Classification of recombinant factor VIII products and implications for clinical practice: A systematic literature review.

INTRODUCTION: Consensus over the definition of recombinant factor VIII (rFVIII) product classification in haemophilia A is lacking. rFVIII products are often classified as standard half-life (SHL) or extended half-life (EHL); despite this, no universally accepted definition currently exists. One proposed definition includes half-life, area under the curve, and technology designed to extend half-life; however, the International Society on Thrombosis and Haemostasis defines activity over time as the most intuitive information for building treatment regimens and the World Federation of Hemophilia describes rFVIII product classification in terms of infusion frequency. AIM: To summarise published data on the clinical and pharmacokinetic criteria used to define rFVIII product classification. METHODS: PubMed and EMBASE database searches of English-language articles (2002-2022) were conducted using search strings to identify the relevant population, intervention, and outcomes (e.g., clinical and pharmacokinetic parameters). Articles then underwent title/abstract and full-text screens. RESULTS: Among 1147 identified articles, 62 were included. Half-life was the most widely reported outcome with no clear trends or product groupings observed. No clear groupings emerged among other outcomes, including infusion frequency, consumption, and efficacy. As activity over time was reported in few articles, further investigation of its relevance to rFVIII product classification is warranted. CONCLUSION: The findings of this systematic literature review suggest that parameters other than half-life might be important for the development of a comprehensive and clinically relevant rFVIII product classification definition. There seems to be an opportunity to consider parameters that are clinically meaningful and useful for shared decision-making in haemophilia A treatment.

Pharmacokinetic Parameters of Recombinant Human Cyclophilin A in Mice.

BACKGROUND AND OBJECTIVE: Cyclophilin A (CypA) is an isomerase that functions as a chaperone, housekeeping protein, and cyclosporine A (CsA) ligand. Secreted CypA is a proinflammatory factor, chemoattractant, immune regulator, and factor of antitumor immunity. Experimental data suggest clinical applications of recombinant human CypA (rhCypA) as a biotherapeutic for cancer immunotherapy, stimulation of tissue regeneration, treatment of brain pathologies, and as a supportive treatment for CsA-based therapies. The objective of this study is to analyze the pharmacokinetics of rhCypA in a mouse model. METHODS: rhCypA was isotope-labeled with 125I and injected intraperitoneally (i.p.) or subcutaneously (s/c) into female mice as a single dose of 100 µg per mouse, equivalent to the estimated first-in-human dose. Analysis of 125I-rhCypA biodistribution and excretion was performed by direct radiometry of the blood, viscera, and urine of mice 0.5-72 h following its administration. RESULTS: rhCypA showed rapid and even tissue-organ distribution, with the highest tropism (fT = 1.56) and accumulation (maximum concentration, Cmax = 137-167 µg/g) in the kidneys, its primary excretory organ. rhCypA showed the lowest tropism to the bone marrow and the brain (fT = 0.07) but the longest retention in these organs [mean retention time (MRT) = 25-28 h]. CONCLUSION: This study identified promising target organs for rhCypA's potential therapeutic effects. The mode of rhCypA accumulation and retention in organs could be primarily due to the expression of its receptors in them. For the first time, rhCypA was shown to cross the blood-brain barrier and accumulate in the brain. These rhCypA pharmacokinetic data could be extrapolated to humans as preliminary data for possible clinical trials.

[Pharmacokinetics of Recombinant Human Coagulation Factor â § Preparations in Patients with Severe Hemophilia A].

OBJECTIVE: To calculate the pharmacokinetic parameters of recombinant human coagulation factor Ⅷ using myPKFiT in patients with severe hemophilia A, and provide an individualized treatment plan for patients. METHODS: A total of 42 patients with severe hemophilia A who were treated with recombinant human coagulation factor Ⅷ were included from January 2021 to December 2021. myPKFiT was used to calculate the pharmacokinetic parameters of FⅧ, and the individualized treatment plan for hemophilia A patients was formulated. RESULTS: The median age of 42 patients with severe hemophilia A was 31（16-50） years old, the average weight was 54.0±9.9 kg, the half-life of FⅧ was 12.05±1.6 h, the time to more than 1% of the baseline was 62.3±15.3 h, and the 0 bleeding rate after the guidance of myPKFiT was significantly increased from 39% to 49%, the Annual bleeding rate was reduced from 3.6±2.5 to 2.1±2.0, and the Annual joint bleeding rate was reduced from 3.2±2.2 to 1.9±0.9, all of which were statistically different (P<0.05). CONCLUSION: Individualized therapy in patients with severe hemophilia A who were guided by myPKFiT assay of pharmacokinetics parameters can significantly reduce the annual bleeding rate and annual joint bleeding rate of patients.

Phase 1 Trial of Antibody NI006 for Depletion of Cardiac Transthyretin Amyloid.

BACKGROUND: Transthyretin amyloid (ATTR) cardiomyopathy is a progressive and fatal disease caused by misfolded transthyretin. Despite advances in slowing disease progression, there is no available treatment that depletes ATTR from the heart for the amelioration of cardiac dysfunction. NI006 is a recombinant human anti-ATTR antibody that was developed for the removal of ATTR by phagocytic immune cells. METHODS: In this phase 1, double-blind trial, we randomly assigned (in a 2:1 ratio) 40 patients with wild-type or variant ATTR cardiomyopathy and chronic heart failure to receive intravenous infusions of either NI006 or placebo every 4 weeks for 4 months. Patients were sequentially enrolled in six cohorts that received ascending doses (ranging from 0.3 to 60 mg per kilogram of body weight). After four infusions, patients were enrolled in an open-label extension phase in which they received eight infusions of NI006 with stepwise increases in the dose. The safety and pharmacokinetic profiles of NI006 were assessed, and cardiac imaging studies were performed. RESULTS: The use of NI006 was associated with no apparent drug-related serious adverse events. The pharmacokinetic profile of NI006 was consistent with that of an IgG antibody, and no antidrug antibodies were detected. At doses of at least 10 mg per kilogram, cardiac tracer uptake on scintigraphy and extracellular volume on cardiac magnetic resonance imaging, both of which are imaging-based surrogate markers of cardiac amyloid load, appeared to be reduced over a period of 12 months. The median N-terminal pro-B-type natriuretic peptide and troponin T levels also seemed to be reduced. CONCLUSIONS: In this phase 1 trial of the recombinant human antibody NI006 for the treatment of patients with ATTR cardiomyopathy and heart failure, the use of NI006 was associated with no apparent drug-related serious adverse events. (Funded by Neurimmune; NI006-101 ClinicalTrials.gov number, NCT04360434.).

Model-informed pediatric dose selection of marzeptacog alfa (activated): An exposure matching strategy.

Marzeptacog alfa (activated) (MarzAA) is an activated recombinant human rFVII variant intended for subcutaneous (s.c.) administration to treat or prevent bleeding in individuals with hemophilia A (HA) or B (HB) with inhibitors, and other rare bleeding disorders. The s.c. administration provides benefits over i.v. injections. The objective of the study was to support the first-in-pediatric dose selection for s.c. MarzAA to treat episodic bleeding episodes in children up through 11 years in a registrational phase III trial. Assuming the same exposure-response relationship as in adults, an exposure matching strategy was used with a population pharmacokinetics model. A sensitivity analysis evaluating the impact of doubling in absorption rate and age-dependent allometric exponents on dose selection was performed. Subsequently, the probability of trial success, defined as the number of successful trials for a given pediatric dose divided by the number of simulated trials (n = 1000) was studied. A successful trial was defined as outcome where four, three, or two out of 24 pediatric subjects per trial were allowed to fall outside the adult exposures after s.c. administration of 60 µg/kg. A dose of 60 µg/kg in children with HA/HB was supported by the clinical trial simulations to match exposures in adults. The sensitivity analyses further supported selection of the 60 µg/kg dose level in all age groups. Moreover, the probability of trial success evaluations given a plausible design confirmed the potential of a 60 µg/kg dose level. Taken together, this work demonstrates the utility of model-informed drug development and could be helpful for other pediatric development programs for rare diseases.

Safety, Tolerability, Pharmacokinetics, and Immunogenicity of a Novel Recombination Human Nerve Growth Factor in Healthy Chinese Subjects.

BACKGROUND: Nerve growth factor (NGF), the first-discovered member of the neurotrophin family, has long been regarded as a potential drug to combat acute and chronic neurodegenerative processes. However, the pharmacokinetic profile of NGF is poorly described. OBJECTIVES: The aim of this study was to investigate the safety, tolerability, pharmacokinetics, and immunogenicity of a novel recombinant human NGF (rhNGF) in healthy Chinese subjects. METHOD: The study randomized 48 and 36 subjects to receive (i) single-ascending dose (SAD group; 7.5, 15, 30, 45, 60, 75 µg or placebo) and (ii) multiple-ascending dose (MAD group; 15, 30, 45 µg, or placebo) rhNGF intramuscular injections, respectively. In the SAD group, all participants received rhNGF or placebo only once. In the MAD group, participants were randomly assigned to receive multiple doses of rhNGF or placebo once a day for 7 consecutive days. Adverse events (AEs) and anti-drug antibodies (ADAs) were monitored throughout the study. Recombinant human NGF serum concentrations were determined using a highly sensitive enzyme-linked immunosorbent assay. RESULTS: All AEs were mild, except for some injection-site pain and fibromyalgia, which were experienced as moderate AEs. Only one moderate AE was observed in the 15 µg cohort throughout the study and resolved within 24 hours of stopping dosing. Many participants (10% in 30 µg, 50% in 45 µg, and 50% in 60 µg in the SAD group; 10% in 15 µg, 30% in 30 µg, and 30% in 45 µg in the MAD group) experienced moderate fibromyalgia. However, all moderate fibromyalgia were resolved by the end of the subject's participation in the study. No severe AEs or clinically significant abnormalities were reported. All subjects in the 75 µg cohort experienced positive ADA in the SAD group, and one subject in the 30 µg dose and four subjects in the 45 µg dose also experienced positive ADA in the MAD group. Recombinant human nerve growth factor was absorbed (median Tmax, 4.0-5.3 h) and eliminated biexponentially (mean t1/2, 4.53-6.09 h) with a moderate speed. The Cmax and AUC increased in an approximately dose-proportional manner over the dose range of 7.5-45 µg, and at doses higher than 45 µg these parameters increased more than dose proportionally. There was no obvious accumulation after 7 days of daily dosing of rhNGF. CONCLUSION: The favorable safety and tolerability and predictable pharmacokinetic profile of rhNGF in healthy Chinese subjects support its continuing clinical development for the treatment of nerve injury and neurodegenerative diseases. The AEs and immunogenicity of rhNGF will continue to be monitored in future clinical trials. TRIAL REGISTRATION: This study was registered with Chinadrugtrials.org.cn (ChiCTR2100042094) on January 13th, 2021.

Pharmacokinetics of Recombinant Human Coagulation Factor Ⅷ Preparations in Patients with Severe Hemophilia A / 中国实验血液学杂志

OBJECTIVE@#To calculate the pharmacokinetic parameters of recombinant human coagulation factor Ⅷ using myPKFiT in patients with severe hemophilia A, and provide an individualized treatment plan for patients.@*METHODS@#A total of 42 patients with severe hemophilia A who were treated with recombinant human coagulation factor Ⅷ were included from January 2021 to December 2021. myPKFiT was used to calculate the pharmacokinetic parameters of FⅧ, and the individualized treatment plan for hemophilia A patients was formulated.@*RESULTS@#The median age of 42 patients with severe hemophilia A was 31（16-50） years old, the average weight was 54.0±9.9 kg, the half-life of FⅧ was 12.05±1.6 h, the time to more than 1% of the baseline was 62.3±15.3 h, and the 0 bleeding rate after the guidance of myPKFiT was significantly increased from 39% to 49%, the Annual bleeding rate was reduced from 3.6±2.5 to 2.1±2.0, and the Annual joint bleeding rate was reduced from 3.2±2.2 to 1.9±0.9, all of which were statistically different (P<0.05).@*CONCLUSION@#Individualized therapy in patients with severe hemophilia A who were guided by myPKFiT assay of pharmacokinetics parameters can significantly reduce the annual bleeding rate and annual joint bleeding rate of patients.

Pharmacokinetic parameter driven outcomes model predicts a reduction in bleeding events associated with BAY 81-8973 versus antihemophilic factor (recombinant) plasma/albumin-free method in a Chinese healthcare setting.

BACKGROUND: Long-term prophylactic therapy is considered the standard of care for hemophilia A patients. This study models the long-term clinical and cost outcomes of two factor VIII (FVIII) products using a pharmacokinetic (PK) simulation model in a Chinese population. METHODS: Head-to-head PK profile data of BAY 81-8973 (KOVALTRY®) and antihemophilic factor (recombinant) plasma/albumin-free method (rAHF-PFM, ADVATE®) were applied to a two-state (alive and dead) Markov model to simulate blood FVIII concentrations at a steady state in prophylactically-treated patients with hemophilia A. Worsening of the Pettersson score was simulated and decline was associated with the probability of having orthopaedic surgery. The only difference between the compounds was FVIII concentration at a given time; each subject was treated with 25 IU/kg every 3 days. The model used a lifetime horizon, with cycle lengths of 1 year. RESULTS: Cumulative bleeding events, joint bleeding events, and major bleeding events were reduced by 19.3% for BAY 81-8973 compared to rAHF-PFM. Hospitalizations and hospitalization days were also reduced by 19.3% for BAY 81-8973 compared to rAHF-PFM. BAY 81-8973 resulted in both cost savings and a gain in quality adjusted life years (QALYs) compared to rAHF-PFM. CONCLUSION: Based on modeled head-to-head comparisons, differences in PK-properties between BAY 81-8973 and rAHF-PFM result in a reduced number of bleeding events, leading to reduced costs and increased quality of life for BAY 81-8973. These results should be used to inform clinical practice in China when caring for patients with severe hemophilia A.

Revised terminal half-life of nonacog alfa as derived from extended sampling data: A real-world study involving 64 haemophilia B patients on nonacog alfa regular prophylaxis.

BACKGROUND: Nonacog alfa, a standard half-life recombinant factor IX (FIX), is used as a prophylactic treatment in severe haemophilia B (SHB) patients. Its half-life determined in clinical studies involving a limited sampling (72 h) was shown to be rather short. In our clinical practice, we suspected that its half-life could have been underestimated. OBJECTIVES: We aimed to evaluate nonacog alfa pharmacokinetics in real world clinical practice based on FIX levels in patients receiving prophylaxis. METHODS: We retrospectively collected data on patients with SHB receiving prophylaxis from eight centres across France. The terminal half-life (THL), time to reach 5-2 IU/dl and FIX activity at 48, 72 and 96 h were derived by Bayesian estimations using NONMEM analysis. RESULTS AND CONCLUSIONS: Infusion data (n = 455) were collected from 64 patients with SHB. The median THL measured in 92 pharmacokinetic (PK) studies was 43.4 h. In 26 patients ≤12 years of age, 51 PK studies showed a median time to reach 5 IU/dl of FIX of 70.5 h and a median time to reach 2 IU/dl of 121.5 h. In 38 patients 13-75 years of age, 41 PK studies showed a median time to reach 5 IU/dl of FIX of 92.0 h and a median time to reach 2 IU/dl of 167.5 h. Extending the sampling beyond 72 h makes it possible to observe a plateau, with FIX remaining between 2 and 5 IU/dl for several days and shows that the THL of nonacog alfa might be longer than previously described. ESSENTIALS: Nonacog alfa terminal half-life (THL) in patients receiving regular prophylaxis was evaluated in clinical practice. The median THL was estimated to be 36.9 h for patients aged .8-12 years. The median THL was estimated to be 49.9 h for patients aged 13-75 years. For patients aged ≤12 and >12 years, the median times to reach 5 IU/dl were 70.5 and 92 h, respectively; to reach 3 IU/dl, 95.5 and 131.5 h, respectively; to reach 2 IU/dl, 121.5 and 167.5 h, respectively. We suggest that the half-life of nonacog alfa might be longer than previously described in both younger and older patients.

Chitosan and Hyaluronic Acid Nanoparticles as Vehicles of Epoetin Beta for Subconjunctival Ocular Delivery.

Neuroprotection in glaucoma using epoetin beta (EPOß) has yielded promising results. Our team has developed chitosan-hyaluronic acid nanoparticles (CS/HA) designed to carry EPOß into the ocular globe, improving the drug's mucoadhesion and retention time on the ocular surface to increase its bioavailability. In the present in vivo study, we explored the possibility of delivering EPOß to the eye through subconjunctival administration of chitosan-hyaluronic acid-EPOß (CS/HA-EPOß) nanoparticles. Healthy Wistar Hannover rats (n = 21) were split into 7 groups and underwent complete ophthalmological examinations, including electroretinography and microhematocrit evaluations before and after the subconjunctival administrations. CS/HA-EPOß nanoparticles were administered to the right eye (OD), and the contralateral eye (OS) served as control. At selected timepoints, animals from each group (n = 3) were euthanized, and both eyes were enucleated for histological evaluation (immunofluorescence and HE). No adverse ocular signs, no changes in the microhematocrits (≈45%), and no deviations in the electroretinographies in both photopic and scotopic exams were observed after the administrations (p < 0.05). Intraocular pressure remained in the physiological range during the assays (11-22 mmHg). EPOß was detected in the retina by immunofluorescence 12 h after the subconjunctival administration and remained detectable until day 21. We concluded that CS/HA nanoparticles could efficiently deliver EPOß into the retina, and this alternative was considered biologically safe. This nanoformulation could be a promising tool for treating retinopathies, namely optic nerve degeneration associated with glaucoma.

Impact of anti-PEG antibody affinity on accelerated blood clearance of pegylated epoetin beta in mice.

Antibodies that bind polyethylene glycol (PEG) can be induced by pegylated biomolecules and also exist in a significant fraction of healthy individuals who have never received pegylated medicines. The binding affinity of antibodies against PEG (anti-PEG antibodies) likely varies depending on if they are induced or naturally occurring. Anti-PEG antibodies can accelerate the clearance of pegylated medicines from the circulation, resulting in loss of drug efficacy, but it is unknown how accelerated blood clearance is affected by anti-PEG antibody affinity. We identified a panel of anti-PEG IgG and IgM antibodies with binding avidities ranging over several orders of magnitude to methoxy polyethylene glycol-epoetin beta (PEG-EPO), which is used to treat patients suffering from anemia. Formation of in vitro immune complexes between PEG-EPO and anti-PEG IgG or IgM antibodies was more obvious as antibody affinity increased. Likewise, high affinity anti-PEG antibodies produced greater accelerated blood clearance of PEG-EPO as compared to low affinity antibodies. The molar ratio of anti-PEG antibody to PEG-EPO that accelerates drug clearance in mice correlates with antibody binding avidity. Our study indicates that the bioactivity of PEG-EPO may be reduced due to rapid clearance in patients with either high concentrations of low affinity or low concentrations of high affinity anti-PEG IgG and IgM antibodies.

Recombinant Human HPS Protects Mice and Nonhuman Primates from Acute Liver Injury.

Acute liver injury shares a common feature of hepatocytes death, immune system disorders, and cellular stress. Hepassocin (HPS) is a hepatokine that has ability to promote hepatocytes proliferation and to protect rats from D-galactose (D-Gal)- or carbon tetrachloride (CCl4)-induced liver injury by stimulating hepatocytes proliferation and preventing the high mortality rate, hepatocyte death, and hepatic inflammation. In this paper, we generated a pharmaceutical-grade recombinant human HPS using mammalian cells expression system and evaluated the effects of HPS administration on the pathogenesis of acute liver injury in monkey and mice. In the model mice of D-galactosamine (D-GalN) plus lipopolysaccharide (LPS)-induced liver injury, HPS treatment significantly reduced hepatocyte death and inflammation response, and consequently attenuated the development of acute liver failure. In the model monkey of D-GalN-induced liver injury, HPS administration promoted hepatocytes proliferation, prevented hepatocyte apoptosis and oxidation stress, and resulted in amelioration of liver injury. Furthermore, the primary pharmacokinetic study showed natural HPS possesses favorable pharmacokinetics; the acute toxicity study indicated no significant changes in behavioral, clinical, or histopathological parameters of HPS-treated mice, implying the clinical potential of HPS. Our results suggest that exogenous HPS has protective effects on acute liver injury in both mice and monkeys. HPS or HPS analogues and mimetics may provide novel drugs for the treatment of acute liver injury.

Fabrication of a Silk Sericin Hydrogel System Delivering Human Lactoferrin Using Genetically Engineered Silk with Improved Bioavailability to Alleviate Chemotherapy-Induced Immunosuppression.

Chemotherapy is one of the main treatments for cancer; however, it usually causes severe atrophy of immune organs and self-immunity damage to patients. Human lactoferrin (hLF) is a multiple biofunctional protein in regulating the immune response and thus holds great promise to alleviate chemotherapy-caused immunosuppression. However, a sufficient hLF resource and efficient delivery of hLF remain a challenge. Here, we provide a useful strategy to simultaneously solve these two problems. A silk sericin hydrogel system delivering recombinant hLF (SSH-rhLF) was fabricated to alleviate the chemotherapeutic drug-caused side effects by rhLF-carrying silk cocoons, which were cost-effectively produced by a transgenic silkworm strain as the resource. SSH-rhLF with a uniform porous microstructural morphology, a dominant ß-sheet internal structure, adjustable concentration and sustainable release of the rhLF, and non-cytotoxicity properties was demonstrated. Interestingly, the sericin hydrogel showed effective protection of the rhLF from degradation in the stomach and small intestine, thus prolonging the bioactivity and bioavailability of rhLF. As a result, the oral administration of SSH-rhLF with a low rhLF dose showed significant therapeutic effects on enhancing the immune organs of cyclophosphamide (CTX)-treated mice by protecting the splenic follicles, promoting the expression of immunoregulatory factors, and recovering the intestinal flora family from CTX-induced imbalance, which were similar to those achieved by oral administration of a high dose of free hLF in the solution form. The results suggest that the strategy of producing rhLF silk cocoons via feeding transgenic silkworms overcomes well the shortage of rhLF resources, improves the bioavailability of oral rhLF, and alleviates the side effects of chemotherapeutic drugs on immune organs. The oral SSH-rhLF will be promising for applications in cancer chemotherapy and immunity enhancement of patients.

Discovery of a Long-Acting Parathyroid Hormone 1-34 Analogue to Treat Hypoparathyroidism.

Hypoparathyroidism (HP) is a rare disease with clinical manifestations of hypocalcemia and hyperphosphatemia, resulting from deficient or absent parathyroid hormone (PTH) secretion. Conventional treatment for patients with HP involves extensive calcium and vitamin D supplementation. In 2015, PTH1-84 was approved by the United States Food and Drug Administration as an adjunct for HP patients who cannot be well-controlled on conventional treatment. However, PTH1-84 therapy requires a daily injection, leading to poor patient compliance. The purpose of this study was to develop a long-acting PTH1-34 analogue by increasing its affinity to albumin. Three PTH1-34 variants were generated by substituting two of the three lysine (Lys) residues with arginine, reserving a single Lys as the modification site in each sequence. A series of side chains, containing fatty acid, deoxycholic acid, or biotin groups, were synthesized to modify these PTH1-34 variants by using a solid-liquid phase synthesis approach. In vitro bioactivity and albumin affinity tests were used to screen these new PTH1-34 analogues. Finally, Lys27-AAPC was selected from 69 synthesized analogues as a candidate therapeutic compound because it retained potency and exhibited a high albumin-binding capacity. In pharmacodynamic experiments, Lys27-AAPC demonstrated enhanced and prolonged efficacy in serum calcium elevating relative to PTH1-84. Moreover, a lyophilized powder for injection containing Lys27-AAPC was developed for further testing and represented a potential long-acting HP treatment.

Ackr3-Venus knock-in mouse lights up brain vasculature.

The atypical chemokine receptor 3, ACKR3, is a G protein-coupled receptor, which does not couple to G proteins but recruits ßarrestins. At present, ACKR3 is considered a target for cancer and cardiovascular disorders, but less is known about the potential of ACKR3 as a target for brain disease. Further, mouse lines have been created to identify cells expressing the receptor, but there is no tool to visualize and study the receptor itself under physiological conditions. Here, we engineered a knock-in (KI) mouse expressing a functional ACKR3-Venus fusion protein to directly detect the receptor, particularly in the adult brain. In HEK-293 cells, native and fused receptors showed similar membrane expression, ligand induced trafficking and signaling profiles, indicating that the Venus fusion does not alter receptor signaling. We also found that ACKR3-Venus enables direct real-time monitoring of receptor trafficking using resonance energy transfer. In ACKR3-Venus knock-in mice, we found normal ACKR3 mRNA levels in the brain, suggesting intact gene transcription. We fully mapped receptor expression across 14 peripheral organs and 112 brain areas and found that ACKR3 is primarily localized to the vasculature in these tissues. In the periphery, receptor distribution aligns with previous reports. In the brain there is notable ACKR3 expression in endothelial vascular cells, hippocampal GABAergic interneurons and neuroblast neighboring cells. In conclusion, we have generated Ackr3-Venus knock-in mice with a traceable ACKR3 receptor, which will be a useful tool to the research community for interrogations about ACKR3 biology and related diseases.

Enhancing the Pharmacokinetic Profile of Interleukin 2 through Site-Specific Conjugation to a Selective Small-Molecule Transthyretin Ligand.

Protein drugs hold great promise as therapeutics for a wide range of diseases. Unfortunately, one of the greatest challenges to be addressed during clinical development of protein therapeutics is their short circulation half-life. Several protein conjugation strategies have been developed for half-life extension. However, these strategies have limitations and there remains room for improvement. Here, we report a novel nature-inspired strategy for enhancing the in vivo half-life of proteins. Our strategy involves conjugating proteins to a hydrophilic small molecule that binds reversibly to the plasma protein, transthyretin. We show here that our strategy is effective in enhancing the pharmacokinetic and pharmacodynamic properties of human interleukin 2 in rats, potentially opening the door for more effective and safer cancer immunotherapies. To our knowledge, this is the first example of successful use of a small-molecule that not only extends the half-life but also maintains the smaller size, binding potency, and hydrophilicity of proteins.

Safety, Tolerability, and Pharmacokinetics of High-Volume Subcutaneous Crenezumab, With and Without Recombinant Human Hyaluronidase in Healthy Volunteers.

Compared with intravenous formulations, subcutaneous (s.c.) formulations of therapeutic monoclonal antibodies may provide increased patient access and more convenient administration options, although historically high-volume s.c. administration (> 10-15 mL) has been challenging. We report results from two phase I studies in healthy participants (GP29523 and GP40201) that evaluated s.c. crenezumab, an anti-Aß monoclonal antibody in development for individuals at risk for autosomal-dominant Alzheimer's disease. GP29523 assessed safety, tolerability, and pharmacokinetics (PK) in 68 participants (aged 50-80 years) who received single ascending doses (600-7,200 mg) of crenezumab or placebo (4-40 mL). GP40201 assessed safety, tolerability, and PK in 72 participants (aged 18-80 years) who received different combinations of dose (1,700-6,800 mg), infusion volume (10-40 mL), and flow rate (2-4 mL/minute), with/without recombinant human hyaluronidase (rHuPH20). There were no serious or dose-limiting adverse events in either study. There were no meaningful differences in pain scores among reference placebo (4 mL), test placebo (4-40 mL), or crenezumab (600-7,200 mg) in GP29523, or across treatments with varying infusion volume, flow rate, dose, or rHuPH20 co-administration or concentration in GP40201. Transient erythema was the most common infusion site reaction in both studies. In GP40201 at volumes of ≥ 20 mL, rHuPH20 co-administration appeared to reduce infusion site swelling incidence, but, in some cases, was associated with larger areas of infusion site erythema. Crenezumab exhibited approximately dose-proportional PK, and s.c. bioavailability was 66% and independent of dose or rHuPH20 co-administration. High-dose, high-concentration, high-volume s.c. crenezumab formulated with/without rHuPH20 was well-tolerated in healthy participants, with an acceptable safety profile.

A selective and sensitive UPLC-ESI-MS/MS method for quantification of Pegylated Interferon Alfa-2b in human serum using signature peptide-based quantitation.

A sensitive method for determination of PEG-IFN-α-2b in human serum was developed using ultra performance liquid chromatography aligned with tandem mass spectrometric detection. A two-treatment, two-period, cross over study was conducted to establish bioequivalence between a test and reference formulation and the method was successfully applied to the quantification of PEG-IFN-α-2b in serum samples of this clinical study. The sample concentrations obtained from LC-MS/MS technique were compared with the concentrations obtained from ELISA technique. PEG-IFN-α-2b was isolated from serum using protein precipitation technique with isopropyl alcohol followed by overnight tryptic digestion. The signature peptide formed as result of tryptic digestion was separated on a chromatograph and detected using a mass detector. The mass transition ion-pair of m/z 741.3 → 1047.1 for PEG-IFN-α-2b and m/z 387.4 → 205.2 for internal standard were used for MS/MS detection. The sample extraction involves a simple protein precipitation method followed by tryptic digestion of the supernatant and further sample cleanup was not needed. The method has been validated over a linear range of 1.028-3200 ng/mL with a correlation coefficient ≥ 0.99. The precision (%RSD) was 5.52 to 7.90 and accuracy (%RE) was within -1.80 to 1.68. The total run time was 22.0 min. The sensitivity of LC-MS/MS method was 1.0 ng/ml which was found to be more sensitive than ELISA and resulted in improving the overall study data by being able to quantify all the samples without any below LOQ results helping to further improve the pharmacokinetic modeling. This improved method is a promising anti-body free LC-MS/MS based methodology for estimation of PEG-IFN-α-2b in human serum and may be applied for other such pegylated molecules.

Safety, PK/PD and preliminary anti-tumor activities of pegylated recombinant human arginase 1 (BCT-100) in patients with advanced arginine auxotrophic tumors.

Background The study determined the safety, pharmacokinetics/pharmacodynamics (PK/PD), and recommended Phase II dose of BCT-100 for arginine auxotrophic tumours in a non-Chinese population. Methods This is a Phase I, 3 + 3 dose-escalation, open-label, multi-centre study in two arginine auxotrophic cancers-Malignant Melanoma (MM) and Castration Resistant Prostate Cancer (CRPC). Patients were enrolled to receive weekly intravenous BCT-100. The dose cohorts were respectively 0.5 mg/kg, 1.0 mg/kg, 1.7 mg/kg and 2.7 mg/kg. Results There were 14 MM and 9 CRPC patients, 16 males and 7 females with a median age of 71. No dose-limiting toxicities were reported. Among all the AEs, 18 were drug-related (mostly were Grade 1). Although there were individual variations in PKs amongst the patients in each cohort, the median arginine level was maintained at 2.5 µM (lower limit of quantification) in all 4 cohorts of patients after the second BCT-100 injection. Therapeutic Arginine Depletion was found in the 1.7 and 2.7 mg/kg/week cohorts when anti-tumor activities were observed. The two cohorts had a similar AUC (20,947 and 19,614 h*µg/ml respectively). Since the 2.7 mg/kg/week cohort had a more sustained arginine depletion for 2 weeks, the 2.7 mg/kg/week dose is chosen as the future phase II dose. There were two complete remissions (1 MM & 1 CRPC), 1PR (MM) and 2 stable diseases with a disease control rate (CR + PR + SD) of 5/23 (22%). Conclusions BCT-100 is safe in a non-Chinese population and has anti-tumor activities in both MM and CRPC. Weekly BCT-100 at 2.7 mg/kg is defined as the optimal biological dose for future clinical phase II studies.

Dispersive effects and focused biodistribution of recombinant human hyaluronidase PH20: A locally acting and transiently active permeation enhancer.

BACKGROUND: Recombinant human hyaluronidase PH20 (rHuPH20) facilitates the dispersion and absorption of subcutaneously administered therapeutic agents. This study aimed to characterize the transient, local action of rHuPH20 in the subcutaneous (SC) space using focused biodistribution and dye dispersion studies conducted in mice. MATERIALS AND METHODS: To evaluate the biodistribution of rHuPH20, mice were intradermally administered rHuPH20 (80 U). The enzymatic activity of rHuPH20 was analyzed in the skin, lymph nodes, and plasma. Animal model sensitivity was determined by intravenous administration of rHuPH20 (80 U) to the tail vein. To evaluate local dispersion, mice received an intradermal injection of rHuPH20 followed by an intradermal injection of Trypan Blue dye at a contralateral site 45 minutes later. Dye dispersion was measured using a digital caliper. RESULTS: After intradermal rHuPH20 injection, enzymatic activity was detected within the skin near the injection site with levels decreasing rapidly after 15 minutes. There was no clear evidence of systemic exposure after administration of rHuPH20, and no discernible rHuPH20 activity was observed in lymph or plasma as a function of time after dosing. In the dye dispersion study, delivery of rHuPH20 at one site did not impact dye dispersion at a distal skin site. CONCLUSION: These observations support the classification of rHuPH20 as a transiently active and locally acting permeation enhancer.