A block copolymer functionalized with an arylazopyrazole photoswitch with photoswitchable anticoagulant activity and the use of the block polymer

Abstract

The subject of the invention is a block copolymer of poly(sodium styrene sulphonate) and poly(acrylic acid) functionalized with a phenylazopyrazole substituent defined by formula (I). The invention also comprises the use of a copolymer for the preparation of a drug for use in controlling the blood clotting process, and its use in the treatment or prevention of diseases requiring control of the blood clotting process.

Description

[0001] A block copolymer functionalized with an arylazopyrazole photoswitch with photoswitchable anticoagulant activity and the use of the block polymer

[0002] The invention relates to a method for controlling blood clotting by the use of a photosensitive polymer, the anticoagulant effect of which can be reversibly activated and inactivated by exposure to light of a suitable wavelength.

[0003] Blood clotting is a physiological process that plays a key role in maintaining the integrity of the circulatory system and preventing excessive blood loss as a result of bleeding from a damaged blood vessel. As a result of blood clotting at the site of vessel damage, a blood clot forms, which stops the outflow of blood from the vessel and facilitates wound healing. The clot formed at the site of the injury also forms a physical barrier preventing the entry of pathogenic microorganisms into the body. In the course of many therapies used in medicine, it is necessary to reduce blood clotting by the use of drugs known as anticoagulants. They are widely used in the treatment and prevention of thromboembolic diseases, i.e., myocardial infarctions, strokes, and coagulation disorders in the course of oncological diseases, and many others. Their use is also necessary during cardiac surgery, hemodialysis, and other procedures that require extracorporeal circulation. Commonly used anticoagulants are heparins, including unfractionated heparin (UFH) and low molecular weight heparins (LMWHs), warfarin, and direct oral anticoagulants (DOACs) such as dabigatran, rivaroxaban, apixaban, and edoxaban. However, the use of anticoagulants has been associated with numerous side effects, including life-threatening hemorrhages. In such situations, suitable antidotes are used to neutralize their anticoagulant activity, e.g., protamine is used in the case of UFH. Unfortunately, in many cases there are no specific antidotes for anticoagulants, or they are only partially effective (e.g., protamine as an LMWH inhibitor is effective in about 60%). A significant limitation in the use of drugs neutralizing the effect of anticoagulants is their serious side effects, e.g., anaphylactic shock, thrombocytopenia, or allergic reactions.

[0004] Anticoagulants are commonly used in medicine to prevent and treat thromboembolic complications, but they have serious adverse effects, of which life-threatening hemorrhages are most dangerous. There is a need to develop and use new anticoagulants with a better safety profile, with an available antidote that will quickly neutralize their action, especially in situations when local neutralization is desired, for example, during unplanned surgical intervention. FactorXI inhibitors such as ISIS416858, abelacimab, osocimab, or asundexian are currently being developed in clinical trials. Also, among block copolymers with anionic groups, new candidates for anticoagulants are sought1. Restoring clotting in small blood vessels and capillaries in vascular areas of limited accessibility may be desirable to stop bleeding, e.g., in the area of the central nervous system, and prevent the effects of intracerebral hemorrhage. Currently, the only solutions are discontinuation of the anticoagulant therapy or systemic removal of anticoagulants, which in turn exposes the patient to a thromboembolic risk and life-threatening complications such as myocardial infarction, ischemic stroke, or pulmonary embolism within the next few days.

[0005] Solutions for controlling blood clotting by light, based on photodissociating molecules (photocages), are known in the art, thus allowing for only a single change in anticoagulant activity. Such molecules include nitrobenzene derivatives2-4and a photodissociating linker Cy7 comprising multiple conjugated carbon-carbon double bonds5.

[0006] The solution to the problems described above would be an active substance that would make it possible to control the blood clotting process by light in an easily reversible manner. Exposure of blood comprising such an active substance, depending on the wavelength of light used, would lead to its photoactivation or photoinactivation, resulting in the acquiring and loss of anticoagulant properties, respectively, and a decrease and increase in blood coagulability, respectively. In the prior art, such solutions based on aptamers are known. The disadvantage of aptamers in this application is their inefficient and expensive synthesis and their short half-life in the bloodstream due to degradation by nucleases. The possibility of changing the anticoagulant activity of aptamers was achieved by their functionalization with various molecules capable of reversibly changing their chemical structure under the influence of light, the so-called photoswitches. Azobenzene derivatives were one of such photoswitches^™ |nthe case of azobenzene derivatives, the photoswitching between the cis and trans forms is usually not quantitative, and the lifetime of the thermally unstable cis isomer is relatively short, especially in an aqueous environment and at physiological temperature™™®^9®. Azobenzenes are not resistant to azo bond reduction by cellular thiols, either10. The photoswitch employed for aptamer functionalization was an anthracene derivative™11™. However, achieving reversibility of anticoagulation by means of light has not been proven in this case. It was achieved only thermally by prolonged heating (12 h) and at a very high temperature (80°C), thus, under conditions excluding in vivo applications. Another photoswitch used to functionalize aptamers was the 8-fluorenylvinyl derivative of deoxyguanosine12, which allowed for the change of the anticoagulant activity of the aptamer several times. However, cis-trans photoisomerization of this photoswitch requires the use of toxic UV radiation with a wavelength of 310 nm. In addition, the thermal stability of aptamers in the trans form turned out to be very poor. The above problems are unexpectedly solved by the present invention.

[0007] The first object of the invention is a block copolymer described by formula (I) wherein: n is the degree of polymerization of the poly (acrylic acid) block and ranges from 33 to 66, x is the number of acrylic acid residues functionalized with the photoswitchable (E)-2-(4-((l- methyl-lH-pyrazol-4-yl)diazenyl)phenoxy)ethyl group and ranges from 2 to 6.

[0008] In a preferred embodiment, the degree of polymerization of the poly(acrylic acid) block n is 33 or 66, the number of acrylic acid residues functionalized with (E)-2-(4-((l-methyl-lH- pyrazol-4-yl)diazenyl)phenoxy)ethyl x is 2 or 6.

[0009] In another preferred embodiment the number of acrylic acid residues functionalized with the photoswitchable (E)-2-(4-((l-methyl-lH-pyrazol-4-yl)diazenyl)phenoxy)ethyl group, x, equal to 6 corresponds to the degree of polymerization of the poly(acrylic acid) block, n, equal to 66, or the number of acrylic acid residues functionalized with the photoswitchable (E)-2-(4- ((l-methyl-lH-pyrazol-4-yl)diazenyl)phenoxy)ethyl group, x, equal to 2 corresponds to the degree of polymerization of the poly(acrylic acid) block, n, equal to 33.

[0010] A second object of the invention is the use of the block copolymer defined by formula (I) for the preparation of a drug for use in controlling the blood clotting process.

[0011] The invention also comprises the use of the block copolymer defined by formula (I) in the treatment or prevention of diseases requiring control of the blood clotting process.

[0012] The polymers of the present invention, solving the problem of controlling anticoagulant activity by means of light, comprise in their structure a block of poly(sodium styrene sulphonate) (PSSS) and a block of poly(acrylic acid) (PAA) and were obtained by a reversible addition-fragmentation chain transfer (RAFT) polymerization process (Figure 1). The copolymers used in the invention are selected macromolecules with a well-defined structure (molecular weight, lengths of both blocks, molecular weight dispersity), obtained by a simple, efficient and reproducible method allowing to obtain polymers comprising a PSSS block of the desired length. The length of the sulfonated block is correlated to the anticoagulant potency13. They were then modified by esterification of the carboxyl groups in the PAA block with the PS1-OH photoswitch ((E)-2-(4-((l-methyl-lH-pyrazol-4-yl)diazenyl)phenoxy)ethyl-l-ol) (Figure 2) or its brominated equivalent, PSl-Br ((E)-4-((4-(2-bromoethoxy)phenyl)diazenyl)-l- methyl-lH-pyrazole) (Figure 3), i.e. compounds capable of changing the structure (photoisomerization) as a result of absorption of light of a specific wavelength. This change may be reversed as a result of the absorption of light of a different wavelength. The photoswitches used herein (PS1-OH and PSl-Br), which are derivatives of arylazopyrazole (AAP), undergo trans-cis photoisomerization as a result of the absorption of light with a wavelength in the range of 340-365 nm and cis-trans photoisomerization as a result of the absorption of light with a wavelength of about 530 nm (Figure 4). The important advantages of these photoswitches are:

[0013] - almost quantitative photoisomerization in both directions; irradiating the trans isomer of PS1-OH and PSl-Br with visible light of 400 nm wavelength also results in efficient, although not quantitative (about 80%), trans-cis photoisomerization, so it is practically possible to use only visible light for the photoisomerization of PS1-OH and PSl-Br in both directions, - exceptional thermal stability of the cis isomer of these photoswitches of about 90 days in an organic solvent at room temperature and about 9 days in physiological conditions, i.e., in an aqueous medium at pH 7.4 and at 37°C14

[0014] - the possibility of undergoing repeated reversible photoisomerization, and thus also multiple activation / deactivation of the anticoagulant activity of polymers functionalized with these photoswitches, unlike those used in the solutions known in the prior art, based on photodissociating molecules (photocages), allowing for only a single activation / deactivation of anticoagulation

[0015] - cis-trans photoisomerization occurs under the influence of light with a longer wavelength (530 nm) than in the case of azobenzene derivatives (about 450 nm), i.e., light penetrating deeper into the tissues.

[0016] Two PSSS58-b-P(AAn-x-co-AA / PSlx)n polymers were obtained with the same length of PSSS blocks, while differing in the length of PAA blocks and the degree of substitution of carboxyl groups with a photoswitch in the PAA block (Figures 2 and 3). The polymers were characterized by spectroscopic measurements (UV-Vis and 1H-NMR electron absorption spectroscopy, Figures 5 and 6), which confirmed the effectiveness of substitution of the polymer PSSS-b-PAA with a photoswitchable PSI group and the trans-cis photoisomerization efficiency of the photoswitchable PSI groups attached to it. DLS measurements showed that as a result of photoisomerization, there is a change in the distribution of the hydrodynamic radius of statistical coils formed by the polymer chains (Figure 7). In studies conducted on plasma samples of Wistar rats, it was shown that both PSSS-b-PAA block polymers functionalized with the photoswitchable PSI group significantly prolonged the aPTT coagulation time and that polymers comprising the photoswitchable PSI group in the trans configuration differed in anticoagulation activity from polymers comprising the photoswitchable group in the cis configuration (Figure 8). In a whole blood sample derived from rats, containing polymers with a photoswitchable PSI group in a trans configuration (non-irradiated), as a result of exposing this sample to light with a wavelength of about 340-365 nm, causing trans-cis photoisomerization of the photoswitchable PSI group in polymers, the effect of polymers extending the coagulation time was inhibited compared to the effect of polymers in the unexposed sample (Figure 9, "unexposed" and "340 nm" bars). In turn, exposure to light with a wavelength of about 530 nm (green) of a rat whole blood sample with a polymer comprising a photoswitchable PSI group in the cis configuration (i.e., deactivated by prior exposure of the polymer to light with a wavelength of 340 nm) resulted in cis-trans photoisomerization of the photoswitchable PSI group. It restored the original anticoagulant effect of the polymer, which was visible as an extension of the aPTT time (Figure 9, "530 nm" bars) compared to the aPTT time of the blood with a polymer comprising a photoswitchable PSI group in the cis configuration (Figure 9, "340 nm" bars).

[0017] The results of the study showed that the light of the appropriate wavelength (340-365 nm) can inhibit the anticoagulant effect of PSSS-b-PAA with the photo-switchable PSI group, which may be important in situations where there is a need to restore normal blood clotting reduced by the polymer. On the other hand, the action of the previously inactivated (by illumination with light of 340-365 nm wavelength) polymer can also be (locally) activated by exposure to a different wavelength (530 nm), if it is preferable to (re-)intensify its anticoagulant effect. The obtained block polymers functionalized with the photoswitchable PSI group thus allow for full control of blood clotting using light, which can be used in the diagnosis and therapy of blood clotting disorders and bleeding. An exemplary application of the developed polymer involves its administration into the bloodstream during procedures requiring extracorporeal circulation or hemodialysis. The blood is then irradiated with light of an appropriate wavelength in transparent tubing before returning to the body, thereby deactivating the circulating polymer and restoring normal blood coagulability..

[0018] The invention differs from other solutions in many respects. Synthetic polymers were used instead of prior art aptamers. In the active form, these polymers have a stronger anticoagulant effect than aptamers and our previously developed compounds13. As the results of the studies show, their blood clotting inhibitory effect is comparable to the effect of the strongest anticoagulants, such as heparins. In addition, synthetic copolymers are metabolically stable, which is also important during therapy, although the pharmacokinetics of the polymer- photoswitch conjugate require in vivo studies. The conducted toxicity studies of the PS1-OH photoswitch showed its low in vitro cytotoxicity up to a concentration of 1.3-10’4M in 48-h culture of B16-F10, 4T1, and NMuMG cells and high in vitro biocompatibility up to a concentration of 100 pg / mL in the hemolysis test. The aptamers described in the literature were studied in patients, but due to significant toxicopharmacokinetic problems and insufficient resistance to degradation by nucleases, studies on them were stopped in the first clinical phase. Although attempts have been made to improve their properties, antisense nucleotides have still not been used in anticoagulation therapy due to their chemical nature. Previous studies have followed the example of heparins, widely used in clinical practice. Experiments were performed in which a photoswitch was used in unfractionated heparin and in low molecular weight heparin - enoxaparin, and we observed a decrease in the anticoagulant activity of heparin after modification. There were differences between the cis and trans forms in the cytotoxic effect, but not in the anticoagulant effect14, which emphasizes the non-obviousness of the solution using anticoagulant block polymers, which are the subject of the invention. Previous experiments have shown that intravenous therapeutic doses of the synthetic copolymers are well tolerated in rats13. Based on the experience gained during these experiments, new synthetic polymers were obtained during the current studies, the anticoagulant activity of which was confirmed already after modification with PS1-OH and PSl-Br photoswitches. Compared to proteins and nucleic acids, PSSS-b-PAA block copolymers undergo much slower enzymatic degradation15, which allows their delivery (in addition to intravenous administration) also by oral, sublingual, or ocular route.

[0019] The invention can also be used in the course of hemorrhages, for example, in the treatment of acute bleeding ulcers of the gastrointestinal tract (methods of delivering light-capsules to the stomach and intestines are currently described) or in the prevention of bleeding from superficial ulcers in the course of cancer (e.g., breast cancer). The possibility of photoinactivation of this type of anticoagulants may reduce the need for systemic administration of classical anticoagulants, such as heparins administered in high doses during procedures requiring extracorporeal circulation, for example, in the case of hemodialysis or open heart surgery, and procedures in which the patient's blood is collected, subjected to specific procedures, and returned to the patient's bloodstream, such as apheresis or photopheresis. These methods require reducing blood clotting until it remains outside the patient's body and restoring its normal level immediately before returning it to the patient's bloodstream.

[0020] The embodiments of the invention are shown in the figures, where:

[0021] Figure 1 shows the structures of the starting block polymers (PSSSss-b-PAAss and PSSSss- b-PAAee) used to obtain the photoswitchable polymers.

[0022] Figure 2 shows the synthesis reaction of PSSSss-b-PjAAeo-co-AA / PSle).

[0023] Figure 3 Shows the synthesis of PSSSss-b-PfAAsi-co-AA / PSlz) Figure 4 illustrates the trans-cis and cis-trans photoisomerization reaction of the PS1-OH photoswitch under the influence of radiation at wavelengths of 340-365 nm and 530 nm, respectively. Trans-cis isomerization under the influence of light with a wavelength of 400 nm also occurs efficiently, although not quantitatively.

[0024] Figure 5 shows the UV-Vis spectra of an aqueous solution of PSSSss-b-PfAAeo-co-AA / PSle) having a concentration of 0.15 mg / mL unexposed (solid line) and exposed (dashed line) to light with a wavelength of 365 nm for 5 min, comprising the photoswitchable PSI group in the trans and cis configurations, respectively.

[0025] Figures 6a-6b show the 1H NMR spectra of PSSSss-b-PAAee (Figure 6a) and PSSSss-b- P(AA6o-co-AA / PSle) (Figure 6b) in D2O.

[0026] Figure 7 shows the distribution of the hydrodynamic diameter of the PSSSs8-b-P(AAeo-co- AA / PS1) chains for the polymer comprising the photoswitchable PSI group in the trans and cis configurations (cp= 0.15 g / L, PBS pH 7.4).

[0027] Figure 8 shows the strong concentration-dependent anticoagulant effect of the PSSSss-b- P(AA3i-co-AA / PS12) polymer (A) and PSSSs8-b-P(AAeo-co-AA / PSle) polymer (B).

[0028] Figure 9 shows the inhibition under the influence of light with a wavelength of 340 nm and then the restoration under the influence of light with a wavelength of 530 nm of the anticoagulant effect of the PSSSss-b-P(AA3i-co-AA / PS12) polymer (V) at a concentration of 0.05 mg / mL (A) and of the PSSSs8-b-P(AAeo-co-AA / PSle) polymer (VI) at a concentration of 0.075 mg / mL (B).

[0029] The substrates for the synthesis of the photoswitchable polymers of the invention have been prepared according to the literature known from the prior art. The synthesis of the PS1-OH photoswitch ((E)-2-(4-((l-methyl-lH-pyrazol-4-yl)diazenyl)phenoxy)ethyl-l-ol)(IVa) was carried out according to the procedure described in the paper by Stolarek et. al.14. The synthesis of the brominated photoswitch PSl-Br (E)-4-((4-(2-bromoethoxy)phenyl)diazenyl)- 1-methyl-lH-pyrazole) (IVb) was carried out according to the procedure described in the paper by Zhang et al.16. The synthesis of the PSSSss-b-PAA33 (II) and PSSSss-b-PAAee (III) polymers was carried out according to the procedure described in the paper by Yap et al.17.

[0030] Example 1. Synthesis of the PSSSss-b-PfAAeo-co-AA / PSle) polymer (V)

[0031] PSSSss-b-PAAee (104 mg) (III), PS1-OH (IVa) (101 mg), and dimethylaminopyridine (DMAP, CAS 1122-58-3, Merck) (15.7 mg) were dissolved in 15 mL DMF and stirred at room temperature with a magnetic stirrer. After 1 h, 151 mg of dicyclohexylcarbodiimide (DCC, CAS 538-75-0, Merck) was added, and the resulting solution was stirred for 24 h. The resulting product was precipitated in water, centrifuged (8000 rpm, 10 min), and the supernatant was removed. The product was dialyzed for 5 days in a dialysis tube (MWCO 3.5 kDa) immersed in distilled water and then lyophilized. 48.5 mg of PSSSss-b-PfAAeo-co-AA / PSle) was obtained (V).

[0032] Example 2. Synthesis of the PSSSss-b-PfAAsi-co-AA / PSlz) polymer (VI)

[0033] PSSSss-b-PAAss (103 mg) (II), PSl-Br (IVb) (75.8 mg), and 75 pL of 1,1,3,3-tetramethylguanidine (TMG, CAS 80-70-6) were dissolved in a mixture of 10 mL DMSO and 5 mL DMF. The resulting pH 8-9 mixture was neutralized to pH 7 with acetic acid and then stirred with a magnetic stirrer at room temperature for 24 h. The resulting product was precipitated in water, centrifuged (8000 rpm, 10 min), and the supernatant was removed. The product was dialyzed in a dialysis tube (MWCO 3.5 kDa) immersed for 1 day in 15% v / v acetonitrile in water, for 2 days in 10% v / v acetonitrile in water, and for 2 days in distilled water and then lyophilized. 60.6 mg of PSSSs8-b-P(AA3i-co-AA / PS12) was obtained.

[0034] Example 3. The use of a block polymer functionalized with an arylazopyrazole photoswitch to control blood clotting under the influence of light

[0035] To confirm the possibility of using block polymers functionalized with a photoswitch to control plasma and blood coagulability, two experiments were conducted. The first study was performed by exposing the polymer solution to a beam of light with a wavelength of 340 nm or 530 nm, adding the exposed polymer solution to rat plasma, and then performing a coagulation test - activated partial thromboplastin time (aPTT) (Figure 8). The second experiment consisted of adding a polymer to a whole blood sample from rats, exposing the sample to a beam of light of the appropriate wavelength, centrifuging the sample to obtain plasma, and then performing a coagulation test - activated partial thromboplastin time (aPTT) (Figure 9). The light source was a 340 or 530 nm LED lamp (ThorLabs, United States). Blood was collected from the right ventricle of Wistar rats weighing 250-300 g from the Center for Experimental Medicine of the Medical University of Bialystok. The study was carried out in accordance with the Act of 17 November 2021 amending the Act on the protection of animals used for scientific or educational purposes. Whole blood or plasma samples with 0.9% NaCI constituted the control group. The tests were performed in 2-6 repetitions. A study conducted on plasma samples showed that both PSSS-b-PAA block polymers functionalized with PS1-OH and PSl-Br photoswitches significantly prolonged the coagulation time aPTT (Figure 8). For the PSSSss-b-PfAAsi-co-AA / PSlz) polymer (VI) in the trans form, half of the maximum anticoagulant effect was observed at a concentration of 0.05 mg / mL (Figure 8A), while for the PSSSss-b-PfAAeo-co-AA / PSle) polymer in the trans form at a concentration of 0.075 mg / mL (Figure 8B). The above concentrations were used in the next stage of the study. In whole blood collected from rats, containing both polymers in unexposed form (trans) and after exposure to a beam of light with a wavelength of about 530 nm, the coagulation time is maximally prolonged (Figure 9). As a result of the absorption of light with a wavelength of about 340-365 nm, causing the trans-cis photoisomerization of photoswitchable PSI group in polymers, the anticoagulant effect of the polymers was inhibited and was similar to the control results.

[0036] References:

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Claims

Claims1. A block copolymer described by formula (I)wherein: n is the degree of polymerization of the poly(acrylic acid) block and ranges from 33 to 66, x is the number of acrylic acid residues functionalized with the photoswitchable (E)-2-(4-((l-methyl-lH-pyrazol-4-yl)diazenyl)phenoxy)ethyl group and ranges from 2 to 6.

2. The block copolymer according to claim 1, characterized in that the degree of polymerization of the polyacrylic acid block n is 33 or 66, the number of acrylic acid residues functionalized with the photoswitchable (E)-2-(4-((l-methyl-lH-pyrazol- 4-yl)diazenyl)phenoxy)ethyl group x is 2 or 6.

3. The block copolymer according to claim 1 or 2, characterized in that the number of acrylic acid residues functionalized with the photoswitchable (E)-2-(4-((l-methyl- lH-pyrazol-4-yl)diazenyl)phenoxy)ethyl group, x, equal to 6 corresponds to the degree of polymerization of the poly(acrylic acid) block, n, equal to 66, or the number of acrylic acid residues functionalized with the photoswitchable (E)-2-(4- ((l-methyl-lH-pyrazol-4-yl)diazenyl)phenoxy)ethyl group, x, equal to 2 corresponds to the degree of polymerization of the poly(acrylic acid) block, n, equal to 33.

4. The block copolymer according to claims 1-3 for the preparation of a drug for usein controlling the blood clotting process.

5. The block copolymer according to claims 1-3 for use in the treatment or prevention of diseases requiring control of the blood clotting process.

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