632/f5ttw4mygy.1; Spearman correlation = 0.61, P < .0001). We performed a correlation analysis between the proteomic data and the previously published mRNA data,2Gulati N. Garcet S. Fuentes-Duculan J. et al.Molecular profiling of immune activation associated with regression of melanoma metastases induced by diphencyprone.J Invest Dermatol. 2016; 136: 2101-2103https://doi.org/10.1016/j.jid.20 16.06.611Crossref PubMed Scopus (0) Google Scholar which both were completed on the same tissue samples, to assess the relationship between these 2 “omic” approaches (Fig 2). Correlation was poor (r = −0.1, P = .4012), including for proteins that were significantly upregulated by proteomics (IL-6 and TNFSF14) (both P < .05). Gene expression from mRNA is variably correlated with protein expression, the latter being influenced by many regulatory processes prior to translation. Another explanation for the discrepancy observed between mRNA and protein expression includes the shorter half-life of mRNA molecules compared to proteins.5Mauger D.M. Cabral B.J. Presnyak V. et al.mRNA structure regulates protein expression through changes in functional half-life.Proc Natl Acad Sci U S A. 2019; 116: 24075-24083https://doi.org/10.1073/pnas.1908052116Crossref PubMed Scopus (152) Google Scholar Despite our limited sample size, we were able to observe statistically significant protein expression changes, and this study is, to our knowledge, the first to assess the proteomic signature of successful immune-mediated regression of cutaneous melanoma metastases induced by a topical immunomodulator. Given the lack of correlation between mRNA and protein expression, along with the relatively greater feasibility of measuring protein compared to gene expression levels, our study demonstrates that proteomic analysis can give unique and potentially complementary insights into immune reactions induced by topical immunotherapy. None disclosed.

Lumbar Interbody Fusion Conducted on a Porcine Model with a Bioresorbable Ceramic/Biopolymer Hybrid Implant Enriched with Hyperstable Fibroblast Growth Factor 2.

Many growth factors have been studied as additives accelerating lumbar fusion rates in different animal models. However, their low hydrolytic and thermal stability both in vitro and in vivo limits their workability and use. In the proposed work, a stabilized vasculogenic and prohealing fibroblast growth factor-2 (FGF2-STAB®) exhibiting a functional half-life in vitro at 37 °C more than 20 days was applied for lumbar fusion in combination with a bioresorbable scaffold on porcine models. An experimental animal study was designed to investigate the intervertebral fusion efficiency and safety of a bioresorbable ceramic/biopolymer hybrid implant enriched with FGF2-STAB® in comparison with a tricortical bone autograft used as a gold standard. Twenty-four experimental pigs underwent L2/3 discectomy with implantation of either the tricortical iliac crest bone autograft or the bioresorbable hybrid implant (BHI) followed by lateral intervertebral fixation. The quality of spinal fusion was assessed by micro-computed tomography (micro-CT), biomechanical testing, and histological examination at both 8 and 16 weeks after the surgery. While 8 weeks after implantation, micro-CT analysis demonstrated similar fusion quality in both groups, in contrast, spines with BHI involving inorganic hydroxyapatite and tricalcium phosphate along with organic collagen, oxidized cellulose, and FGF2- STAB® showed a significant increase in a fusion quality in comparison to the autograft group 16 weeks post-surgery (p = 0.023). Biomechanical testing revealed significantly higher stiffness of spines treated with the bioresorbable hybrid implant group compared to the autograft group (p < 0.05). Whilst histomorphological evaluation showed significant progression of new bone formation in the BHI group besides non-union and fibrocartilage tissue formed in the autograft group. Significant osteoinductive effects of BHI based on bioceramics, collagen, oxidized cellulose, and FGF2-STAB® could improve outcomes in spinal fusion surgery and bone tissue regeneration.

In vivo blockade of mononuclear phagocyte system with solid nanoparticles: Efficiency and affecting factors

Smart nanomaterials, contrast nanoparticles and drug nanocarriers of advanced targeting architecture were designed for various biomedical applications. Most of such agents demonstrate poor pharmacokinetics in vivo due to rapid elimination from the bloodstream by cells of the mononuclear phagocyte system (MPS). One of the promising methods to prolong blood circulation of the nanoparticles without their modification is MPS blockade. The method temporarily decreases macrophage endocytosis in response to uptake of a low-toxic non-functional material. The effect of different factors on the efficiency of macrophage blockade in vivo induced by solid nanomaterials has been studied here. Those include: blocker nanoparticle size, ζ-potential, surface coating, dose, mice strain, presence of tumor or inflammation. We found that the blocker particle coating type had the strongest effect on MPS blockade efficiency, which allowed to prolong functional particle blood circulation half-life 18 times. The mechanisms capable of regulation of the MPS blockade have been demonstrated, which can promote application of this phenomenon in medicine for improving delivery of diagnostic and therapeutic nanomaterials.

Global Warming Mitigating Role of Wood Products from Washington State’s Private Forests

Similar to standing trees in the forests, wood products play an important role in enhancing the global sequestered carbon pool, by retaining the atmospheric carbon in a sequestered form for the duration of the functional life of the wood products. This study uses a temporal radiative forcing analysis along with the functional half-life of different wood products to evaluate the impacts of wood products on global warming, including carbon storage and life cycle greenhouse gas production/extraction emissions. The methodology is applied to Washington State’s aboveground biomass and timber harvest data, and to the State’s comprehensive wood products mix. A moderate harvest rate simulation within Washington Biomass Calculator is used to estimate state harvest level, and statewide wood products manufacturing data is used for developing wood product mix estimates. Using this method, we estimate that the temporal carbon storage leads to a global warming mitigation benefit equivalent to 4.3 million tCO2eq. Even after factoring in the greenhouse gas emissions associated with the harvest operations and wood products manufacturing processes, within the temporal model, the results show a net beneficial impact of approximately 1.7 million tCO2eq, on an annual basis. It can further be noted that Washington State’s annual biomass growth in its private forests exceeds its annual harvest, by a significant margin. This net yearly accumulation of biomass in the State’s private forests leads to additional global warming mitigation benefits equivalent to 7.4 million tCO2eq. Based on these results, we conclude that Washington’s private forestry industry is a net global warming mitigator for the State, equivalent to 12% of the State’s greenhouse gas emissions in 2015.

Acceleration of chondrogenic differentiation of human mesenchymal stem cells by sustained growth factor release in 3D graphene oxide incorporated hydrogels

Damaged articular cartilage has limited self-healing capabilities, leading to degeneration that affects millions of people. Although cartilage tissue engineering is considered a promising approach for treatment, robust and long-term chondrogenesis within a 3-dimensional (3D) scaffold remains a major challenge for complete regeneration. Most current approaches involve incorporation of transforming growth factor-β (TGF-β) into the scaffold, but have limited utility owing to the short functional half-life and/or rapid clearance of TGF-β. In this study, we have tested the incorporation of graphene oxide nanosheets (GO) within a photopolymerizable poly-D, l-lactic acid/polyethylene glycol (PDLLA) hydrogel, for its applicability in sustained release of the chondroinductive growth factor TGF-β3. We found that with GO incorporation, the hydrogel scaffold (GO/PDLLA) exhibited enhanced initial mechanical strength, i.e., increased compressive modulus, and supported long-term, sustained release of TGF-β3 for up to 4 weeks. In addition, human bone marrow-derived mesenchymal stem cells (hBMSCs) seeded within TGF-β3 loaded GO/PDLLA hydrogels displayed high cell viability and improved chondrogenesis in a TGF-β3 concentration-dependent manner. hBMSCs cultured in GO/PDLLA also demonstrated significantly higher chondrogenic gene expression, including aggrecan, collagen type II and SOX9, and cartilage matrix production when compared to cultures maintained in GO-free scaffolds containing equivalent amounts of TGF-β3. Upon subcutaneous implantation in vivo, hBMSC-seeded TGF-β3-GO/PDLLA hydrogel constructs displayed considerably greater cartilage matrix than their TGF-β3/PDLLA counterparts without GO. Taken together, these findings support the potential application of GO in optimizing TGF-β3 induced hBMSC chondrogenesis for cartilage tissue engineering. STATEMENT OF SIGNIFICANCE: In this work, we have developed a graphene oxide (GO) incorporated, photocrosslinked PDLLA hybrid hydrogel for localized delivery and sustained release of loaded TGF-β3 to seeded cells. The incorporation of GO in PDLLA hydrogel suppressed the burst release of TGF-β3, and significantly prolonged the retention time of the TGF-β3 initially loaded in the hydrogel. Additionally, the GO improved the initial compressive strength of the hydrogel. Both in vitro analyses and in vivo implantation results showed that the GO/PDLLA constructs seeded with human mesenchymal stem cells (hMSCs) showed significantly higher cartilage formation, compared to GO-free scaffolds containing equivalent amount of TGF-β3. Findings from this work suggest the potential application of the GO-TGF/PDLLA hydrogel as a functional scaffold for hMSC-based cartilage tissue engineering.

MRNA structure regulates protein expression through changes in functional half-life

Messenger RNAs (mRNAs) encode information in both their primary sequence and their higher order structure. The independent contributions of factors like codon usage and secondary structure to regulating protein expression are difficult to establish as they are often highly correlated in endogenous sequences. Here, we used 2 approaches, global inclusion of modified nucleotides and rational sequence design of exogenously delivered constructs, to understand the role of mRNA secondary structure independent from codon usage. Unexpectedly, highly expressed mRNAs contained a highly structured coding sequence (CDS). Modified nucleotides that stabilize mRNA secondary structure enabled high expression across a wide variety of primary sequences. Using a set of eGFP mRNAs with independently altered codon usage and CDS structure, we find that the structure of the CDS regulates protein expression through changes in functional mRNA half-life (i.e., mRNA being actively translated). This work highlights an underappreciated role of mRNA secondary structure in the regulation of mRNA stability.

Site-specific PEGylation of micro-plasmin for improved thrombolytic therapy through engineering enhanced resistance against serpin mediated inhibition.

The relatively rapid inhibition of microplasmin by α2-AP leads to short functional half-life of the molecule in vivo, causing inefficient clot dissolution, even after site-specific, local catheter-based delivery. Here, we describe a PEGylation approach for improving the therapeutic potential via improving the survival of microplasmin in presence of its cognate inhibitor, α2-AP, wherein a series of strategically designed cysteine analogs of micro-plasminogen were prepared and expressed in E. coli, and further modified by covalent grafting in vitro with PEG groups of different molecular sizes so as to select single or double PEG chains that increase the molecular weight and hydrodynamic radii of the conjugates, but with a minimal discernible effect on intrinsic plasmin activity and structural framework, as explored by amidolytic activity and CD-spectroscopy, respectively. Interestingly, some of the purified PEG-coupled proteins after conversion to their corresponding proteolytically active forms were found to exhibit significantly reduced inhibition rates (up to 2-fold) by α2-AP relative to that observed with wild-type microplasmin. These results indicate an interesting, and not often observed, effect of PEG groups through reduced/altered dynamics between protease and inhibitor, likely through a steric hindrance mechanism. Thus, the present study successfully identifies single- and double-site PEGylated muteins of microplasmin with significantly enhanced functional half-life through enhanced resistance to inactivation by its in vivo plasma inhibitor. Such an increased survival of bioactivity in situ, holds unmistakable potential for therapeutic exploitation, especially in ischemic strokes where a direct, catheter-based deposition within the cranium has been shown to be promising, but is currently limited by the very short in vivo bioactive half-life of the fibrin dissolving agent/s.

Local delivery of stabilized chondroitinase ABC degrades chondroitin sulfate proteoglycans in stroke-injured rat brains

Central nervous system (CNS) injuries, such as stroke and spinal cord injuries, result in the formation of a proteoglycan-rich glial scar, which acts as a barrier to axonal regrowth and limits the regenerative capacity of the CNS. Chondroitinase ABC (ChABC) is a potent bacterial enzyme that degrades the chondroitin sulfate proteoglycan (CSPG) component of the glial scar and promotes tissue recovery; however, its use is significantly limited by its inherent instability at physiological temperatures. Here, we demonstrate that ChABC can be stabilized using site-directed mutagenesis and covalent modification with poly(ethylene glycol) chains (i.e. PEGylation). Rosetta protein structure modeling was used to screen >20,000 single point mutations, and four potentially stabilizing mutations were tested in vitro. One of the mutations, N1000G (asparagine ➔ glycine at residue 1000), significantly improved the long-term activity of the protein, doubling its functional half-life. PEGylation of this ChABC mutant inhibited unfolding and aggregation and resulted in prolonged bioactivity with a 10-fold increase in activity compared to the unmodified protein after two days. Local, affinity-controlled release of the modified protein (PEG-N1000G-ChABC) was achieved by expressing it as a fusion protein with Src homology 3 (SH3) and delivering the protein from a methylcellulose hydrogel modified with SH3 binding peptides. This affinity-based release strategy provided sustained PEG-N1000G-ChABC-SH3 release over several days in vitro. Direct implantation of the hydrogel delivery vehicle containing stabilized PEG-N1000G-ChABC-SH3 onto the rat brain cortex in a sub-acute model of stroke resulted in significantly reduced CSPG levels in the penumbra of 49% at 14 and 40% at 28 days post-injury compared to animals treated with the vehicle alone.

A strategy based on thermal flexibility to design triosephosphate isomerase proteins with increased or decreased kinetic stability

Kinetic stability of proteins determines their susceptibility to irreversibly unfold in a time-dependent process, and therefore its half-life. A residue displacement analysis of temperature-induced unfolding molecular dynamics simulations was recently employed to define the thermal flexibility of proteins. This property was found to be correlated with the activation energy barrier (Eact) separating the native from the transition state in the denaturation process. The Eact was determined from the application of a two-state irreversible model to temperature unfolding experiments using differential scanning calorimetry (DSC). The contribution of each residue to the thermal flexibility of proteins is used here to propose multiple mutations in triosephosphate isomerase (TIM) from Trypanosoma brucei (TbTIM) and Trypanosoma cruzi (TcTIM), two parasites closely related by evolution. These two enzymes, taken as model systems, have practically identical structure but large differences in their kinetic stability. We constructed two functional TIM variants with more than twice and less than half the activation energy of their respective wild-type reference structures. The results show that the proposed strategy is able to identify the crucial residues for the kinetic stability in these enzymes. As it occurs with other protein properties reflecting their complex behavior, kinetic stability appears to be the consequence of an extensive network of inter-residue interactions, acting in a concerted manner. The proposed strategy to design variants can be used with other proteins, to increase or decrease their functional half-life.

NS5A inhibitors unmask differences in functional replicase complex half-life between different hepatitis C virus strains

NS5A inhibitors unmask differences in functional replicase complex half-life between different hepatitis C virus strains

An S116R Phosphorylation Site Mutation in Human Fibroblast Growth Factor-1 Differentially Affects Mitogenic and Glucose-Lowering Activities

Fibroblast growth factor-1 (FGF-1), a potent human mitogen and insulin sensitizer, signals through both tyrosine kinase receptor–mediated autocrine/paracrine pathways as well as a nuclear intracrine pathway. Phosphorylation of FGF-1 at serine 116 (S116) has been proposed to regulate intracrine signaling. Position S116 is located within a ∼17 amino acid C-terminal loop that contains a rich set of functional determinants including heparin∖heparan sulfate affinity, thiol reactivity, nuclear localization, pharmacokinetics, functional half-life, nuclear ligand affinity, stability, and structural dynamics. Mutational targeting of specific functionality in this region without perturbing other functional determinants is a design challenge. S116R is a non-phosphorylatable variant present in bovine FGF-1 and other members of the human FGF family. We show that the S116R mutation in human FGF-1 is accommodated with no perturbation of biophysical or structural properties, and is therefore an attractive mutation with which to elucidate the functional role of phosphorylation. Characterization of S116R shows reduction in NIH 3T3 fibroblast mitogenic stimulation, increase in fibroblast growth factor receptor-1c activation, and prolonged duration of glucose lowering in ob/ob hyperglycemic mice. A novel FGF-1/fibroblast growth factor receptor-1c dimerization interaction combined with non-phosphorylatable intracrine signaling is hypothesized to be responsible for these observed functional effects.

PEGylation of a High-Affinity Anti-(+)Methamphetamine Single Chain Antibody Fragment Extends Functional Half-Life by Reducing Clearance.

Methamphetamine (METH) abuse is a worldwide drug problem, yet no FDA-approved pharmacological treatments are available for METH abuse. Therefore, we produced an anti-METH single chain antibody fragment (scFv7F9Cys) as a pharmacological treatment for METH abuse. ScFv's have a short half-life due to their small size, limiting their clinical use. Thus, we examined the pharmacokinetic effects of conjugating poly(ethylene) glycol (-PEG) to scFv7F9Cys to extend its functional half-life. The affinity of scFv7F9Cys and PEG conjugates to METH was determined in vitro via equilibrium dialysis saturation binding. Pharmacokinetic and parameters of scFv7F9Cys and scFv7F9Cys-PEG20K (30mg/kg i.v. each) and their ability to bind METH in vivo were determined in male Sprague-Dawley rats receiving a subcutaneous infusion of METH (3.2mg/kg/day). Of three PEGylated conjugates, scFv7F9Cys-PEG20K was determined the most viable therapeutic candidate. PEGylation of scFv7F9Cys did not alter METH binding functionality in vitro, and produced a 27-fold increase in the in vivo half-life of the antibody fragment. Furthermore, total METH serum concentrations increased following scFv7F9Cys or scFv7F9Cys-PEG20K administration, with scFv7F9Cys-PEG20K producing significantly longer changes in METH distribution than scFv7F9Cys. PEGylation of scFv7F9Cys significantly increase the functional half-life of scFv7F9Cys, suggesting it may be a long-lasting pharmacological treatment option for METH abuse.

Regulation of mRNA Decay in Bacteria.

Gram-negative and gram-positive bacteria use a variety of enzymatic pathways to degrade mRNAs. Although several recent reviews have outlined these pathways, much less attention has been paid to the regulation of mRNA decay. The functional half-life of a particular mRNA, which affects how much protein is synthesized from it, is determined by a combination of multiple factors. These include, but are not necessarily limited to, (a) stability elements at either the 5' or the 3' terminus, (b) posttranscriptional modifications, (c) ribosome density on individual mRNAs, (d) small regulatory RNA (sRNA) interactions with mRNAs, (e) regulatory proteins that alter ribonuclease binding affinities, (f) the presence or absence of endonucleolytic cleavage sites, (g) control of intracellular ribonuclease levels, and (h) physical location within the cell. Changes in physiological conditions associated with environmental alterations can significantly alter the impact of these factors in the decay of a particular mRNA.

Improved antithrombotic activity and diminished bleeding side effect of a PEGylated αIIbβ3 antagonist, disintegrin

IntroductionThe applicability of protein drugs is confined by protein degradation and rapid elimination. PEGylation of polypeptides improves protein stability by sterically obstructing the degradation by serum proteases and reduces renal clearance by the increased mass. Experimental approachWe compared the antithrombotic activities of intact rhodostomin (Rn) and PEGylated rhodostomin (PRn) both in vitro and in vivo systems. In addition, the functional half-life in inhibiting platelet aggregation and the tendency in causing bleeding side effect were investigated. ResultsRn and PRn both potently inhibited human and mouse platelet aggregation induced by collagen, thrombin or ADP in vitro with a similar IC50 around 60–100nM. Rotational thromboelastometry assay indicated that PRn was more effective than Rn in preventing clot formation in human whole blood. In platelet-rich plasma from mice injected with Rn or PRn, the inhibitory effects on collagen-induced platelet aggregation were also comparable, but Rn caused higher bleeding tendency. In ferric chloride-induced arterial thrombosis, Rn and PRn significantly prolonged occlusion time at high dosage (0.2μg/g). However, PRn obviously prolonged the occlusion time even given at a lower dosage (0.06μg/g). The functional half-life assay revealed that PEGylation prolonged the in vivo half-life of Rn. ConclusionsPRn exhibits higher antithrombotic potency and longer half-life in vivo as compared with native Rn on a molar basis. In addition, PRn exhibits a better safety profile at an efficacious antithrombotic dose in vivo. Therefore, PEGylation may be one of the ideal options in modifying disintegrin derivatives as the safe therapeutic agents for integrin-related diseases.