Thrombolytics Development for Stroke

Ischemic stroke is caused by occlusion of cerebral arteries that are inaccessible by intra-arterial catheters. Therefore, reperfusion with thrombolytics will remain an important treatment for hyperacute ischemic stroke. Ace Therapeutics is a stroke-focused preclinical CRO company, offering clients one-stop thrombolytics development services.

Introduction to the Development of Thrombolytics for Stroke

Thrombolytic agents were first discovered from bacteria (streptokinase, staphylococcal kinase), tissues (fibrin kinase), urine (urokinase), or substances from bat saliva that can activate the fibrinolytic system. Recombinant tissue plasminogen activator (rt-PA) is the only established acute thrombolytic treatment option for ischemic stroke. However, rt-PA still often fails to achieve rapid reperfusion, has relatively low recanalization rates, and is associated with an increased risk of bleeding. There is an unmet need for novel thrombolytic agents with better benefit-risk than rt-PA. Pharmaceutical companies need to step up their efforts to develop new compounds faster to bring hope to more stroke patients.

Fig. 1. Molecular structure and fibrinolytic function of tissue plasminogen activator (tPA). (Nikitin et al., 2021)

Our Services

Ace Therapeutics has a team of experts in biochemistry, pharmacology and drug development with a comprehensive understanding of the preclinical research pipeline in stroke and the current state of thrombolytics. Our strong capabilities and technologies in the field of stroke contribute to the development of novel thrombolytics. We offer comprehensive services covering every stage of the stroke drug development process, from target identification to preclinical evaluation.

Identification of Molecules with Thrombolytic Activity

Ace Therapeutics offers a variety of molecular biology methods and computational approaches to find molecules with thrombolytic activity in different organisms.

• The fibrin-specific thrombolytics of bacterial origin, such as Streptococcus uberis, Bacillus, or Stenotrophomonas.
• Thrombolytics of various eukaryotic origin, such as Chlorella alga, venomous snakes, and polychaetes.
• Thrombolytics of non-mammalian origin.

Our Approach to Developing Novel Thrombolytics

We can use a combination of methods to help our clients screen a wider range of molecules and increase the likelihood of finding good targets, while testing the biophysical, biochemical and biological properties of thrombolytic candidates.

Development of Novel Thrombolytics Through Protein Engineering

We provide rational protein design services to determine the crystal structure of thrombolytic enzymes and use them for computer modeling. We aim to improve the half-life, potency, solubility, and fibrin affinity of thrombolytics.

• Glycosylation Engineering: Modification of glycosylation patterns to improve pharmacokinetics, circulating half-life and brain targeting properties.
• Polyethylene Glycollization: Addition of polyethylene glycol polymers to enhance solubility and stability, and reduce immunogenicity.

In Vitro Testing of Novel Thrombolytics

We offer a variety of in vitro assays to evaluate individual properties affected by structural modifications of thrombolytics.

• Enzymatic activity assays: We offer a variety of methods to assess the fibrinolytic potential of thrombolytics, including radiolabeling, euglobulin cleavage assays, the Clot Formation and Lysis (CloFAL) assay, and streptokinase-activated lysis time (SALT) assay. These assays monitor not only the enzymatic activity of thrombolytics but also their overall efficacy.
• Resistance assays: We use enzyme-linked immunosorbent assays (ELISA) or colorimetric/fluorescence assays to determine the amount of free and active thrombolytics. These assays provide information on the kinetics and thermodynamics of thrombolytic inhibition.
• Fibrin affinity assays: We provide fibrin immobilization assay to measure the affinity and kinetics of interactions associated with thrombolytics.
• Receptor binding assays: We offer several methods to determine the affinity of thrombolytics for membrane receptors, including surface plasmon resonance (SPR), ELISA, colorimetric/fluorescence assays, and warm titration calorimetry.

In Vivo Testing of Novel Thrombolytics

Our available thromboembolic models (e.g., clot injection-induced thromboembolic stroke model, in situ thrombosis model, FeCl₃-induced thromboembolic model, photochemical thrombosis model) can be used to test the efficacy and safety of thrombolytics. We offer a range of stroke-related endpoint measurements.

• Measure the area of brain tissue damage using techniques such as TTC staining or MRI.
• Foot fault test, grip strength test, rotating rod test.
• We can characterize and manage heterogeneity in mouse models of embolic stroke by combining in vivo cerebral blood flow (CBF) monitoring and ex vivo near-infrared fluorescence imaging to visualize thrombi.
• We use micro-computed tomography (CT) and fibronectin-targeted glycol-chitosan-coated gold nanoparticles (GCAuNPs) in mice to continuously, non-invasively, and quantitatively assess thromboembolic burden and efficacy of pharmacological treatments.

Ace Therapeutics offers customized thrombolytics development services to help clients develop safe and effective thrombolytics for stroke. Whether it is target identification or preclinical experimental design, we work closely with our partners to develop a comprehensive strategy that meets their goals and timelines. If you are interested in our services, please do not hesitate to contact us!

1. Nikitin, D., et al. (2021). Development and testing of thrombolytics in stroke. Journal of Stroke, 23(1), 12.