scholarly journals PASylated Thymosin α1: A Long-Acting Immunostimulatory Peptide for Applications in Oncology and Virology

2020 ◽  
Vol 22 (1) ◽  
pp. 124
Author(s):  
Uli Binder ◽  
Arne Skerra

Thymosin α1 (Tα1) is an immunostimulatory peptide for the treatment of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections and used as an immune enhancer, which also offers prospects in the context of COVID-19 infections and cancer. Manufacturing of this N-terminally acetylated 28-residue peptide is demanding, and its short plasma half-life limits in vivo efficacy and requires frequent dosing. Here, we combined the PASylation technology with enzymatic in situ N-acetylation by RimJ to produce a long-acting version of Tα1 in Escherichia coli at high yield. ESI-MS analysis of the purified fusion protein indicated the expected composition without any signs of proteolysis. SEC analysis revealed a 10-fold expanded hydrodynamic volume resulting from the fusion with a conformationally disordered Pro/Ala/Ser (PAS) polypeptide of 600 residues. This size effect led to a plasma half-life in rats extended by more than a factor 8 compared to the original synthetic peptide due to retarded kidney filtration. Our study provides the basis for therapeutic development of a next generation thymosin α1 with prolonged circulation. Generally, the strategy of producing an N-terminally protected PASylated peptide solves three major problems of peptide drugs: (i) instability in the expression host, (ii) rapid degradation by serum exopeptidases, and (iii) low bioactivity because of fast renal clearance.

Pharmaceutics ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 904
Author(s):  
Irin Tanaudommongkon ◽  
Asama Tanaudommongkon ◽  
Xiaowei Dong

Most antiretroviral medications for human immunodeficiency virus treatment and prevention require high levels of patient adherence, such that medications need to be administered daily without missing doses. Here, a long-acting subcutaneous injection of lopinavir (LPV) in combination with ritonavir (RTV) using in situ self-assembly nanoparticles (ISNPs) was developed to potentially overcome adherence barriers. The ISNP approach can improve the pharmacokinetic profiles of the drugs. The ISNPs were characterized in terms of particle size, drug entrapment efficiency, drug loading, in vitro release study, and in vivo pharmacokinetic study. LPV/RTV ISNPs were 167.8 nm in size, with a polydispersity index of less than 0.35. The entrapment efficiency was over 98% for both LPV and RTV, with drug loadings of 25% LPV and 6.3% RTV. A slow release rate of LPV was observed at about 20% on day 5, followed by a sustained release beyond 14 days. RTV released faster than LPV in the first 5 days and slower than LPV thereafter. LPV trough concentration remained above 160 ng/mL and RTV trough concentration was above 50 ng/mL after 6 days with one subcutaneous injection. Overall, the ISNP-based LPV/RTV injection showed sustained release profiles in both in vitro and in vivo studies.


Author(s):  
Alex E. Roher ◽  
Kenneth C. Palmer ◽  
John Capodilupo ◽  
Arun R. Wakade ◽  
Melvyn J. Ball

ABSTRACT:Purification of amyloid plaque core proteins (APCP) from Alzheimer's disease brains to complete homogeneity and in high yield permitted its chemical fractionation and characterization of its components. APCP is mainly made of β-amyloid (βA) and an assortment of glycoproteins (accounting for 20%) rich in carbohydrates compatible with N-and O-linked saccharides. When added to tissue culture of sympathetic and sensory neurons APCP and βA inhibited neuritic sprouting, a reversible phenomenon at low doses. Higher concentrations of both substances kill the neurons in culture. APCP is significantly more toxic than βA, suggesting the minor components may play an important role in increasing the toxicity of βA. If the observed toxic effects of APCP in situ are occurring in vivo during the course of AD, then the accumulation of these extracellular proteins could be largely responsible for some of the neuronal death observed in this neuropathology.


2017 ◽  
Vol 62 (2) ◽  
Author(s):  
Alexander J. Lepak ◽  
Miao Zhao ◽  
Brian VanScoy ◽  
Paul G. Ambrose ◽  
David R. Andes

ABSTRACT Echinocandins are important in the prevention and treatment of invasive candidiasis but limited by current dosing regimens that include daily intravenous administration. The novel echinocandin CD101 has a prolonged half-life of approximately 130 h in humans, making it possible to design once-weekly dosing strategies. The present study examined the pharmacodynamic activity of CD101 using the neutropenic invasive candidiasis mouse model against select Candida albicans (n = 4), C. glabrata (n = 3), and C. parapsilosis (n = 3) strains. The CD101 MIC ranged from 0.03 to 1 mg/liter. Plasma pharmacokinetic measurements were performed using uninfected mice after intraperitoneal administration of 1, 4, 16, and 64 mg/kg. The elimination half-life was prolonged at 28 to 41 h. Neutropenic mice were infected with each strain by lateral tail vein injection, treated with a single dose of CD101, and monitored for 7 days, at which time the organism burden was enumerated from the kidneys. Dose-dependent activity was observed for each organism. The pharmacokinetic/pharmacodynamic (PK/PD) index of the area under the concentration-time curve over 24 h in the steady state divided by the MIC (AUC/MIC index) correlated well with efficacy (R 2, 0.74 to 0.93). The median stasis 24-h free-drug AUC/MIC targets were as follows: for C. albicans, 2.92; for C. glabrata, 0.07; and for C. parapsilosis, 2.61. The PK/PD targets for 1-log10 kill endpoint were 2- to 4-fold higher. Interestingly, the aforementioned PK/PD targets of CD101 were numerically lower for all three species than those of other echinocandins. In summary, CD101 is a promising, novel echinocandin with advantageous pharmacokinetic properties and potent in vivo pharmacodynamic activity.


Peptides ◽  
1994 ◽  
pp. 837-839
Author(s):  
C. Basava ◽  
L. M. Selk ◽  
R. Basava ◽  
M. Gardner ◽  
S. Parker ◽  
...  

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2333-2333
Author(s):  
Pamela R. Westmark ◽  
Pansakorn Tanratana ◽  
John P. Sheehan

Abstract Introduction Hemophilia B is an X-linked genetic disorder characterized by defective factor IX activity. Recombinant factor IX (rFIX) is employed as protein replacement for the treatment and prophylaxis of bleeding episodes. Antithrombin is the primary plasma inhibitor of activated factor IX (FIXa), and inhibition is enhanced by heparin/heparan sulfate. We hypothesize that selective disruption of protease interactions with heparin and antithrombin via mutations in the respective heparin- and antithrombin-binding exosites may enhance rFIX(a) efficacy by prolonging protease half-life in vivo. Aim To assess the effect of mutations in the FIX(a) heparin- and antithrombin-binding exosites on traditional coagulant activity, thrombin generation, and protease half-life in human plasma. Methods Human FIX cDNA constructs with alanine substitutions (chymotrypsinogen numbering) in the heparin exosite (K126A, K132A, K126A/K132A), antithrombin exosite (R150A), or both (K126A/R150A, K132A/R150A, K126A/K132A/R150A) were expressed in HEK293 cell lines. Recombinant zymogens were purified from conditioned media, and a portion activated to protease with human factor XIa. Zymogen and protease forms were characterized in APTT-based clotting assays, and tissue factor (TF) and FIXa-initiated thrombin generation (TG) assays in pooled human FIX-deficient plasma, respectively. Comparisons were made with human plasma-derived factor IX (pFIX) and recombinant FIX wild type (WT). Protease half-life in pooled, citrated human plasma was determined using a novel assay that detects FIXa activity by TG response. Results Zymogen coagulant activities (% WT ± S.E) were: pFIX 105.2 ± 2.8, WT 100 ± 7.1, K132A/R150A 75.8 ± 3.4, K126A 63.3 ± 2.3, R150A 62.4 ± 4.0, K132A 30.9 ± 1.0, K126A/R150A 27.0 ± 2.1, K126A/K132A 20.6 ± 9.2, and K126A/K132A/R150A 7.3 ± 3.8. Similarly, protease coagulant activities were: WT 100 ± 6.1, pFIXa 98.4 ± 11.4, K132A 91.4 ± 1.6, K132A/R150A 84.9 ± 2.8, R150A 77.1 ± 5.8, K126A 39.5 ± 2.4, K126A/R150A 25.3 ± 2.8, K126A/K132A/R150A 10.9 ± 0.6, and K126A/K132A 9.3 ± 0.6. In contrast to their relative coagulant activities, FIX K126A (1.9-fold), R150 (1.6-fold), and K132A/R150A (1.3-fold) supported increased peak thrombin concentrations during TF-triggered TG; pFIX, FIX K132A and K126A/R150A were similar to WT; and FIX K126A/K132A/R150A (0.6-fold) and K126A/K132A (0.2-fold) demonstrated marked reductions in peak thrombin relative to WT. In the FIXa-initiated TG assay, FIXa K126A/R150A and K132A/R150A (1.5-fold) demonstrated significantly increased peak thrombin concentrations; pFIXa, FIXa K132A, R150A, and K126A (0.8-1.0 fold) were similar to WT; while FIXa K126A/K132A and K126A/K132A/R150A demonstrated markedly reduced (0.2-0.3 fold) and delayed peak thrombin concentrations. In pooled, citrated FIX-deficient plasma, FIXa WT (40.9 ± 1.4 min) and K126A/K132A (37.2 ± 0.7 min) demonstrated similar half-lives, while FIXa R150A, K126A/R150A, and K132A/R150A all had half-lives > 2 hr. Conclusions Single exosite mutations resulted in mild to moderate reductions in coagulant activity, while the double mutation in the heparin exosite (K126A/K132A) markedly reduced activity, likely due to a synergistic effect on cofactor binding. Traditional coagulant activity did not accurately represent the ability of the mutant proteins to support thrombin generation. Despite variable reductions in coagulant activity, FIX K126A, K132A, R150A, K126A/R150A and K132A/R150A supported levels of plasma thrombin generation that were equal to or greater than FIX WT. The plasma half-life of FIXa WT activity was remarkably lengthy, and while mutations in the heparin exosite had negligible effects, R150A in the antithrombin exosite substantially increased protease half-life, consistent with a primary role for antithrombin in the plasma inhibition of FIXa. Thus, single exosite mutations did not significantly disrupt the procoagulant function of human FIX(a), and combined exosite mutations (K126A/R150A and K132A/R150A) maintain or enhance plasma thrombin generation while disrupting exosite-mediated regulatory mechanisms. The combination of intact procoagulant function with disruption of antithrombin- and heparin-mediated regulation of FIX(a) will potentially enhance in vivo recovery, prolong plasma half-life, and enhance the efficacy of hemophilia B replacement therapy. Disclosures: Sheehan: Novo Nordisk Access to Insight Basic Research Grant: Research Funding; Bayer Hemophilia Awards Program: Research Funding; Diagnostica Stago: reagents, reagents Other.


Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4670-4670 ◽  
Author(s):  
Lior Binder ◽  
Ahuva Bar-Ilan ◽  
Malka Hoffman ◽  
Gili Hart

Abstract Introduction: OPKO Biologics is a clinical-stage public company developing bio-better long-acting versions of existing therapeutic proteins, utilizing a technology termed CTP. The technology involves fusion of the C-terminal peptide of hCG to a target protein. The aim of this work was to comprehensively assess the feasibility of intravenous (IV) or subcutaneous (SC) administration of FVIIa-CTP (MOD-5014) utilizing the most relevant in vivo pre-clinical models, and to characterize the FVIIa-CTP mechanism of action in preparation for an on-going clinical study. Methods: FVII-CTP was expressed in CHO cells, purified and activated utilizing a CTP-specific purification process. FVIIa-CTP's pharmacokinetics (PK), pharmacodynamics (PD), long-term hemostatic effect and safety parameters were extensively characterized following SC and IV administration in transient FVII-/- rats and FVIII-/- mice. In addition, the long-term hemostatic effect of FVIIa-CTP was evaluated following a bleeding challenge and compared to commercial rFVIIa. Finally, interaction with co-factors, activity, and the off-target effect of FVIIa-CTP was comprehensively characterized. Results: The studies demonstrated that FVIIa-CTP provides long-term exposure (AUC) and half-life that are significantly superior to those of rFVIIa, and consistent with the prolonged half-life of FVIIa-CTP (at an average of 3- and 5-fold, respectively) when compared to IV or SC administration of FVIIa. In addition, a 30% increase in bioavailability was observed relative to commercial FVIIa. A profound improvement in clotting parameters and survival rate following TVT, as well as a reduction of bleeding duration and intensity in tail-clip studies were obtained for both routes of administration for up to 48 hours. Moreover, the safety profile of FVIIa-CTP was further confirmed. Conclusion: Attachments of CTP to FVIIa led to a pronounced enhancement of PK and PD, increased exposure as reflected by AUC, elevated half-life, and improved recovery in mice, rats and pigs following SC and IV administration. FVIIa-CTP injection resulted in an improved bioavailability that translated to a marked in vivo hemostatic effect. Our data suggest that CTP-fused FVIIa can potentially provide a novel approach for IV or SC prophylactic treatment of hemophilic patients (both pediatric and adult), with the major benefit of significant improvement in quality of life. Disclosures No relevant conflicts of interest to declare.


Author(s):  
Bo Liang ◽  
Xudong Yuan ◽  
Gang Wei ◽  
Wei Wang ◽  
Ming Zhang ◽  
...  

AbstractTo curb the spread of SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, we characterize the virucidal activity of long-acting Povidone Iodine (PVP-I) compositions developed using an in-situ gel forming technology. The PVP-I gel forming nasal spray (IVIEW-1503) and PVP-I gel forming ophthalmic eye drop (IVIEW-1201) rapidly inactivated SARS-CoV-2, inhibiting the viral infection of VERO76 cells. No toxicity was observed for the PVP-I formulations. Significant inactivation was noted with preincubation of the virus with these PVP-I formulations at the lowest concentrations tested. It has been demonstrated that both PVP-I formulations can inactivate SARS-CoV-2 virus efficiently in both a dose-dependent and a time-dependent manner. These results suggest IVIEW-1503 and IVIEW-1201 could be potential agents to reduce or prevent the transmission of the virus through the nasal cavity and the eye, respectively. Further studies are needed to clinically evaluate these formulations in early-stage COVID-19 patients.


1984 ◽  
Vol 51 (01) ◽  
pp. 012-015 ◽  
Author(s):  
C T Smit Sibinga ◽  
S M G J Daenen ◽  
G W van Imhoff ◽  
A Maas ◽  
P C Das

SummaryNew approaches and techniques for improving source material collection and Factor VIII production at Blood Bank level have been reported recently.Heparin has been shown to be of importance in increasing yields and stability of FVIII in the purification and concentration process. Work has been done to develop on a routine scale the heparin double cold precipitation technique for the production of a freeze-dried high yield purified FVIII concentrate.The product has been tested clinically in 4 severe hemophilia A patients for recovery, half-life and acute side-effects, using two dosages over 8 infusions. There was no significant difference between the two dosages. Mean recovery 99.1% and mean halflife 8 hr, ranging from 6.5 to 10.3 hr.No side-effects were observed.These results justify further exploration of the potential of heparin for high yield purified FVIII production.


1979 ◽  
Author(s):  
P. Han ◽  
A.G.G. Turpie ◽  
E. Genton

It is important to differentiate betwteen extravascular (autoimmune thrombocytopenia, ATP) and intravascular (thrombotic thrombocytopenia, TTP) platelet destruction in thrombocytopenia. Betathromboglobulin (BTG), a platelet-specific protein with a plasma half life of 20 minutes is released in-vivo from platelets by various stimuli and may reflect platelet activation or destruction. BTG concentration can be measured in plasma usin a radioimmunoassay to a sensitivity of 1 ng/ml., (nomal 28.0 ± 8.0 ng/ml., n = 70). Plasma BTG was measured in 3 patients with ATP (platelet counts: 17, 20, 16 x 109/L) and 2 patients with TTP (platelet counts: 20, 40 x 109/L). In ATP, BTG was normal (22, 11, 17 ng/ml.) and in TTP, BTG was elevated (80, 72 ng/ml.). Plasma BTG remained normal in ATP after treatment. BTG remained elevated in TTP (120 ng/ml.) even when the platelet count became normal (220 x 109/L) but while fragmented RBC were still present and became normal (21 ng/ml.) on complete recovery. These data suggest that plasma BTG may be useful in differenfiating extravascu1ar from intravascular platelet destruction by detecting increased concentrations of BTG in plasma.


2019 ◽  
Vol 103 ◽  
pp. 109730 ◽  
Author(s):  
Monika Yadav ◽  
Ana Guzman-Aranguez ◽  
Maria J. Perez de Lara ◽  
Mandeep Singh ◽  
Joga Singh ◽  
...  

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