Prolonged Half-life Research Articles

Age Regulates Desmopressin Responses in Patients with Low Von Willebrand Factor and Type 1 Von Willebrand Disease in the Lovic and Win Studies

Introduction Desmopressin is the treatment of choice in many patients with Low VWF and type 1 VWD. However, not all patients respond well to desmopressin. Moreover, desmopressin is contra-indicated in patients > 60 years in some countries. Interestingly however, previous studies have demonstrated that increases in plasma VWF levels seen with ageing are attributable to enhanced VWF biosynthesis coupled with reduced VWF clearance. Therefore, we investigated the hypothesis that Weibel-Palade body stores of VWF and clearance of VWF, and hence desmopressin responses, may vary with ageing. Methods We investigated datasets collected through the previously described Low VWF in Ireland Cohort (LoVIC) and Willebrand in the Netherlands (WiN) studies. All patients included in the LoVIC study had a personal bleeding history and VWF levels in the 30-50 IU/dL range. Conversely, WiN patients had either a personal bleeding history or positive family history, combined with VWF levels ≤ 30 IU/dL. Desmopressin was administered intravenously at a dosage of 0.3 μg/kg (maximum dose capped at 28 μg/kg) or intranasally at a total dosage of 300 μg. Complete desmopressin response was defined according to the 2021 ASH/ISTH/NHF/WFH guideline (i.e. increase of 2 times the baseline and sustained VWF and FVIII activity > 50 IU/dL for at least 4 hours). Results In total, 261 patients were included (178 WiN patients with type 1 VWD and 83 LoVIC patients with Low VWF). Based upon levels at inclusion in the WiN study versus original diagnosis, type 1 VWD patients were categorized into three groups - (i) 69 patients with persistent VWF levels < 30 IU/dL; (ii) 55 patients with partial correction in VWF levels into the 30-50 IU/dL range; and (iii) 54 patients with normalization of VWF levels > 50 IU/dL. Importantly, a complete response to desmopressin was observed in only 58% of WiN patients with persistent VWF levels < 30 IU/dL, whereas 100% of WiN patients with partially corrected or normalized levels and 100% of LoVIC patients had a complete response to desmopressin (p<0.001). Type 1 patients with persistent VWF levels < 30 IU/dL had significantly lower VWF:Ag at all time points after desmopressin compared to the other groups (Fig 1, p<0.001). Unexpectedly, VWF:Ag at 1, 4 and 24 hours after desmopressin was significantly higher in WiN normalized compared to LoVIC patients (Fig 1, p<0.001). Importantly however, the WiN normalized cohort was significantly older at the time of their desmopressin trial than the LoVIC group (41.9 ±13.8 years versus 32.9 ±10.7 years, p=0.002). Therefore, we hypothesized that the difference in VWF responses might be age-dependent. To investigate this concept, desmopressin responses for different age groups were assessed in LoVIC patients and WiN partially corrected and WiN normalized patients. Interestingly, an age-dependent effect on VWF responses at 1, 4 and 24 hours after desmopressin administration was observed (Fig 2). Older patients had both a significantly better initial desmopressin response at 1 hour after desmopressin (Fig 2, p=0.018), and a significantly prolonged half-life of VWF at 4 hours after desmopressin (Fig 2, p=0.004). Together, these data suggest that aging leads to both increased VWF synthesis/secretion and reduced clearance of VWF. Finally, we performed a retrospective cohort study to investigate whether desmopressin response could be utilized to predict which type 1 VWD patients would normalize their plasma VWF levels with aging. We included 94 LoVIC and 185 type 1 WiN patients with a mean follow-up of 9.7 ±3.9 years and 16.9 ±7.4 years respectively. We demonstrate that desmopressin response predicted the normalization of VWF levels with aging with a 100% accuracy (p<0.001). None of the patients with an incomplete response to desmopressin normalized during follow-up, whereas the median time of normalization was 13 years [7-26] in patients with complete response to desmopressin (p<0.001). Conclusion Cumulatively, we demonstrate that desmopressin responses increase significantly in VWD patients and Low VWF patients as they get older. Given our findings, future studies assessing the clinical efficacy and safety of using attenuated desmopressin doses in older patients should be considered. Alternatively, developing additional therapies that can be safely used to trigger secretion of enhanced Weibel-Palade body stores of VWF in older patients may provide novel treatment opportunities.

Inpatient transition of methadone to Suboxone via rapid micro-induction in sepsis: a case report

ABSTRACT Buprenorphine is often preferred over methadone for medication-assisted treatment of opioid use disorder (OUD) because of its superior accessibility and less severe side effect and drug interaction profile; however, a transition from methadone to buprenorphine is made challenging by the prolonged half-life of methadone. Rapid micro-induction of buprenorphine is an up-and-coming strategy that allows the transition from a full opioid agonist to take place within 48 hours and without a traditionally required preceding period of opioid withdrawal syndrome. We report a case of successful inpatient transition from methadone to Suboxone in a patient with OUD and sepsis using a 48-hour rapid micro-induction protocol with pre-induction discontinuation of methadone and discuss the feasibility of this protocol in situations where the transition is time-critical.

Long-Term Suppressive Therapeutic-Drug-Monitoring-Guided Dalbavancin Therapy for Cardiovascular Prosthetic Infections.

Dalbavancin represents a promising treatment for cardiovascular prosthetic infections due to its prolonged half-life, bactericidal activity, large spectrum of activity, and excellent biofilm penetration. However, the use of dalbavancin in this setting is limited, and only a few cases have performed therapeutic drug monitoring (TDM) analysis to optimize dosage in suppressive treatments longer than 4 weeks. Our retrospective case series reports the use of dalbavancin in a small cohort of patients with cardiovascular prosthetic infections (cardiac implantable electronic device infections (CEDIs), prosthetic valve endocarditis (PVE), prosthetic vascular graft infections (PVGIs)) treated with dalbavancin as sequential therapy. From May 2019 to May 2023, 14 patients were included: eight cases of PVE (57.1%), seven cases of PVGI (50%), three cases of CEDI (21.4%), and four cases with overlap of infection sites (28.6%). The main pathogen was Staphylococcus aureus (35.7%). Prosthesis replacement was obtained in four patients (28.6%). The median time between symptom onset and the end of treatment was 15 weeks (IQR 7-53), with a median duration of dalbavancin therapy of 8 weeks (IQR 1 to 45 weeks) and 3.5 doses per patient. Among patients managed with TDM-guided strategy, dalbavancin infusion intervals ranged from 4 to 9 weeks. The median length of follow-up was 65 weeks (IQR 23 to 144 weeks). Clinical success was achieved in 10 cases (76.9%); all clinical failures occurred in patients with the implant retained. Among patients monitored by TDM, clinical success was 87.5% vs. 60% in patients treated without TDM. Because of pharmacokinetic individual variability, dalbavancin TDM-guided administration could improve clinical outcomes by individualizing dosing and selecting dosing intervals. This case series seems to suggest a promising role of long-term suppressive dalbavancin treatment for difficult-to-treat cardiovascular prosthesis infection, also with limited surgical indications.

Co-delivery of combretastatin A4 and docetaxel with pegylated nanostructured lipid carriers in tumor cells

Purpose: To investigate a novel co-delivery system using nanostructured lipid carriers (NLCs) for simultaneous administration of two potent anti-cancer drugs, combretastatin A-4 (CA-4) and docetaxel (DTX), against tumor cells and vasculature.
 Methods: The CA-4 and DTX co-loaded NLCs (C-D-NLC) were formulated and investigated for physical properties, stability, and drug release. Safety and efficacy of C-D-NLC were investigated on Lewis Lung Carcinoma (LLC) tumor cells in vitro and in vivo using cytotoxicity and anti-tumor assays. The pharmacokinetics of CA-4 and DTX in rats after intravenous injection of C-D-NLC were also studied to evaluate potential drug interactions.
 Results: The C-D-NLC was successfully prepared with a spherical shape, mean size of 130 nm, negative charge, high encapsulation efficiency and drug loading of 94.89, 88.16, 2.44, and 4.52 for DTX and CA-4, respectively. Also, C-D-NLC had a significant inhibitory effect on LLC cells, superior to a single drug or solution group. Combretastatin A4 did not affect the pharmacokinetics of DTX, but combretastatin–docetaxel nanostructured lipid carriers (C-D-NLC) reduced plasma clearance of CA-4 and DTX, prolonged half-life, mean residence time, and increased area under concentration curves (AUC) values. Furthermore, combretastatin–docetaxel nanostructured lipid carriers (C-D-NLC) inhibited the growth of LLC tumors in mice and reduced drug toxicity.
 Conclusion: Combretastatin–docetaxel nanostructured lipid carriers (C-D-NLC) sustain drug release and enhance tumor growth inhibition of CA-4 and DTX by targeting both tumor cells and vasculature. The co-delivery system prolongs drug circulation compared to solution administration. Thus, nanostructured lipid carriers (NLCs) with dual drug loading may be a promising strategy for clinical combination chemotherapy in future.

Classical Complement Inhibition By SAR445088 (BIVV020) in Adults with Cold Agglutinin Disease: Safety, Tolerability and Activity Results from the Open-Label, Non-Randomized, Single-Dose Phase 1b Study

Background:Cold agglutinin disease (CAD) is a rare, chronic autoimmune hemolytic anemia characterized by complement-mediated hemolysis. The classical complement inhibitor sutimlimab, is the first approved pharmacotherapy for treating patients with CAD. The second-generation complement inhibitor SAR445088 (BIVV020) is a humanized monoclonal antibody that selectively inhibits the activated form of C1s (sutimlimab inhibits total C1s). Furthermore, SAR445088 contains mutations that increase FcRn binding, preventing its degradation and allowing it to be recycled back into the system, resulting in reduced drug clearance and a prolonged half-life, which may allow a longer dosing interval than that of sutimlimab. Aims:This Phase 1b open-label study evaluated the safety, tolerability, effect on hemolysis, and pharmacodynamics (PD)/pharmacokinetics (PK) of a single intravenous (IV) dose of SAR445088. The study aimed to assess the impact of SAR445088 on complement-mediated hemolysis to support proof of concept for SAR445088 as a treatment for patients with CAD. Methods:The study design included up to three single-dose cohorts with a maximum of six patients per cohort. SAR445088 was administered on Day 1 with follow-up visits from Day 2 to Day 71; end of study (EOS) visit was on Day 106. The first cohort received a dose of 30 mg/kg IV with decisions about the selection of the dose for the next cohort being based on safety, biomarker activity, and PD. In the second cohort, patients received a lower dose of 15 mg/kg IV; a third, higher-dose cohort was not enrolled. To be enrolled in the study, participants needed to be aged ≥18 years with a confirmed diagnosis of CAD and a hemoglobin level ≤11.0 g/dL at time of screening. The primary endpoint was assessment of safety, with secondary endpoints of hematologic biomarkers and PD/PK. Results:Twelve patients received a single IV dose of SAR445088 (n=6, 30 mg/kg; n=6, 15 mg/kg). Patients enrolled in the study were mainly female (91.7%, n=11) with a median age (range) of 69 (54-80) years, and median (range) hemoglobin at baseline was 9.60 (4.8-10.9) g/dL. There were no treatment-emergent serious adverse events (AEs), discontinuations or deaths reported in the study. Fifty-eight treatment-emergent AEs (TEAEs) were reported in 11 patients (91.7%). There was one reported pre-treatment severe AE (Grade 3) of hemolytic anemia in a patient in the 15 mg/kg IV cohort which began prior to treatment and continued during the treatment period; the remainder of TEAEs were mild or moderate in severity (Grade 1 or 2). The most frequently reported AE was hematuria. No serious infections, meningococcal infections, hypersensitivity, or thromboembolic events were reported. Nine patients (n=4, 30 mg/kg IV cohort; n=5, 15 mg/kg IV cohort) reached an increase of hemoglobin ≥1.5 g/dL from baseline to Day 106 (EOS visit); eight patients (n=4 for both cohorts) reached an increase in hemoglobin >1.0 g/dL from baseline to Day 15 (Figure A). Total mean bilirubin decreased by Day 1 and this was sustained to Day 106 (EOS visit; Figure B).The decrease was more pronounced in the 30 mg/kg IV cohort. Improvements in clinical markers correlated with sustained reductions in CH50 in both treatment cohorts. Reductions in mean percent classical complement pathway (CP) activity were noted 1 hour after a single dose of 30 or 15 mg/kg IV, followed by gradual rebound of CP activity over the 15-week study period. Conclusion:SAR445088 was generally well tolerated; no safety concerns were identified in patients with CAD. A single IV dose of SAR445088 led to classical complement inhibition, control of hemolysis, and improvement in anemia, which was sustained for 15 weeks.

Development of a bioengineered Erwinia chrysanthemi asparaginase to enhance its anti-solid tumor potential for treating gastric cancer

Asparaginase has been traditionally applied for only treating acute lymphoblastic leukemia due to its ability to deplete asparagine. However, its ultimate anticancer potential for treating solid tumors has not yet been unleashed. In this study, we bioengineered Erwinia chrysanthemi asparaginase (ErWT), one of the US Food and Drug Administration-approved types of amino acid depleting enzymes, to achieve double amino acid depletions for treating a solid tumor. We constructed a fusion protein by joining an albumin binding domain (ABD) to ErWT via a linker (GGGGS)5 to achieve ABD-ErS5. The ABD could bind to serum albumin to form an albumin-ABD-ErS5 complex, which could avoid renal clearance and escape from anti-drug antibodies, resulting in a remarkably prolonged elimination half-life of ABD-ErS5. Meanwhile, ABD-ErS5 did not only deplete asparagine but also glutamine for ∼2 weeks. A biweekly administration of ABD-ErS5 (1.5 mg/kg) significantly suppressed tumor growth in an MKN-45 gastric cancer xenograft model, demonstrating a novel approach for treating solid tumor depleting asparagine and glutamine. Multiple administrations of ABD-ErS5 did not cause any noticeable histopathological abnormalities of key organs, suggesting the absence of acute toxicity to mice. Our results suggest ABD-ErS5 is a potential therapeutic candidate for treating gastric cancer.

A global comparative field study to evaluate the factor VIII activity of efanesoctocog alfa by one-stage clotting and chromogenic substrate assays at clinical haemostasis laboratories.

Structural and chemical modifications of factor VIII (FVIII) products may influence their behaviour in FVIII activity assays. Hence, it is important to assess the performance of FVIII products in these assays. Efanesoctocog alfa is a new class of FVIII replacement therapy designed to provide both high sustained factor activity levels and prolonged plasma half-life. Evaluate the accuracy of measuring efanesoctocog alfa FVIII activity in one-stage clotting assays (OSAs) and chromogenic substrate assays (CSAs). Human plasma with no detectable FVIII activity was spiked with efanesoctocog alfa or a full-length recombinant FVIII product comparator, octocog alfa, at nominal concentrations of 0.80 IU/mL, 0.20 IU/mL, or 0.05 IU/mL, based on labelled potency. Clinical haemostasis laboratories (N=35) tested blinded samples using in-house assays. Data from 51 OSAs (14 activated partial thromboplastin time [aPTT] reagents) and 42 CSAs (eight kits) were analyzed. Efanesoctocog alfa activity was reliably (±25% of nominal activity) measured across all concentrations using OSAs with Actin FSL and multiple other aPTT reagents. Under- and overestimation of FVIII activity occurred with some reagents. No specific trend was observed for any class of aPTT activators. A two- to three-fold overestimation was consistently observed using CSAs and the OSA with Actin FS as the aPTT reagent across evaluated concentrations. Under- or overestimation occurred with some specific OSAs and most CSAs, which has been previously observed with other modified FVIII replacement products. Efanesoctocog alfa FVIII activity was measured with acceptable accuracy and reliability using several OSA methods and commercial plasma standards.

Lipid-Based Nanoparticles as Oral Drug Delivery Systems: Overcoming Poor Gastrointestinal Absorption and Enhancing Bioavailability of Peptide and Protein Therapeutics.

Delivery and formulation of oral peptide and protein therapeutics have always been a challenge for the pharmaceutical industry. The oral bioavailability of peptide and protein therapeutics mainly relies on their gastrointestinal solubility and permeability which are affected by their poor membrane penetration, high molecular weight and proteolytic (chemical and enzymatic) degradation resulting in limited delivery and therapeutic efficacy. The present review article highlights the challenges and limitations of oral delivery of peptide and protein therapeutics focusing on the application, potential and importance of solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) as lipid-based drug delivery systems (LBDDSs) and their advantages and drawbacks. LBDDSs, due to their lipid-based matrix can encapsulate both lipophilic and hydrophilic drugs, and by reducing the first-pass effect and avoiding proteolytic degradation offer improved drug stability, dissolution rate, absorption, bioavailability and controlled drug release. Furthermore, their small size, high surface area and surface modification increase their mucosal adhesion, tissue-targeted distribution, physiological function and half-life. Properties such as simple preparation, high-scale manufacturing, biodegradability, biocompatibility, prolonged half-life, lower toxicity, lower adverse effects, lipid-based structure, higher drug encapsulation rate and various drug release profile compared to other similar carrier systems makes LBDDSs a promising drug delivery system (DDS). Nevertheless, undesired physicochemical features of peptide and protein drug development and discovery such as plasma stability, membrane permeability and circulation half-life remain a serious challenge which should be addressed in future.

Thrombospondin-1 proteomimetic polymers exhibit anti-angiogenic activity in a neovascular age-related macular degeneration mouse model.

Neovascular age-related macular degeneration (nAMD) is the leading cause of blindness in the developed world. Current therapy includes monthly intraocular injections of anti-VEGF antibodies, which are ineffective in up to one third of patients. Thrombospondin-1 (TSP1) inhibits angiogenesis via CD36 binding, and its down-regulated expression is negatively associated with the onset of nAMD. Here, we describe TSP1 mimetic protein-like polymers (TSP1 PLPs). TSP1 PLPs bind CD36 with high affinity, resist degradation, show prolonged intraocular half-lives (13.1 hours), have no toxicity at relevant concentrations in vivo (40 μM), and are more efficacious in ex vivo choroidal sprouting assays compared to the peptide sequence and Eylea (aflibercept), the current standard of care anti-VEGF treatment. Furthermore, PLPs exhibit superior in vivo efficacy in a mouse model for nAMD compared to control PLPs consisting of scrambled peptide sequences, using fluorescein angiography and immunofluorescence. Since TSP-1 inhibits angiogenesis by VEGF-dependent and independent mechanisms, TSP1 PLPs are a potential therapeutic for patients with anti-VEGF treatment-resistant nAMD.

Insertion of (Bioactive) Equatorial Ligands into Platinum(IV) Complexes.

Platinum(IV) prodrugs are highly interesting alternatives to platinum(II) anticancer therapeutics due to their increased tumor selectivity and reduced side effects. In contrast to the established theory, we recently observed that the equatorial ligand(s) of e.g. oxaliplatin(IV) complexes can be hydrolyzed with formation of [(DACH)Pt(OHeq )2 (OAcax )2 ]. In the work presented here, we investigated the reactivity and synthetic usability of this complex to be exploited as a precursor for the development of novel platinum(IV) complexes, not able to be synthesized by conventional protocols. Indeed, we could substitute the equatorial hydroxido ligand(s) e.g. by one or two monodentate biotin ligands (which would be oxidized under standard methods). The formed complexes turned out to be very stable with slow ligand release after reduction, ideal for long-circulating tumor-targeting strategies. Therefore, two platinum(IV) complexes with equatorial maleimides, capable of exploiting serum albumin as a natural nanocarrier, were synthesized as well. The complexes showed massively prolonged plasma half-life and distinctly improved anticancer activity in vivo compared to oxaliplatin. Taken together, the newly developed synthetic platform allows the simple and specific insertion of equatorial ligands into platinum(IV) complexes. This will enable the attachment of three different (bioactive) moieties generating targeted triple-action platinum(IV) prodrugs within one single platinum complex.

Preclinical safety, toxicokinetics and metabolism of BIIB131, a novel prothrombolytic agent for acute stroke

BIIB131, a small molecule, is currently in Phase 2 for the treatment of acute ischemic stroke. Safety and metabolism of BIIB131 were evaluated following intravenous administration to rats and monkeys. Exposure increased dose-proportionally in rats up to 60 mg/kg and more than dose-proportionally in monkeys at greater than 10 mg/kg accompanied by prolonged half-life and safety findings. The BIIB131 was poorly metabolized in microsomes with no inhibition of CYPs. BIIB131-glucuronide, formed by UGT1A1, accounted for 21.5% metabolism in human hepatocytes and 28–40% in rat bile. In rats, excretion was primarily via the bile. BIIB131 inhibited the hERG and Nav1.5 cardiac channels by 39% but showed no effect on cardiovascular parameters in monkeys. Toxicology findings were limited to reversable hematuria, changes in urinary parameters and local effects. A MTD of 30 mg/kg was established in monkeys, the most sensitive species, at total plasma Cmax and AUC of 6- and 14-fold, respectively, greater than the NOAEL. The Phase 1 study started with intravenous 0.05 mg/kg and ascended to 6.0 mg/kg which corresponded to safety margins of 147- to 0.9-fold (for Cmax) within the linear drug exposure. Thus, the preclinical profile of BIIB131 has been appropriately characterized and supports its further clinical development.

A Narrative Review on the Role of Dalbavancin in the Treatment of Bone and Joint Infections.

Bone and joint infections (BJI) require prolonged antimicrobial treatment, leading to lengthy hospitalizations, high costs, the risk of nosocomial infections, and the development of antimicrobial resistance. Dalbavancin is a novel semisynthetic lipoglycopeptide approved for the treatment of adults and children with acute bacterial skin and skin structure infections. This narrative review aims to summarize the characteristics of dalbavancin and the current scientific evidence regarding its clinical efficacy and safety in the treatment of BJI. A literature search until June 2023 was performed to identify all published research about the role of dalbavancin in the management of BJI. Due to its unique pharmacokinetics characterized by prolonged half-life, high bactericidal activity against most Gram-positive bacteria, a good safety profile, and high tissue penetration, dalbavancin can be a valuable alternative to the treatment of BJI. Clinical studies have shown its non-inferiority compared to conventional therapies in BJI, offering potent activity against key pathogens and an extended dosing interval that may shorten hospitalization. In conclusion, dalbavancin represents a promising treatment option for BJI with a favorable safety profile, but further research in both adults and particularly children, who are ideal candidates for long-acting antibiotics, is necessary to evaluate the role of dalbavancin in BJI.

Biodistribution of Agmatine to Brain and Spinal Cord after Systemic Delivery.

Agmatine, an endogenous polyamine, has been shown to reduce chronic pain behaviors in animal models and in patients. This reduction is due to inhibition of the GluN2B subunit of the N-methyl-D-aspartate receptor (NMDAR) in the central nervous system (CNS). The mechanism of action requires central activity, but the extent to which agmatine crosses biologic barriers such as the blood-brain barrier (BBB) and intestinal epithelium is incompletely understood. Determination of agmatine distribution is limited by analytical protocols with low sensitivity and/or inefficient preparation. This study validated a novel bioanalytical protocol using high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) for quantification of agmatine in rat biologic matrices. These protocols were then used to determine the plasma pharmacokinetics of agmatine and the extent of distribution to the CNS. Precision and accuracy of the protocol met US Food and Drug Administration (FDA) standards in surrogate matrix as well as in corrected concentrations in appropriate matrices. The protocol also adequately withstood stability and dilution conditions. Upon application of this protocol to pharmacokinetic study, intravenous agmatine showed a half-life in plasma ranging between 18.9 and 14.9 minutes. Oral administration led to a prolonged plasma half-life (74.4-117 minutes), suggesting flip-flop kinetics, with bioavailability determined to be 29%-35%. Intravenous administration led to a rapid increase in agmatine concentration in brain but a delayed distribution and lower concentrations in spinal cord. However, half-life of agmatine in both tissues is substantially longer than in plasma. These data suggest that agmatine adequately crosses biologic barriers in rat and that brain and spinal cord pharmacokinetics can be functionally distinct. SIGNIFICANCE STATEMENT: Agmatine has been shown to be an effective nonopioid therapy for chronic pain, a significantly unmet medical necessity. Here, using a novel bioanalytical protocol for quantification of agmatine, we present the plasma pharmacokinetics and the first report of agmatine oral bioavailability as well as variable pharmacokinetics across different central nervous system tissues. These data provide a distributional rationale for the pharmacological effects of agmatine as well as new evidence for kinetic differences between brain and spinal cord.

Effect of food and polymorphisms in SLCO2B1, CYP3A4 and UGT1A4 on pharmacokinetics of abiraterone and its metabolites in Chinese volunteers.

Abiraterone acetate, a prodrug of abiraterone (ABI), provides an efficient therapeutic option for metastatic castration-resistant prostate cancer patients. ABI undergoes extensive metabolism in vivo and is transformed into active metabolites Δ4 -abiraterone and 3-keto-5α-abiraterone as well as inactive metabolites abiraterone sulfate and abiraterone N-oxide sulfate. We aimed to examine the effect of polymorphisms in SLCO2B1, CYP3A4 and UGT1A4 on the pharmacokinetics of ABI and its metabolites. In this study, 81 healthy Chinese subjects were enrolled and divided into 2 groups for fasted (n = 45) and fed (n = 36) studies. Plasma samples were collected after administering a 250 mg abiraterone acetate tablet followed by liquid chromatography-tandem mass spectrometry analysis. Genotyping was performed on a MassARRAY system. The association between SLCO2B1, CYP3A4, UGT1A4 genotype and pharmacokinetic parameters of ABI and its metabolites was assessed. Food effect study demonstrated high fat meal remarkedly increased systemic exposure of ABI and its metabolites. The geometric mean ratio and 90% confidence interval of area under the plasma concentration-time curve from time 0 to the time of the last quantifiable concentration(AUC0-t ) and maximum plasma concentration (Cmax ) of ABI in fed state vs. fasted state were 351.64% (286.86%-431.04%) and 478.45% (390.01%-586.94%), respectively, while the corresponding results were ranging from 145.11% to 269.42% and 150.10% to 478.45% for AUC0-t and Cmax of ABI metabolites in fed state vs. fasted state, respectively. The SLCO2B1 rs1077858 had a significant influence on AUC0-t and Cmax , while 7 other SLCO2B1 variants prolonged half-life of ABI under both fasted and fed conditions. As for ABI metabolites, the systemic exposure of Δ4 -abiraterone, abiraterone sulfate and abiraterone N-oxide sulfate as well as the elimination of 3-keto-5α-abiraterone were significantly affected by SLCO2B1 polymorphisms. Polymorphisms in CYP3A4 and UGT1A4 did not significantly affect pharmacokinetics of ABI and its metabolites. Polymorphisms in SLCO2B1 were significantly related to the pharmacokinetic variability of ABI and its metabolites under both fasted and fed conditions.

Molecular evolution of Cypridina noctiluca secretory luciferase for production of spectrum-shifted luminescence-emitting mutants and their application in nuclear receptor-reporter assays.

Luciferase is a popular enzyme used for biological analyses, such as reporter assays. In addition to a conventional reporter assay using a pair of firefly and Renilla luciferases, a simple multicolor reporter assay using multiple firefly or beetle luciferases emitting different color luminescence with a single substrate has been reported. Secretory luciferases have also been used for convenient sample preparation in reporter assays; however, reporter assay using secretory luciferase mutants that emit spectrum-shifted luminescence have not yet been reported. In this study, we generated blue- and red-shifted (-16 and 12 nm) luminescence-emitting Cypridina secretory luciferase (CLuc) mutants using multiple cycles of random and site-directed mutagenesis. Even for red-shifted CLuc mutant, which exhibited relatively low activity and stability, its enzymatic activity was sufficiently high for a luciferase assay (3.26 × 106 relative light unit/s), light emission was sufficiently prolonged (half-life is 3 min), and stability at 37°C was high. We independently determined the luminescence of these CLuc mutants using a luminometer with an optical filter. Finally, we replaced the commonly used reporters, firefly and Renilla luciferases used in a conventional nuclear receptor-reporter assay with these CLuc mutants and established a secretory luciferase-based single-substrate dual-color nuclear receptor-reporter assay.

Characterization of a recombinant factor IX molecule fused to coagulation factor XIII-B subunit.

Severe haemophilia B (HB) is characterized by spontaneous bleeding episodes, mostly into joints. Recurrent bleeds lead to progressive joint destruction called haemophilic arthropathy. The current concept of prophylaxis aims at maintaining the FIX level >3-5IU/dL, which is effective at reducing the incidence of haemophilic arthropathy. Extended half-life FIX molecules make it easier to achieve these target trough levels compared to standard FIX concentrates. We previously reported that the fusion of a recombinant FIX (rFIX) to factor XIII-B (FXIIIB) subunit prolonged the half-life of the rFIX-LXa-FXIIIB fusion molecule in mice and rats 3.9- and 2.2-fold, respectively, compared with rFIX-WT. However, the mechanism behind the extended half-life was not known. Mass spectrometry and ITC were used to study interactions of rFIX-LXa-FXIIIB with albumin. Pharmacokinetic analyses in fibrinogen-KO and FcRn-KO mice were performed to evaluate the effect of albumin and fibrinogen on in-vivo half-life of rFIX-LXa-FXIIIB. Finally saphenous vein bleeding model was used to assess in-vivo haemostatic activity of rFIX-LXa-FXIIIB. We report here the key interactions that rFIX-LXa-FXIIIB may have in plasma are with fibrinogen and albumin which may mediate its prolonged half-life. In addition, using the saphenous vein bleeding model, we demonstrate that rFIX-FXIIIB elicits functional clot formation that is indistinguishable from that of rFIX-WT.

Co-administration of an effector antibody enhances the half-life and therapeutic potential of RNA-encoded nanobodies

The incidence of Clostridioides difficile infection (CDI) and associated mortality have increased rapidly worldwide in recent years. Therefore, it is critical to develop new therapies for CDI. Here we report on the development of mRNA-LNPs encoding camelid-derived VHH-based neutralizing agents (VNAs) targeting toxins A and/or B of C. difficile. In preclinical models, intravenous administration of the mRNA-LNPs provided serum VNA levels sufficient to confer protection of mice against severe disease progression following toxin challenge. Furthermore, we employed an mRNA-LNP encoded effector antibody, a molecular tool designed to specifically bind an epitopic tag linked to the VNAs, to prolong VNA serum half-life. Co-administration of VNA-encoding mRNA-LNPs and an effector antibody, either provided as recombinant protein or encoded by mRNA-LNP, increased serum VNA half-life in mice and in gnotobiotic piglets. Prolonged serum half-life was associated with higher concentrations of serum VNA and enhanced prophylactic protection of mice in challenge models.

Oral cytarabine ocfosfate pharmacokinetics and assessment of leukocyte biomarkers in normal dogs.

Cytosine arabinoside (Ara-C) is a nucleoside analog prodrug utilized for immunomodulatory effects mediated by its active metabolite Ara-CTP. Optimal dosing protocols for immunomodulation in dogs have not been defined. Cytarabine ocfosfate (CO) is a lipophilic prodrug of Ara-C that can be administered PO and provides prolonged serum concentrations of Ara-C. Provide pharmacokinetic data for orally administered CO and determine accumulation and functional consequences of Ara-CTP within peripheral blood leukocytes. Three healthy female hound dogs and 1 healthy male Beagle. Prospective study. Dogs received 200 mg/m2 of CO PO q24h for 7 doses. Serum and cerebrospinal fluid (CSF) CO and Ara-C concentrations were measured by liquid chromatography-tandem mass spectroscopy (LC-MS/MS). Complete blood counts, flow cytometry, and leukocyte activation assays were done up to 21 days. Incorporation of Ara-CTP within leukocyte DNA was determined by LC-MS/MS. Maximum serum concentration (Cmax ) for Ara-C was 456.1-724.0 ng/mL (1.88-2.98 μM) and terminal half-life was 23.3 to 29.4 hours. Cerebrospinal fluid: serum Ara-C ratios ranged from 0.54 to 1.2. Peripheral blood lymphocyte concentrations remained within the reference range, but proliferation rates poststimulation were decreased at 6 days. Incorporation of Ara-CTP was not saturated and remained >25% of peak concentration at 13 days. Oral CO may produce prolonged serum Ara-C half-lives at concentrations sufficient to induce functional changes in peripheral leukocytes and is associated with prolonged retention of DNA-incorporated Ara-CTP. Application of functional and active metabolite assessment is feasible and may provide more relevant data to determine optimal dosing regimens for Ara-C-based treatments.

Intelligent Responsive Nanoparticles with Multilevel Triggered Drug Penetration for Tumor Photochemotherapy.

Nanomedicines have contradictory size requirements to overcome systemic barriers and penetrate the tumor extracellular matrix (ECM). Larger-sized nanoparticles (50-200 nm) exhibit prolonged blood circulation half-life and improved tumor enrichment, while small-sized nanoparticles (4-20 nm) easily penetrate deep tumor tissues. Therefore, the development of intelligent responsive nanomedicine systems can not only increase nanodrug tumor accumulation but also improve their penetration into the ECM. Herein, we propose an intelligent responsive nanoparticle triggered by near-infrared light (NIR). The nanoparticle was constructed by a temperature-sensitive liposome (TSL) encapsulating ultrasmall melanin nanoparticles (MNPs) loaded with doxorubicin (MNP/doxorubicin (DOX)@TSL). When exposed to NIR irradiation, the tailor-made nanoparticles not only effectively ablated the tumor cells around blood vessels but also destroyed the structural integrity and released loaded ultrasmall MNP/DOX (<10 nm) to promote deep tumor penetration and enhance interior tumor cell killing. This NIR-triggered intelligent nanoparticle successfully integrated photothermal therapy (PTT) for perivascular tumor cells and chemotherapy for deep tumor cell inhibition. The in vivo results showed remarkable tumor regression in 4T1 breast tumor-bearing mice by 74.2%. This controllable size switchable nanosystem with efficient tumor accumulation and penetration has shown great potential in improving synergistic antitumor effects of photochemotherapy.

Insulin concentrations following termination of high-dose insulin euglycemic therapy

Introduction High-dose insulin therapy is used in patients with calcium channel blocker and beta-adrenergic antagonist overdoses. The pharmacokinetics of insulin are scantly reported following high-dose insulin therapy. We present two cases of persistently elevated insulin concentrations following high-dose insulin therapy. Case reports A 50-year-old woman and a 45-year-old man experienced hypotension after overdosing on amlodipine and atenolol. They were treated with high-dose insulin therapy for 54 hours at 2 units/kilogram/hour and 48 hours at 10 units/kilogram/hour, respectively. Following termination, serum insulin elimination was studied. Insulin concentrations remained greater than 1,000 µU/mL (fasting reference 2.6–24.9 µU/mL) for longer than 4 hours (case 1) and 11 hours (case 2) and greater than 300 µU/mL for longer than 8 hours and 21 hours, respectively. Insulin concentrations decreased with apparent first-order elimination half-lives of 13.0 hours and 6.0 hours. Discussion Following high-dose insulin therapy, insulin concentrations remained elevated for longer than expected based on normal pharmacokinetics in therapeutic dosing. Three previous cases reported insulin half-lives of between 2.2 hours and 18.7 hours. The current cases add to the existing data that insulin has a variable but prolonged half-life following high-dose insulin therapy. Conclusions These findings suggest that patients are at prolonged risk of hypoglycemia following cessation of high-dose insulin infusions.