Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Targeted Thrombolysis for Treatment of Microvascular Thrombosis

a thrombolytic and microvascular technology, applied in the field of medicine and pharmacy, can solve the problems of organ damage, multi-organ failure with lethal consequences, obstructing the microvasculature with life-threatening consequences, etc., and achieve the effect of promoting recombination

Pending Publication Date: 2021-01-28
UMC UTRECHT HLDG BV
View PDF0 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention is about a new way to treat a condition called TTP (thrombotic thrombocytopenic purpura) by targeting the formation of blood clots in small blood vessels. The invention involves using modified plasminogen activators that can bind to the protein VWF, which is involved in the formation of blood clots. By stimulating the cleavage of VWF by plasmin, the invention aims to clear the blood clots and improve the treatment of TTP. The invention also provides a method for preventing or treating diseases or conditions associated with thrombi, including but not limited to TTP. The modified plasminogen activators used in the invention have been designed to have improved flexibility and solubility, and can be used in pharmaceutical compositions and gene therapy vectors for the treatment or prevention of thrombotic diseases.

Problems solved by technology

This becomes clear in thrombotic thrombocytopenic purpura (TTP), where platelet- and VWF-rich, but fibrin-poor microthrombi obstruct microvasculature with life-threatening consequences.
In severe cases of MVT, multi-organ failure can occur with lethal consequences.
Even in less severe cases, organ damage may form and reducing both the quality of life, as well as the life expectancy of the patient.
This is ultimately thought to cause heart failure, in particular in females.
However, persistent autoantibodies impede elimination of microthrombi.
This makes therapy time-consuming and very costly (Fijnheer et al., Ned Tijdsch Hematol 2016; 13 (1): 18-24).
Although plasmin(ogen) can directly bind to unrolled VWF, natural plasminogen activators—such as tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA)—cannot.
Furthermore, microthrombi in TTP are fibrin-poor and it is uncertain whether fibrin is a prerequisite for other types of MVT.
This renders these molecules (i.e. tPA, uPA) that are generally used as thrombolytic agents in the treatment of macrovascular disease ineffective in treating MVT.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Targeted Thrombolysis for Treatment of Microvascular Thrombosis
  • Targeted Thrombolysis for Treatment of Microvascular Thrombosis
  • Targeted Thrombolysis for Treatment of Microvascular Thrombosis

Examples

Experimental program
Comparison scheme
Effect test

example 1

Methods and Materials

[0118]Nanobody-mUPA construction

[0119]The cDNA sequence for both human and mouse urokinase (PLAU) was obtained from the NCBI database (NM_002658.4 and NM_008873.3 respectively). The sequence for the signal peptide, EGF-like and Kringle domain were removed as well as the first part of the connecting peptide. To the remaining connecting peptide and S1 peptidase domain (Catalytic domain) a N-terminal sequence coding for a Tobacco Etch Virus cleavage site followed by an GGGGS linker was added. In the GGGGS linked a Pstl and BamHI digestion site were incorporated without disturbing the amino acid sequence. At the 5′ side an EcoRl digestion site was added and at the 3′ side and Notl digestion was added after the STOP codon of PLAU. The construct was obtained from IDT (Integrated DNA Technologies, Leuven, Belgium) as a custom gene construct.

[0120]Coding sequences for nanobodies (also known as VHH) were codon optimized via IDT for expression in a human host cells. At th...

example 2

Micro-Thrombolysis of VWF-Platelet on Endothelial Cells in Flow Perfusion Materials and Methods

Human Vascular Endothelial Cell (Huvecs) Culture on Cover Glasses

[0134]Huvecs (passage 0) stored in liquid nitrogen were thawed at 37° C. and added to medium 1:10 (EBM-2 Lonza or Promocel supplemented with huvec growth factors EGM2) and spun down 100 g for 5 minutes. Supernatant was discarded and cells were taken up in 5 mL medium and cultured in T25 flasks at 37 degrees Celsius, 5% CO2. The following day the cells were passed to 3× T75 flasks. On day 6 the cells are passed 1:6 to the cover glasses pre-treated with 1,25% glutaraldehyde in HT-buffer pH 7.4 (HEPES Tyrode buffer: 10 mM HEPES, 0.5 mM Na2HPO4, 145 mM NaCl, 5 mM KCl, 1 mM MgSO4).

[0135]To coat cover glasses with glutaraldehyde, coverglasses were rinsed with demi water and with ethanol. Cover glasses were then incubated in HCL 37% : methanol (1:1) for 30 minutes followed by a rinse with demi water for 5 minutes. Next cover glasses...

example 3

Methods and Materials

Caplacizumab Production

Cloning

[0142]Caplacizumab (a bi-valent variant of the Cablivi VHH) was produced in E.coli and purified via HIS-tag affinity chromatography. The Caplacizumab protein sequence was derived from its EMA assessment report (EMA / 490172 / 2018; Procedure No. EMEA / H / C / 004426 / 0000) and codon optimized for E.coli expression via the integrated DNA technologies (IDT) codon optimized tool. N-terminal BamHl and C-terminal Notl digestion sites were added to the constructs and ordered as a double stranded DNA fragment from IDT (SEQ ID NO:40).

[0143]The DNA fragment were dissolved in 5 mM Tris-buffer (pH=8.5) and heated at 50° C. for 20 minutes. Hereafter the DNA fragment was ligated into the pJET1.2 vector (CloneJET PCR Cloning Kit; Thermo Fisher) according to manufacturer instructions. The ligated product was transformed into chemically competent E.coli TOP10 (Thermo Fisher) via heat shock according to manufacturer instructions. Transformed bacteria were cul...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
Energyaaaaaaaaaa
Capacitanceaaaaaaaaaa
Login to View More

Abstract

The present invention provides fusion proteins for targeted delivery of plasminogen activators to platelet-VWF complexes, or alternatively to the site where these are located, in a fibrin-independent manner. The fusion protein of the invention are for use in methods for the prevention or treatment of diseases or conditions associated with such platelet-VWF complexes, which may cause microvascular thrombosis in diseases such as e.g. thrombotic thrombocytopenic purpura. Preferred targeting agents for incorporation into the fusion proteins are e.g. nanobodies against VWF or platelets. Preferred plasminogen activators for use in the fusion proteins comprise the protease domains of uPA or tPA. The invention further pertains to nucleic acid molecule encoding the fusion proteins of the invention, e.g. a gene therapy vector, and to pharmaceutical compositions comprising the fusion proteins of the invention or such gene therapy vectors.

Description

FIELD OF THE INVENTION[0001]The present invention relates to the field of medicine and pharmacy, in particular to the field of biopharmaceuticals for use in the prevention or treatment of a disease or condition associated with microvascular thrombosis, such as e.g. thrombotic thrombocytopenic purpura. More specifically, the invention relates to fusion proteins comprising a targeting agent and a plasminogen activator, wherein the targeting agent targets the plasminogen activator to at least one of VWF, platelets and activated vascular endothelium with the aim to enzymatically degrade vascular obstructions in a fibrin-independent manner. The invention further relates to gene therapy vectors encoding such fusion proteins.BACKGROUND ART[0002]Microvascular thrombosis (MVT) is characterized by the formation of microvascular platelet aggregates. They are minimally composed of platelets and VWF. This becomes clear in thrombotic thrombocytopenic purpura (TTP), where platelet- and VWF-rich, b...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): A61K38/49A61K38/17A61P9/10
CPCA61K38/49A61P9/10A61K38/1777C07K14/00C07K16/00C07K19/00C07K2317/92C07K2319/00A61K38/16
Inventor MAAS, COENDE MAAT, STEVEN
Owner UMC UTRECHT HLDG BV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com