Mod-5014, a Novel Long-Acting FVIIa Proposing An Improved Prophylactic and On Demand Treatment For Hemophilic Patients Following SC and IV Administration – Comprehensive In -Vitro and In -Vivo evaluation In Preparation For Clinical Studies

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3578-3578
Author(s):  
Gili Hart ◽  
Oren Hershkovitz ◽  
Ahuva Bar Iilan ◽  
Miri Zakar ◽  
Lior Binder ◽  
...  
Keyword(s):  
Clinical Studies ◽  
Hemostatic Effect ◽  
On Demand ◽  
Long Acting ◽  
Tail Clip ◽  
In Vitro Interactions ◽  
Physiological Inhibitors

Abstract Introduction Prolor Biotech Inc. is a clinical stage public company developing biobetter long acting versions of existing therapeutic proteins utilizing a technology termed CTP. The technology involves fusion of the C terminus peptide of hCG to one or both ends of the target protein. The technology was clinically validated and proven as a safe and efficient way for prolonging the half-lives of several therapeutic proteins while maintaining their biological activity. The aim of this extensive study was to characterize the in vitro potency as well as the in –vitro interactions of MOD-5014 (FVIIa-CTP ) with physiological inhibitors and cofactors and to determine its pharmacokinetic (PK), pharmacodynamic (PD) and long term hemostatic effects in the relevant hemophilic animal models following IV and SC administrations as moving forward into clinical studies . Methods FVII-CTP was expressed in CHO cells, purified and activated utilizing a CTP specific purification process. MOD-5014 interactions with physiological inhibitors and cofactors was characterized in –vitro utilizing SPR and ex-vivo assays ( TEG and TG). In order to assess the long term in vivo effect , MOD-5014 was administered to warfarin treated rats or FVIII-/- mice, and following IV and SC injection the PK and PD profiles were determined as well as coagulation parameters (PT,aPTT ,TG and FVIIa activity) at a time dependent manner. In addition, the long term hemostatic effect was evaluated following bleeding challenge by tail clip assay and tail vein transection as compared to commercial rFVIIa. Results MOD-5014 in vitro activity was comparable to commercial FVIIa. PT , thrombin generation , PK and PD parameters following IV and SC administration were superior to those of rFVIIa. PT values of warfarin treated rats were maintained normal for significantly longer time post injection. MOD-5014 half-life and AUC following IV administration were 5 and 3.5 fold higher, respectively and were also significantly superior following SC injection. Following SC administration, MOD-5014's bioavailability, was shown to be superior to commercial rFVIIa in both rats and hemophilic mice models as well. In a tail vein transection studies, MOD-5014 had a profound effect on survival rate, which was maintained for more than 24 and significantly reduced duration and intensity of bleeding was also observed in tail clip studies in warfarin treated rats as well as in hemophilic mice in both routes. Finally, toxicological studies in rodents demonstrated that MOD-5014 is safe and tolerable at relatively high doses. Conclusion Attachments of CTP to FVIIa led to a markedly enhanced PK, increased exposure as reflected by AUC, improved recovery and a prolonged hemostatic effect in hemophilic mice and rats further supporting the comparable specific activity of MOD-5014 to rFVIIa. In addition, SC administration of MOD-5014 resulted in improved bioavailability and exposure was significantly prolonged relative to IV administration which was also translated to superior-vivo efficacy. CTP attachment to FVIIa had no significant impact on the in-vitro interactions with physiological inhibitors and cofactors proposing a similar mechanism of action and comparable activity. Our data suggest that CTP fused FVIIa is safe and tolerable in rodents and has the potential to significantly the frequency of injection given on demand as well as potentially enable prophylactic treatment for hemophilic patients. Disclosures: No relevant conflicts of interest to declare.

Factor VIIa-CTP, a Novel Long-Acting Coagulation Factor, Displays a Prolonged Hemostatic Effect and Augmented Pharmacokinetics and pharmacodynamics Following IV and SC Administration in Hemophilic Animal Models

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1114-1114 ◽  
Author(s):  
Gili Hart ◽  
Oren Hershkovitz ◽  
Ahuva Bar-Ilan ◽  
Uri Seligsohn ◽  
Eyal Fima
Keyword(s):  
Half Life ◽  
Specific Activity ◽  
Therapeutic Proteins ◽  
Coagulation Factor ◽  
Tail Vein ◽  
Hemostatic Effect ◽  
Long Acting ◽  
Tail Clip

Abstract Abstract 1114 Background: Prolor Biotech Inc. is a clinical stage public company developing biobetter long acting versions of existing therapeutic proteins utilizing a technology termed CTP. The technology involves fusion of the C terminus peptide of hCG to one or both ends of the target protein. The technology was clinically validated and proven as a safe and efficient way for prolonging the half-lives of several therapeutic proteins while maintaining their biological activity. Aims: To determine the pharmacokinetic (PK), pharmacodynamic (PD) and long term hemostatic effects of FVIIa-CTP in murine FVIII−/− mice following IV and SC administrations and to characterize FVIIa-CTP in vitro mechanism of action. Methods: FVIIa-CTP was expressed in CHO cells, purified and activated utilizing a purification process involving a CTP specific step. FVIIa-CTP was administered to FVIII−/− mice, and following IV and SC injection the PK and PD profiles were determined. In addition, the long term hemostatic effect was evaluated following bleeding challenge (tail clip assay and in a Tail Vein Transection) as compared to commercial rFVIIa. FVIIa-CTP in-vitro characteristics were also evaluated. Results: FVIIa-CTP PK parameters following IV administration, as assessed by a clotting assay, were superior to those of rFVIIa. Its half-life and AUC were 5 and 3.5 fold higher, respectively. A 2 fold improvement in the recovery was observed 15–30 minutes post dosing. Thrombin generation parameters were also superior in these studies. In a Tail Vein Transection study, FVIIa-CTP had a profound effect on survival, which was maintained for more than 24 hours. Reduced duration and intensity of bleeding was also observed in the tail clip study. Following SC administration, FVIIa-CTP's bioavailability, Cmax and half life were superior to commercial rFVIIa in both rats and hemophilic mice models. Correction of thromboelastograply in FVIII −/− mice plasma was maintained longer with FVIIa-CTP than with rFVIIa. Conclusion: Attachments of CTP to FVIIa led to a markedly enhanced PK, increased exposure, as reflected by AUC,improved recovery and a prolonged hemostatic effect in hemophilic mice with a comparable specific activity to rFVIIa. In addition, SC administration of FVIIa-CTP resulted in improved bioavailability and exposure was significantly prolonged relative to IV administration. Our data suggest that CTP fused FVIIa has the potential to significantly improve the prophylactic and on demand treatment of hemophilic patients. Disclosures: Seligsohn: prolor biotech: Consultancy, clinical advisor Other.

scholarly journals Long-Acting Risperidone Dual Control System: Preparation, Characterization and Evaluation In Vitro and In Vivo

Pharmaceutics ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1210
Author(s):  
Xieguo Yan ◽  
Shiqiang Wang ◽  
Kaoxiang Sun
Keyword(s):  
Drug Loading ◽  
Entrapment Efficiency ◽  
In Vitro Dissolution ◽  
Dissolution Behavior ◽  
In Vivo Evaluation ◽  
Long Term Treatment ◽  
Long Acting

Schizophrenia, a psychiatric disorder, requires long-term treatment; however, large fluctuations in blood drug concentration increase the risk of adverse reactions. We prepared a long-term risperidone (RIS) implantation system that can stabilize RIS release and established in-vitro and in-vivo evaluation systems. Cumulative release, drug loading, and entrapment efficiency were used as evaluation indicators to evaluate the effects of different pore formers, polymer ratios, porogen concentrations, and oil–water ratios on a RIS implant (RIS-IM). We also built a mathematical model to identify the optimized formulation by stepwise regression. We also assessed the crystalline changes, residual solvents, solubility and stability after sterilization, in-vivo polymer degradation, pharmacokinetics, and tissue inflammation in the case of the optimized formulation. The surface of the optimized RIS microspheres was small and hollow with 134.4 ± 3.5 µm particle size, 1.60 SPAN, 46.7% ± 2.3% implant drug loading, and 93.4% entrapment efficiency. The in-vitro dissolution behavior of RIS-IM had zero-order kinetics and stable blood concentration; no lag time was released for over three months. Furthermore, the RIS-IM was not only non-irritating to tissues but also had good biocompatibility and product stability. Long-acting RIS-IMs with microspheres and film coatings can provide a new avenue for treating schizophrenia.

Mod-5014, a Long-Acting FVIIa-CTP, Proposing a Novel Prophylactic Treatment Supporting Less Frequent Subcutaneous or Intravenous Injections with a Similar Mechanism of Action to rFVIIa: Proof-of-Concept in Hemophilic Animal Models

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4670-4670 ◽  
Author(s):  
Lior Binder ◽  
Ahuva Bar-Ilan ◽  
Malka Hoffman ◽  
Gili Hart
Keyword(s):  
Mechanism Of Action ◽  
Half Life ◽  
Prophylactic Treatment ◽  
Conflicts Of Interest ◽  
Clinical Stage ◽  
Routes Of Administration ◽  
Hemostatic Effect ◽  
Long Acting

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.

scholarly journals In Vitro–In Vivo Correlation (IVIVC) Population Modeling for the In Silico Bioequivalence of a Long-Acting Release Formulation of Progesterone

Pharmaceutics ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 255
Author(s):  
Elena M. Tosca ◽  
Maurizio Rocchetti ◽  
Elena Pérez ◽  
Conchi Nieto ◽  
Paolo Bettica ◽  
...  
Keyword(s):  
Clinical Studies ◽  
In Vitro Release ◽  
Release Formulation ◽  
Mixed Effect ◽  
Ring Model ◽  
Long Acting ◽  
Vitro Release ◽  
Release Profiles

Health authorities carefully evaluate any change in the batch manufacturing process of a drug before and after regulatory approval. In the absence of an adequate in vitro–in vivo correlation (Level A IVIVC), an in vivo bioequivalence (BE) study is frequently required, increasing the cost and time of drug development. This study focused on developing a Level A IVIVC for progesterone vaginal rings (PVRs), a dosage form designed for the continuous delivery in vivo. The pharmacokinetics (PK) of four batches of rings charged with 125, 375, 750 and 1500 mg of progesterone and characterized by different in vitro release rates were evaluated in two clinical studies. In vivo serum concentrations and in vitro release profiles were used to develop a population IVIVC progesterone ring (P-ring) model through a direct differential-equation-based method and a nonlinear-mixed-effect approach. The in vivo release, Rvivo(t), was predicted from the in vitro profile through a nonlinear relationship. Rvivo(t) was used as the input of a compartmental PK model describing the in vivo serum concentration dynamics of progesterone. The proposed IVIVC P-ring model was able to correctly predict the in vivo concentration–time profiles of progesterone starting from the in vitro PVR release profiles. Its internal and external predictability was carefully evaluated considering the FDA acceptance criteria for IVIVC assessment of extended-release oral drugs. Obtained results justified the use of the in vitro release testing in lieu of clinical studies for the BE assessment of any new PVRs batches. Finally, the possible use of the developed population IVIVC model as a simulator of virtual BE trials was explored through a case study.

THYROID STIMULATORS AND THYROID STIMULATION

1971 ◽  
Vol 66 (3) ◽  
pp. 558-576 ◽  
Author(s):  
Gerald Burke
Keyword(s):  
Regulatory System ◽  
Control Site ◽  
Thyroid Cell ◽  
Primary Control ◽  
Physico Chemical ◽  
Site Of Action ◽  
Long Acting

ABSTRACT A long-acting thyroid stimulator (LATS), distinct from pituitary thyrotrophin (TSH), is found in the serum of some patients with Graves' disease. Despite the marked physico-chemical and immunologic differences between the two stimulators, both in vivo and in vitro studies indicate that LATS and TSH act on the same thyroidal site(s) and that such stimulation does not require penetration of the thyroid cell. Although resorption of colloid and secretion of thyroid hormone are early responses to both TSH and LATS, available evidence reveals no basic metabolic pathway which must be activated by these hormones in order for iodination reactions to occur. Cyclic 3′, 5′-AMP appears to mediate TSH and LATS effects on iodination reactions but the role of this compound in activating thyroidal intermediary metabolism is less clear. Based on the evidence reviewed herein, it is suggested that the primary site of action of thyroid stimulators is at the cell membrane and that beyond the(se) primary control site(s), there exists a multifaceted regulatory system for thyroid hormonogenesis and cell growth.

The depressing (negative) effect of oestradiol benzoate on the in vitro secretion of LH and FSH by the pituitary gland of the LRH-pretreated long-term ovariectomized rat changes into the augmentative (positive) effect after discontinuation of the LRH-pretreatment

1985 ◽  
Vol 110 (3) ◽  
pp. 329-337 ◽  
Author(s):  
G. A. Schuiling ◽  
H. Moes ◽  
T. R. Koiter
Keyword(s):  
Depressing Effect ◽  
Ovx Rats ◽  
Gonadotrophin Secretion ◽  
Oestradiol Benzoate ◽  
Negative Effect ◽  
Positive Effect ◽  
Perifusion System

Abstract. The effect of pretreatment in vivo with oestradiol benzoate on in vitro secretion of LH and FSH was studied in long-term ovariectomized (OVX) rats both at the end of a 5-day continuous in vivo pretreatment with LRH and 4-days after cessation of such LRH pretreatment. Rats were on day 0 sc implanted with osmotic minipumps which released LRH at the rate of 250 ng/h. Control rats were implanted with a piece of silicone elastomer with the dimensions of a minipump. On days 2 and 4 the rats were injected with either 3 μg EB or with oil. On day 5 part of the rats were decapitated and the in vitro autonomous (i.e. non-LRH-stimulated) and 'supra-maximally' LRHstimulated release of LH and FSH was studied using a perifusion system. From other rats the minipumps were removed on day 5 and perifusion was performed on day 9. On the 5th day of the in vivo LRH pretreatment the pituitary LH/FSH stores were partially depleted; the pituitaries of the EB-treated rats more so than those of the oil-injected rats. EB alone had no significant effect on the content of the pituitary LH- and FSH stores. On day 9, i.e. 4 days after removal of the minipumps, the pituitary LH and FSH contents had increased in both the oil- and the EB injected rats, but had not yet recovered to control values. In rats not subjected to the 5-days pretreatment with LRH EB had a positive effect on the supra-maximally LRH-stimulated secretion of LH and FSH as well as on the non-stimulated secretion of LH. EB had no effect on the non-stimulated secretion of FSH. After 5 days of in vivo pretreatment with LRH only, the in vitro non-stimulated and supra-maximally LRH-stimulated secretion of both LH and FSH were strongly impaired, the effect correlating well with the LRH-induced depletion of the pituitary LH/FSH stores. In such LRH-pretreated rats EB had on day 5 a negative effect on the (already depressed) LRH-stimulated secretion of LH (not on that of FSH). EB had no effect on the non-stimulated LH/FSH secretion. It could be demonstrated that the negative effect of the combined LRH/EB pretreatment was mainly due to the depressing effect of this treatment on the pituitary LH and FSH stores: the effect of oestradiol on the pituitary LRH-responsiveness (release as related to pituitary gonadotrophin content) remained positive. In LRH-pretreated rats, however, this positive effect of EB was smaller than in rats not pretreated with LRH. Four days after removal of the minipumps there was again a positive effect of EB on the LRH-stimulated secretion of LH and FSH as well as on the non-stimulated secretion of LH. The positive effect of EB on the pituitary LRH-responsiveness was as strong as in rats which had not been exposed to exogenous LRH. The non-stimulated secretion of FSH was again not affected by EB. The results demonstrate that the effect of EB on the oestrogen-sensitive components of gonadotrophin secretion consists of two components: an effect on the pituitary LRH-responsiveness proper, and an effect on the pituitary LH/FSH stores. The magnitude of the effect of EB on the LRH-responsiveness is LRH dependent: it is very weak (almost zero) in LRH-pretreated rats, but strong in rats not exposed to LRH as well as in rats of which the LRH-pretreatment was stopped 4 days previously. Similarly, the effect of EB on the pituitary LH and FSH stores is LRH-dependent: in the absence of LRH, EB has no influence on the contents of these stores, but EB can potentiate the depleting effect of LRH on the LH/FSH-stores. Also this effect disappear after cessation of the LRH-pretreatment.

THE ROLE OF POLYETHYLENIMINE IN ENHANCING PERFORMANCE OF GLUTAMATE BIOSENSORS

2018 ◽  
Vol 8 (3) ◽  
pp. 36-41
Author(s):  
Diep Do Thi Hong ◽  
Duong Le Phuoc ◽  
Hoai Nguyen Thi ◽  
Serra Pier Andrea ◽  
Rocchitta Gaia
Keyword(s):  
First Generation ◽  
Good Reproducibility ◽  
Complex Matrices ◽  
Long Term Stability ◽  
In Vitro Characterization ◽  
Glutamate Oxidase

Background: The first biosensor was constructed more than fifty years ago. It was composed of the biorecognition element and transducer. The first-generation enzyme biosensors play important role in monitoring neurotransmitter and determine small quantities of substances in complex matrices of the samples Glutamate is important biochemicals involved in energetic metabolism and neurotransmission. Therefore, biosensors requires the development a new approach exhibiting high sensibility, good reproducibility and longterm stability. The first-generation enzyme biosensors play important role in monitoring neurotransmitter and determine small quantities of substances in complex matrices of the samples. The aims of this work: To find out which concentration of polyethylenimine (PEI) exhibiting the most high sensibility, good reproducibility and long-term stability. Methods: We designed and developed glutamate biosensor using different concentration of PEI ranging from 0% to 5% at Day 1 and Day 8. Results: After Glutamate biosensors in-vitro characterization, several PEI concentrations, ranging from 0.5% to 1% seem to be the best in terms of VMAX, the KM; while PEI content ranging from 0.5% to 1% resulted stable, PEI 1% displayed an excellent stability. Conclusions: In the result, PEI 1% perfomed high sensibility, good stability and blocking interference. Furthermore, we expect to develop and characterize an implantable biosensor capable of detecting glutamate, glucose in vivo. Key words: Glutamate biosensors, PEi (Polyethylenimine) enhances glutamate oxidase, glutamate oxidase biosensors

Strategies to Protect Hematopoietic Stem Cells from Culture-Induced Stress Conditions

2020 ◽  
Vol 15 ◽  
Author(s):  
Fatima Aerts-Kaya
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Ex Vivo ◽  
Stress Conditions ◽  
Stress Factors ◽  
Hematopoietic Stem ◽  
Induced Stress

: In contrast to their almost unlimited potential for expansion in vivo and despite years of dedicated research and optimization of expansion protocols, the expansion of Hematopoietic Stem Cells (HSCs) in vitro remains remarkably limited. Increased understanding of the mechanisms that are involved in maintenance, expansion and differentiation of HSCs will enable the development of better protocols for expansion of HSCs. This will allow procurement of HSCs with long-term engraftment potential and a better understanding of the effects of the external influences in and on the hematopoietic niche that may affect HSC function. During collection and culture of HSCs, the cells are exposed to suboptimal conditions that may induce different levels of stress and ultimately affect their self-renewal, differentiation and long-term engraftment potential. Some of these stress factors include normoxia, oxidative stress, extra-physiologic oxygen shock/stress (EPHOSS), endoplasmic reticulum (ER) stress, replicative stress, and stress related to DNA damage. Coping with these stress factors may help reduce the negative effects of cell culture on HSC potential, provide a better understanding of the true impact of certain treatments in the absence of confounding stress factors. This may facilitate the development of better ex vivo expansion protocols of HSCs with long-term engraftment potential without induction of stem cell exhaustion by cellular senescence or loss of cell viability. This review summarizes some of available strategies that may be used to protect HSCs from culture-induced stress conditions.

scholarly journals An ARF GTPase module promoting invasion and metastasis through regulating phosphoinositide metabolism

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Marisa Nacke ◽  
Emma Sandilands ◽  
Konstantina Nikolatou ◽  
Álvaro Román-Fernández ◽  
Susan Mason ◽  
...  
Keyword(s):  
Metastatic Cancer ◽  
Cellular Responses ◽  
Signalling Pathways ◽  
External Signal ◽  
Phosphoinositide Metabolism ◽  
Invasion And Metastasis ◽  
3 Dimensional

AbstractThe signalling pathways underpinning cell growth and invasion use overlapping components, yet how mutually exclusive cellular responses occur is unclear. Here, we report development of 3-Dimensional culture analyses to separately quantify growth and invasion. We identify that alternate variants of IQSEC1, an ARF GTPase Exchange Factor, act as switches to promote invasion over growth by controlling phosphoinositide metabolism. All IQSEC1 variants activate ARF5- and ARF6-dependent PIP5-kinase to promote PI(3,4,5)P3-AKT signalling and growth. In contrast, select pro-invasive IQSEC1 variants promote PI(3,4,5)P3 production to form invasion-driving protrusions. Inhibition of IQSEC1 attenuates invasion in vitro and metastasis in vivo. Induction of pro-invasive IQSEC1 variants and elevated IQSEC1 expression occurs in a number of tumour types and is associated with higher-grade metastatic cancer, activation of PI(3,4,5)P3 signalling, and predicts long-term poor outcome across multiple cancers. IQSEC1-regulated phosphoinositide metabolism therefore is a switch to induce invasion over growth in response to the same external signal. Targeting IQSEC1 as the central regulator of this switch may represent a therapeutic vulnerability to stop metastasis.

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