IL-8 was radiolabeled with 125I via Bolten-Hunter agent and the distribution of 125I-IL-8 in mice was determined in order to understand IL-8 behaviors in vivo. The percentage of 125I-IL-8 in blood, heart, liver, lung, kidneys, bone and spleen was obtained. The half-clearance of fast phase T1/2 alpha and slow phase T1/2 beta were 0.32 h and 8.01 h respectively. Most of 125I-IL-8 was excluded by kidneys.
Animals ; Interleukin-8 ; pharmacokinetics ; Iodine Radioisotopes ; Male ; Mice ; Recombinant Proteins ; pharmacokinetics ; Tissue Distribution

Interleukin-1 receptor antagonist (IL-1ra), a member of IL-1 family, is a naturally occurring IL-1 inhibitor as "receptor antagonist", which blocks biological responses mediated by IL-1. Recombinant human IL-1ra (rhIL-1ra, Kineret) was introduced in clinical trials involving patients with RA. Between 2001 to approximately 2002, rhIL-1 ra was approved by the US Food and Drug Administration and the European Agency for the Evaluation of Medicine Procedure. Unfortunately, 10,000 to 100,000-fold excess amounts of IL-1ra are needed to relieve disease because minimal IL-1 can induce complete biological responses, and the dosage of 100 to approximately 150mg/day in a RA patient is so big that it greatly influence patients' physical, psychological and economical situation. In this study, IL-1ra mutants were established by site-specific mutagenesis to improve its stability. The sites of mutagenesis included R6 K7-AA,R93 K94-AA and K97 R98-AA. IL-1ra and its mutants were expressed in E. coli BL21 (DE3) using pTIG-Trx expressing system with the induction of IPTG. The recombinant proteins were purified by Ni2+ chelate chromatography and Sephadex G75 gel filtration chromatography. The activity of mutants is as high as IL-1ra. We characterized the pharmacokinetic profile of IL-1ra and its mutants. The third mutant's half life is 2.26 times than wt IL-1ra. The study has provided some approaches and experience for further research to improve the metabolism stability of IL-1ra.
Animals ; Escherichia coli ; genetics ; metabolism ; Female ; Humans ; Interleukin 1 Receptor Antagonist Protein ; biosynthesis ; genetics ; pharmacokinetics ; Mutagenesis, Site-Directed ; methods ; Mutant Proteins ; biosynthesis ; pharmacokinetics ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacokinetics

There are many factors that can influence the pharmacokinetics (PK) of a mAb or Fc-fusion molecule with the primary determinant being FcRn-mediated recycling. Through Fab or Fc engineering, IgG-FcRn interaction can be used to generate a variety of therapeutic antibodies with significantly enhanced half-life or ability to remove unwanted antigen from circulation. Glycosylation of a mAb or Fc-fusion protein can have a significant impact on the PK of these molecules. mAb charge can be important and variants with pI values of 1-2 unit difference are likely to impact PK with lower pI values being favorable for a longer half-life. Most mAbs display target mediated drug disposition (TMDD), which can have significant consequences on the study designs of preclinical and clinical studies. The PK of mAb can also be influenced by anti-drug antibody (ADA) response and off-target binding, which require careful consideration during the discovery stage. mAbs are primarily absorbed through the lymphatics via convection and can be conveniently administered by the subcutaneous (sc) route in large doses/volumes with co-formulation of hyaluronidase. The human PK of a mAb can be reasonably estimated using cynomolgus monkey data and allometric scaling methods.
Absorption, Physiological ; Animals ; Antibodies, Monoclonal ; pharmacokinetics ; Dose-Response Relationship, Immunologic ; Humans ; Receptors, Fc ; metabolism ; Recombinant Fusion Proteins ; pharmacokinetics ; Tissue Distribution

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.
Adult ; Humans ; Middle Aged ; Blood Coagulation Factors ; Factor VIII/pharmacokinetics* ; Hemophilia A ; Hemorrhage ; Recombinant Proteins/pharmacokinetics* ; Adolescent ; Young Adult

PEGylation is considered one of the most successful techniques to improve the characteristics of protein drugs including to increase the circulating half-life of proteins in blood and to decrease their immunogenicity and antigenicity. One known PEG modification method is to attach PEG to the free amino group, typically at lysine residues or at the N-terminal amino acid with no selectivity, resulting in a heterogeneous product mixture. This lack of selectivity can present problems when a therapeutic PEGylated protein is being developed, because predictability of activity and manufacturing reproducibility are needed for regulatory approval. Enzymatic PEGylation of proteins is one route to overcome this limitation. Transglutaminases (TGase) are enzyme candidates for site-specific PEGylation. We use human interferon alpha 2a (IFN α2a) as a test case, and predict that the potential modification residues are Gln101 by computational approach as it contains 12 potential PEGylation sites. IFN α2a was PEGylated by Y shaped PEG40k-NH2 mediated by microbial transglutaminase. Our results show that the microbial transglutaminase mediated PEGylation of IFN α2a was site-specific only at the site of Gln101 in IFN α2a, yielding the single mono-conjugate PEG-Gln101-IFN α2a with a mass of 59 374.66 Da. Circular dichroism studies showed that PEG-Gln101-IFN α2a preserved the same secondary structures as native IFN α2a. As expected, the bioactivity and pharmacokinetic profile in rats of PEG-Gln101-IFN α2a revealed a significant improvement to unmodified IFN α2a, and better than PEGASYS.
Animals ; Antiviral Agents ; Humans ; Interferon alpha-2 ; metabolism ; Interferon-alpha ; biosynthesis ; pharmacokinetics ; Polyethylene Glycols ; pharmacokinetics ; Protein Structure, Secondary ; Rats ; Recombinant Proteins ; biosynthesis ; pharmacokinetics ; pharmacology ; Reproducibility of Results ; Transglutaminases ; metabolism

OBJECTIVETo investigate the intracellular trafficking pathway of fusion protein epidermal growth factor-adenovirus early region 4 open reading frame 4 protein (EGF-E4orf4) internalized via epidermal growth factor (EGF) receptor and its affectivity on extracellular signal-regulated kinase (ERK) phosphorylation.

METHODSMDA-MB-231 and BGC823 cells were incubated with fluorescein isothiocyanate-EGF-E4orf4 or EGF at different time points. The specific molecular mark of early endosome or late lysosome was labeled by indirect immunofluorescence, and then colocalization staining was observed using confocal laser microscopy. The levels of ERK phosphorylation were detected by Western blot.

RESULTSThe fluorescent signal of fusion protein EGF-E4orf4 accumulated within the cells and congregated to the perinuclear region. A nucleus localization of the fusion protein was only at MDA-MB-231 cell. Colocalization of EGF-E4orf4 with early endosome/late lysosome was observed. EGF-E4orf4 stimulated ERK phosphorylation, which was most obvious 10 minutes after stimulation, and then gradually attenuated, which was similar to EGF stimulation but with a less decrease.

CONCLUSIONSInternalized EGF-E4orf4 can be slowly degraded via endosome-lysosome pathway. The action features of EGF-E4orf4 are remarkably different between MDA-MB-231 and BGC823 cells, which may help explain the differences in its anti-tumor potency and in the special selectivity toward different tumor cells.

Adenovirus E4 Proteins ; pharmacokinetics ; Cell Line, Tumor ; Epidermal Growth Factor ; pharmacokinetics ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Humans ; Phosphorylation ; drug effects ; Protein Transport ; Recombinant Fusion Proteins ; pharmacokinetics ; Viral Proteins ; pharmacokinetics

To reduce the serum clearance of interferon alpha2b, a chimeric gene encoding an human serum albumin(HSA)--human interferon alpha2b(IFNalpha2b) fusion protein was overexpressed in Pichia pastoris. After fermentation in a 5L bioreactor, the fusion protein, capable of cross-reacting with anti-IFN alpha and anti-HSA antibody, was purified from the culture of the recombinant yeast by ultrafiltration, blue Sepharose affinity, phenyl hydrophobic interaction and Q ion exchange chromatography. Its IFNa2b moiety exhibits antiviral activity similar to that of recombinant human IFNa2b. In Cynomolgus monkeys model, The fusion protein was detectable in plasma, even 336h after a single does of 90 microg/kg injection intravenously or subcutaneously. The elimination phase half-life of the fusion protein was 101h after intravenous injection and 68.2h after subcutaneous injection. Its Subcutaneous bioavailability was 67.9%. The enhanced pharmacokinetics of interferon a2b fused to human serum albumin suggest its promissing application in clinic medicine.
Animals ; Bioreactors ; microbiology ; Fermentation ; Humans ; Interferon-alpha ; biosynthesis ; genetics ; Macaca fascicularis ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacokinetics ; Recombinant Proteins ; Serum Albumin ; biosynthesis ; genetics

Double antibody sandwich-type ELISA was used to detect rhG-CSF in serum to study the pharmacokinetics of rhG-CSF, PEG-rhG-CSF and rHSA-hG-CSF in mice and to confirm that PEGlyation and albumin fusion of rhG-CSF technology can prolong half-life of G-CSF. Pharmacokinetic parameters were calculated with 3P87 software. T1/2 s of rhG-CSF, PEG-rhG-CSF and rHSA-hG-CSF are 2. 1 , 14.2 and 10. 6 h, respectively. T1/2 s of PEG- rhG-CSF and rHSA-hG-CSF are 7, 5 times than T1/2 s of rhG-CSF, respectively. Tpeak s of PEG-rhG-CSF and rHSA-hG-CSF are 15, 13 times than Tpeak of rhG-CSF, respectively. The result of ELISA indicates that PEGlyation and albumin fusion of rhG-CSF technology can prolong half-life of G-CSF.
Animals ; Area Under Curve ; Enzyme-Linked Immunosorbent Assay ; methods ; Granulocyte Colony-Stimulating Factor ; blood ; chemistry ; pharmacokinetics ; Half-Life ; Humans ; Male ; Mice ; Mice, Inbred ICR ; Polyethylene Glycols ; chemistry ; Recombinant Fusion Proteins ; blood ; chemistry ; pharmacokinetics ; Recombinant Proteins ; Serum Albumin ; chemistry

To prolong serum half-life of human Erythropoietin for better efficacy, a new form of recombinant human erythropoietin (rhEpo-L-Fc) was generated by fusion of a full length human erythropoietin gene and the Fc fragment of human IgG1 with flexible linker sequence. The fusion gene rhEPO-L-Fc was constructed by PCR, then inserted into expression vector pOptiVEC-TOPO, and expressed in Chinese Hamster Ovary cells deficient in the DHFR enzyme(CHO-dhfr-). The chimeric protein was purified by Protein A affinity chromatography, showed expected molecular weight and demonstrated a similar bioactivity compared to that of the native recombinant human erythropoietin (rhEPO) in an EPO-dependent cell-based assay. In vivo pharmacokinetic studies showed that the rhEPO-L-Fc had an elimination half-life of 27 h. In vivo efficacy studies showed that a single dose administration of rhEPO-L-Fc in rats increased the reticulocyte number in the peripheral blood significantly. These results demonstrated that the new engineered rhEPO-L-Fc may become alternative therapeutic approach to extend the half-time of rhEPO to treat anemia.
Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Erythropoietin ; chemistry ; genetics ; metabolism ; pharmacokinetics ; Female ; Humans ; Immunoglobulin Fc Fragments ; biosynthesis ; genetics ; Male ; Rats ; Rats, Sprague-Dawley ; Recombinant Fusion Proteins ; biosynthesis ; chemistry ; genetics ; pharmacokinetics ; Recombinant Proteins

AIMTo investigate the pharmacokinetic characteristics of recombinant human acidic fibroblast growth factor (rhaFGF) after external use in rabbits.

METHODS125I-rhaFGF 180 U.cm-2 was daubed to normal skin and scathed skin in rabbits. The radioactivity and paper chromatography were used to determine the 125I-concentrations and distribution in plasma and organs at different times.

RESULTSThe plasma concentration of 125I-rhaFGF increased rapidily, and reach peak plasma level (73.03 pg.mL-1) thirty minutes after administration. Then the concentration of 125I-rhaFGF decreased quickly after thirty minutes, and approached to zero after three hours. Highest radioactivity accumulated in the skin, followed by kidney, lowest in the brain 96 h after administration.

CONCLUSIONrhaFGF can not be absorbed from the normal skin, whereas a small amount of rhaFGF can be absorbed through scathed skin. The t1/2 of rhaFGF in plasma was very short. Cumulative effect of rhaFGF was not observed. Absorbed rhaFGF showed high affinity to skin, and can be distributed to skin far from the site of administration.

Administration, Cutaneous ; Animals ; Female ; Fibroblast Growth Factor 1 ; administration & dosage ; pharmacokinetics ; Male ; Rabbits ; Recombinant Proteins ; administration & dosage ; pharmacokinetics ; Skin ; injuries ; metabolism ; Skin Absorption ; Tissue Distribution