NZ748309B2 - Cyclodextrins as procoagulants - Google Patents

Abstract

The invention relates to substituted cyclodextrins comprising at least one substituent -S-(Cn-alkylene)-R and pharmaceutically acceptable salts thereof, pharmaceutical compositions, kits of parts and their use as procoagulants. The invention further relates to said cyclodextrins for use in methods of reversing an anticoagulant effect of an anticoagulant in a subject, in methods for reducing or preventing bleeding in a subject and in methods for the treatment or prevention of a blood coagulation disorder. f reversing an anticoagulant effect of an anticoagulant in a subject, in methods for reducing or preventing bleeding in a subject and in methods for the treatment or prevention of a blood coagulation disorder.

Description

P110697PC00 Title: Cyclodextrins as procoagulants Field of the invention The invention relates to the field of medicine, in particular to substituted cyclodextrins and their use as procoagulants.

Background of the invention Thromboembolic disorders such as myocardial tion, stroke, and venous thromboembolism are the most common causes of mortality and morbidity in Western societies. These thromboembolic events can be red by excessive activation of coagulation, and in plays a major role in these processes. The most widely used agents for antithrombotic therapy are heparins (including low molecular weight heparins, LMWH) and oral indirect thrombin inhibitors such as vitamin K antagonists (VKA) (warfarin, acenocoumarol and phenprocoumon).

However, e of the need for frequent monitoring and the desire for safer anticoagulants, several novel non-vitamin K-dependent oral anticoagulants (NOACs) have been developed. These newer agents include the factor Xa inhibitors (such as rivaroxaban, apixaban, edoxaban), along with the direct thrombin inhibitors (dabigatran). Unlike the vitamin K antagonists, these new anticoagulants do not require routine (INR) monitoring and possess favourable pharmacological properties. NOACs act rapidly, and have a stable and table dose-related anticoagulant effect with few clinically relevant drug-drug interactions. Despite these improvements in ent, anticoagulation therapy in general is associated with an increased risk of bleeding.

The traditional anticoagulants, unfractionated heparin (UFH) and the vitamin K antagonists (VKA), have antidotes to reverse their intended therapeutic effect. Protamine sulfate largely reverses the antithrombotic effect of UHF, but not t some potential s side effects. Vitamin K reverses the impaired coagulation induced by the VKAs by re-establishing synthesis of the reduced vitamin K-dependent coagulation factors, but not without the ck of ing 12–24 h to achieve significant levels of these factors to improve coagulation. LMWHs and arinux, both further refinements on the heparin le, have a more limited ability of reversal by protamine sulfate.

Fondaparinux has only anti-Xa activity and is not reversed by protamine sulphate, which is also the case for danaparoid. In on, also argatroban and bivalirudin (intravenous anticoagulants) have no reversal agent, but they have a very short ife of 30–50 min, their indications are limited and their use is usually for relatively short durations.

The more recent non-vitamin K-dependent oral anticoagulants are small molecules that bind directly to their intended target, either activated factor II (IIa or thrombin) or activated factor X (Xa) and antagonize their activity. These drugs are intended for long-term, outpatient use for many of the same indications as the VKAs. Consequently, not only are there millions of users for these drugs, but for many tions they are taken over a significant portion of one’s me. This results in many patient-years where duals are at risk for bleeding, either spontaneously or following trauma or surgical procedures. Only for one anti-IIa inhibitor (dabigatran) a selective antidote is currently available. For the other NOACs (rivaroxaban, edoxaban and an) tes have not yet been developed. There is general sus that the lack of a reversal agent for the NOACs is a major barrier to their more widespread use, and even with their short half-life, being able to rapidly reverse agulation in the face of major or life threatening bleeding would certainly be beneficial. Reversal might also be valuable in the setting of overdose or in preparation for emergency surgery or interventions.

For elective interventions, a reversal agent might also allow for shorter intervals when a patient is sub-therapeutic in preparation for the intervention.

Consequently, there remains a need for safe, immediately effective, and easy to administer antidotes for patients taking anticoagulants in the settings of major bleeding, need for emergency surgery, and accidental overdose. A general te that may be used in emergency situations regardless of which type of anticoagulant therapy has been used, would in ular have advantages over more selective antidotes since, especially in emergency situations, the exact type of anticoagulant therapy is often unknown. A preferred general antidote may find additional use as a procoagulant in the treatment of bleeding disorders.

Summary of the invention It is an object of the t ion to provide extrins that have a procoagulant effect. Preferably the cyclodextrins have such procoagulant effect both in the presence and absence of anticoagulants. It is a further object of the present invention to provide methods for reversing an anticoagulant effect of an anticoagulant and/or for reducing or preventing bleeding in a subject and/or for treating or preventing blood coagulation disorders; and/or to provide the public with a useful choice.

In a first aspect the present invention provides a use of a substituted cyclodextrin of formula (I): a (I), wherein n is an integer from 3 to 7 and R is selected from the group consisting of - COOH, -OH and -COO(1-4C)alkyl, and wherein p + q is 6, 7 or 8, whereby p is 5 and q, is 1 or p is 6 and q is 1, or p is 7 and q is 1, or p is 0 and q is 6, or p is 0 and q is 7, or p is 0 and q is 8, or a pharmaceutically acceptable salt or ester thereof, in the manufacture of a medicament for initiating or rating blood clot formation in a subject in need thereof.

In a second aspect the present invention provides a pharmaceutical composition comprising at least one tuted cyclodextrin or pharmaceutically acceptable salt or ester thereof as defined in the first asepct and at least one pharmaceutically acceptable auxiliary, wherein said pharmaceutical composition is formulated for topical administration as a gel, cream, ointment, dressing, compress, plaster, band-aid or patch.

In a third aspect the present invention provides a substituted cyclodextrin of formula (II): Formula (II), wherein: - p is 6, q is 1, m is 5 and R is COOH; - p is 0, q is 7, m is 5 and R is COOH; - p is 7, q is 1, m is 5 and R is COOH; - p is 0, q is 7, m is 4 and R is COOH; - p is 0, q is 7, m is 6 and R is COOH; - p is 0, q is 7, m is 7 and R is COOH; - p is 5, q is 1, m is 5 and R is COOH; - p is 0, q is 6, m is 5 and R is COOH; - p is 6, q is 1, m is 6 and R is COOH; - p is 6, q is 1, m is 4 and R is OH; - p is 7, q is 1, m is 6 and R is COOH; or - p is 7, q is 1, m is 4 and R is OH, or a pharmaceutically acceptable salt or ester of any of these cyclodextrins, or a substituted cyclodextrin of formula (II) wherein p is 0, q is 8, m is 5 and R is COOH.

In a fourth aspect the t invention provides a pharmaceutical composition comprising a substituted cyclodextrin or ceutically acceptable salt or ester thereof ing to the third aspect and at least one pharmaceutically acceptable auxiliary.

In a fifth aspect the present invention provides a kit of parts comprising: - a substituted cyclodextrin or ceutically acceptable salt thereof according to the third aspect, and - a recombinant or isolated coagulation factor.

In a sixth aspect the present invention provides a use of a substituted cyclodextrin or a ceutically acceptable salt thereof as defined in the first aspect for the manufacture of a medicament for reversing an anticoagulant effect of an anticoagulant in a subject.

In a seventh aspect the present invention provides a use of a substituted cyclodextrin or a pharmaceutically acceptable salt thereof as defined in the first aspect for the manufacture of a medicament for reducing or preventing bleeding in a subject.

In an eighth aspect the present invention provides a use of a tuted cyclodextrin or a pharmaceutically acceptable salt thereof as defined in the first aspect in the cture of a medicament for the treatment or prevention of a blood coagulation disorder.

Also described is a substituted cyclodextrin according to formula I, comprising at least one substituent -S-(Cnalkylene)-R, wherein n is 3-7, and R is selected from the group consisting of -COOH, -OH, and -4C)alkyl, or a pharmaceutically acceptable salt thereof.

Formula (I) In a preferred tuted cyclodextrin described herein, p + q is 6, 7 or 8, whereby p is 5 and q is 1, or p is 6 and q is 1, or p is 7 and q is 1, or p is 0 and q is 6, or p is 0 and q is 7, or p is 0 and q is 8.

In preferred substituted cyclodextrin described herein, S-(Cnalkylene)-R is -S-(CH2)m-R, wherein m is an integer from 3 to 7.In a red substituted cyclodextrin bed herein, R is - selected from the group consisting of –COOH and –OH.

In a most preferred substituted cyclodextrin described herein, p is 0, q is 8, m is 5, and R is COOH.

Also described is a pharmaceutical composition comprising a substituted cyclodextrin described herein, and at least one pharmaceutically acceptable auxiliary.

Also described is a kit of parts comprising: - substituted cyclodextrin bed herein, or a pharmaceutically acceptable salt thereof, and - a recombinant or isolated coagulation .

Also described is a tuted cyclodextrin described herein, or a ceutically acceptable salt thereof for use as a procoagulant.

Also described is a substituted cyclodextrin described herein, or a pharmaceutically acceptable salt thereof for use in a method for reversing an anticoagulant effect of an anticoagulant in a subject.

Also described is a method for reversing an anticoagulant effect of an anticoagulant in a subject in need f, the method comprising administering to the subject, which subject has been administered said anticoagulant, a therapeutically effective amount of a substituted cyclodextrin described herein, or a ceutically acceptable salt thereof.

Also described is a substituted cyclodextrin described herein, or a ceutically acceptable salt thereof for the preparation of a medicament for reversing an anticoagulant effect of an agulant in a subject.

Also described is a substituted cyclodextrin described herein, or a pharmaceutically acceptable salt thereof for use in a method for reducing or preventing bleeding in a subject.

Also described is a method for inducing or stimulating coagulation in a subject in need thereof sing administering to the subject a therapeutically effective amount of a substituted cyclodextrin described herein, or a pharmaceutically acceptable salt thereof.

Also described is a method for reducing or preventing bleeding in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a tuted cyclodextrin described herein, or a ceutically acceptable salt thereof.

Also described is a substituted cyclodextrin described herein, or a pharmaceutically acceptable salt thereof for the preparation of a medicament for reducing or preventing bleeding in a subject.

Also described is a substituted cyclodextrin described herein, or a pharmaceutically acceptable salt thereof for use in a method for the treatment or prevention of a blood coagulation disorder.

Also described is a method for the treatment or prevention of a blood coagulation disorder, comprising administering to a subject in need f a therapeutically effective amount of a tuted cyclodextrin described herein, or a pharmaceutically acceptable salt thereof.

Also described is a substituted cyclodextrin described herein, or a ceutically acceptable salt f for the preparation of a medicament for the treatment or prevention of a blood coagulation disorder.

Detailed description The present inventors have identified a set of cyclodextrins having one or more specific substituents that have a procoagulant effect both in vitro and in vivo. Such procoagulant effect of cyclodextrins was previously unknown. The effect of the cyclodextrins on several ters of thrombin generation were inter alia determined. Thrombin generation is one of the final stages in the blood ation process and therefore a particularly important ter when assessing the effect of a compound on the coagulation process. The procoagulant activity of the cyclodextrins is evidenced by a reduction in the lag time for thrombin generation, an increase in the peak thrombin level, a reduction in the time to peak thrombin level or a combination thereof in the presence of the cyclodextrins described herein.

In addition, procoagulant activity of the cyclodextrins was demonstrated in vivo as evidenced by ation of blood clot formation. As is shown in the Examples, the cyclodextrins bed herein are capable of at least partly reversing the anticoagulant effect of a wide array of anticoagulant agents. It has r been shown that specific cyclodextrins described herein exert a procoagulant effect in normal pooled plasma, i.e. they influence normal blood coagulation in the absence of agulants or deficiency of a coagulation factor. Moreover, the cyclodextrins described herein have a procoagulant effect in plasma deficient in a blood coagulation factor.

The use of cyclodextrins as described herein as procoagulants has many advantages over the use of known procoagulants. For ce, the cyclodextrins have the advantage that they can be used to reverse the anticoagulant effect of a wide variety of anticoagulants. The anticoagulant activity of direct acting oral anticoagulants such as factor Xa inhibitors (e.g. rivaroxaban, apixaban, edoxaban), and direct thrombin inhibitors (e.g. dabigatran), of pentasaccharides such as fondaparinux, of low molecular weight heparins such as nadroparin and tinzaparin, of tionated heparin and of vitamin K nists is reversed by the cyclodextrins bed herein. Contrary, many of the currently known procoagulants are specific for one anticoagulant or one class of anticoagulants.

Consequently, the cyclodextrins bed herein can be used to reverse an agulant effect without the need to identify the specific anticoagulant first since, in emergency situations, this is often n. A general procoagulant that may be used regardless of which type of anticoagulant therapy has been used, is preferred over more ive antidotes in emergency situations.

In addition, the cyclodextrins described herein are able to e the anticoagulant of compounds such as argatroban, bivalirudin, rivaroxaban, edoxaban and apixaban for which currently no reversal agents are available.

Further, the cyclodextrins described herein in principle exert their procoagulant activity rapidly after administration, e.g. within minutes, unlike many known specific procoagulant such as vitamin K, which is able to e the anticoagulant effect of vitamin K antagonists only after 12-24 h. However, the half- life of cyclodextrins is dependent on their hydrophilicity. Hence, the half-life of the cyclodextrins described herein can be influenced, typically by the introduction of groups that are more hydrophilic or the introduction of additional hydrophilic . This results in an increase in the ife of the cyclodextrins. This way, the cyclodextrins can be modified to have the optimal half-life for a desired application.

In on to the above, cyclodextrins have been widely used in food products and pharmaceutical compositions. They are associated with little sideeffects.

For instance, cyclodextrins are less immunogenic when administered to humans as compared to proteinaceous procoagulants, such as recombinant coagulation s that are currently used to treat patients suffering from a deficiency in such coagulation factor.

Accordingly described is a substituted cyclodextrin comprising at least one substituent -S-(Cnalkylene)-R, wherein n is 1 or an integer from 3 to 10 and R is selected from the group ting of -COOH, -OH, and -COO(1-4C)alkyl, or a pharmaceutically acceptable salt thereof. Preferably n is an integer from 3 to 7.

Also described is a substituted cyclodextrin comprising at least one substituent -S-(Cnalkylene)-R, wherein n is 1 or an integer from 3 to 10, preferably 3-7, and R is selected from the group consisting of -COOH, -OH, and -COO(1- 4C)alkyl, or a pharmaceutically acceptable salt thereof for use as a procoagulant.

A substituted cyclodextrin comprising at least one substituent -S- (Cnalkylene)-R, wherein n is 1 or an integer from 3 to 10, preferably 3-7, and R is selected from the group consisting of -COOH, -OH, and -4C)alkyl is herein also referred to as “a substituted cyclodextrin bed herein”.

Cyclodextrins are a family of cyclic oligosaccharides. extrins are composed of 6 or more α-D-glucopyranoside units linked 1->4 (see figure 1a).

Cyclodextrins containing 6, 7 and 8 sugar units are referred to as alphacyclodextrins (α-CD), beta-cyclodextrins (β-CD) and gamma-cyclodextrins (γ-CD), respectively. Cyclodextrins contain a somewhat lipophilic central cavity and a hilic outer surface. They are used in food, pharmaceutical and chemical industries and for drug delivery. One or more of the -OH groups can be substituted to provide a wide variety of cyclodextrin derivatives or substituted cyclodextrins.

As used herein, the term "cyclodextrin" refers to a cyclic oligosaccharide moiety composed of 6 or more copyranoside units linked through α-(1 ,4) glucosidic bonds. The term "substituted extrin" as used herein refers to a cyclodextrin moiety which is substituted with at least one substituent -S- ylene)-R group, wherein n and R are as defined herein. Such substituted cyclodextrin is also referred to as a cyclodextrin tive. Preferably, one or more -OH groups d on the primary face of the cyclodextrin moiety (see figure 1b) is ed with an -O-S-(Cnalkylene)-R group, wherein n and R are as defined herein.

Said cyclodextrin moiety does not contain any further substituents.

A substituted cyclodextrin described herein or for use described herein preferably comprises 6-10 glucopyranoside units, more preferably 6-8 units. Hence, a substituted cyclodextrin ably comprises α-cyclodextrin, β-cyclodextrin, γcyclodextrin or mixtures thereof. Further preferred is a mixture of one or more substituted α-cyclodextrins, one ore more substituted β-cyclodextrins and/or one or more substituted γ-cyclodextrins. Hence, a substituted cyclodextrin more preferably comprises α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin or mixtures thereof.

The substituted cyclodextrin is preferably substantially free of an inclusion complex compound, i.e. a compound that forms a complex with the cyclodextrin and is d inside lipophilic central cavity of the cyclodextrin.

A substituted cyclodextrin described herein or for use bed herein ses at least one substituent -S-(Cnalkylene)-R, wherein n is 1 or an integer from 3 to 10 and R is selected from the group consisting of -COOH, -OH, and - COO(1-4C)alkyl. Preferably n is an integer from 3 to 7. Substitutions are ably made through substitution of the primary hydroxyl groups d on the primary face of the glucopyranose units (see figure 1b).

A substituted cyclodextrin described herein or for use described herein preferably has the formula (I): Formula (I), n n is 1 or an integer from 3 to 10, preferably n is an integer from 3 to 7, p is an integer from 0 to 7 and q is an integer from 1 to 8 with the proviso that p + q is 6, 7 or 8. Preferably, p + q is 7 or 8.

As used herein, the term “-Cnalkylene-” refers to a branched or unbranched saturated alkylene group having n carbon atoms. For instance, Cnalkylene n n is 4 can be the following: -(CH2)4-, -C(CH3)2-CH2-, - (CH2)2-. In substituent -S-(Cnalkylene)-R, n is preferably an integer from 3 to 10, more preferably from 3 to 9, more preferably from 3 to 8, more preferably from 3 to 7, or from 3 to 5. In a particularly preferred substituted cyclodextrin, n is an integer from 3 to 7. Said substituent -S-(Cnalkylene)-R preferably is -S-(CH2)m-R, wherein m is 1 or an integer from 3 to 10, and the -(CH2)m- moiety is optionally substituted with 1 to 3 CH3 , with the proviso that the total number of carbon atoms does not exceed 10. In a further preferred embodiment, m is an integer from 3 to 10, more preferably from 3 to 9, more preferably from 3 to 8, more preferably from 3 to 7, more preferably from 3 to 5, and the -(CH2)m- moiety is optionally substituted with 1 to 3 CH3 groups, with the proviso that the total number of carbon atoms does not exceed 10. The total number of carbon atoms preferably does not exceed 8, more preferably 7, more preferably 6, more preferably . In a ularly preferred embodiment, the -(CH2)m- moiety is unsubstituted.

Therefore, a substituted extrin described herein or for use bed herein preferably has the formula (II): a (II), wherein m is 1 or an integer from 3 to 10, preferably m is an integer from 3 to 7, the -(CH2)m- moiety is optionally substituted with 1 to 3 CH3 groups with the proviso that the total number of carbon atoms does not exceed 10, p is an integer from 0 to 7 and q is an r from 1 to 8 with the proviso that p + q is 6, 7 or 8.

Preferably, p + q is 7 or 8. In a particularly preferred embodiment, the -(CH2)mmoiety is unsubstituted and m is an integer from 3 to 7, more preferably from 3 to A tuted cyclodextrin described herein or for use bed herein comprises at least one substituent -S-(Cnalkylene)-R. In a preferred embodiment, a tuted α-cyclodextrin comprises 1 to 6 of such substituents, a substituted βcyclodextrin comprises 1 to 7 of such tuents and/or a substituted γcyclodextrin comprises 1 to 8 of such substituents. In a particularly preferred ment, a substituted cyclodextrin described herein or for use described , preferably α-cyclodextrin, β-cyclodextrin and/or γ-cyclodextrin, is a monosubstituted or per-substituted cyclodextrin.

As used herein, the term “mono-substituted cyclodextrin” refers to a cyclodextrin comprising one substituent -S-(Cnalkylene)-R or - S-(CH2)m-R as defined herein. In a mono-substituted α-cyclodextrin according to formula (I) or formula (II), p is 5 and q is 1. In a mono-substituted β-cyclodextrin according to formula (I) or formula (II), p is 6 and q is 1. In a mono-substituted γ-cyclodextrin according to formula (I) or formula (II), p is 7 and q is 1.

As used herein the term “per-substituted cyclodextrin” refers to a cyclodextrin wherein all primary OH-groups are substituted with a substituent -S- (Cnalkylene)-R or - S-(CH2)m-R as d herein. Hence, a per-substituted αcyclodextrin contains 6 substituents -S-(Cnalkylene)-R or - S-(CH2)m-R as defined herein, a per-substituted β -cyclodextrin ns 7 substituents -S-(Cnalkylene)-R or - S-(CH2)m-R as defined herein and a per-substituted γ-cyclodextrin contains 8 substituents -S-(Cnalkylene)-R or - S-(CH2)m-R as defined herein. In a persubstituted odextrin according to formula (I) or formula (II), p is 0 and q is 6.

In a per-substituted odextrin according to formula (I) or formula (II), p is 0 and q is 7. In a per-substituted γ-cyclodextrin according to formula (I) or formula (II), p is 0 and q is 8.

Hence, in formula (I) and a (II), preferably p + q is either 7, whereby p is 0 and q is 7, or p is 6 and q is 1; or p + q is 8, whereby p is 0 and q is 8,or p is 7 and q is 1.

In substituent -S-(Cnalkylene)-R and in substituent - S-(CH2)m-R, wherein n and m are as defined herein, R is selected from the group consisting of COOH, -OH,and -COO(1-4C)alkyl. Most preferably, R is selected from the group consisting of COOH and OH.

Particularly preferred substituted extrins described herein or for use described herein are cyclodextrins of formula (I) wherein: - p is an integer from 0 to 7 and q is an integer from 1 to 8 with the o that p + q is 7 or 8; - n is 1 or an integer from 3 to 10, preferably n is an r from 3 to 7, more preferably from 3 to 5; and - R is selected from the group consisting of -COOH, -OH,and -COO(1- 4C)alkyl, more preferably from the group consisting of -COOH and -OH, or a pharmaceutically acceptable salt thereof. r preferred substituted cyclodextrins described herein or for use described herein are cyclodextrins of formula (I) wherein: - p + q is either 7, whereby p is 0 and q is 7, or p is 6 and q is 1; or p + q is 8, whereby p is 0 and q is 8, or p is 7 and q is 1; - n is 1 or an integer from 3 to 10, preferably n is an integer from 3 to 7, more preferably from 3 to 5; and - R is selected from the group consisting of -COOH, -OH,and -COO(1- yl, more preferably from the group consisting of -COOH and -OH, or a pharmaceutically acceptable salt thereof.

Further preferred tuted cyclodextrins described herein or for use described herein are cyclodextrins of a (II) wherein: - p + q is either 7, whereby p is 0 and q is 7; or p is 6 and q is 1; or p + q is 8, whereby p is 0 and q is 8, or p is 7 and q is 1; - m is an integer from 3 to 7, ably from 3 to 5; - the m- moiety is optionally substituted with 1 to 3 CH3 groups, preferably wherein the -(CH2)m- moiety is unsubstituted; and - R is ed from the group consisting of -COOH, -OH, and -COO(1- 4C)alkylmore preferably from the group consisting of -COOH and -OH, or a pharmaceutically acceptable salt thereof.

Further preferred substituted cyclodextrins described herein or for use described herein are cyclodextrins of formula (II) wherein: - p + q is either 7, whereby p is 0 and q is 7, or p is 6 and q is 1; or p + q is 8, whereby p is 0 and q is 8, or p is 7 and q is 1; - m is an integer from 3 to 7, preferably from 3 to 5; - the -(CH2)m- moiety is unsubstituted; and - R is ed from the group consisting of -COOH and -OH, or a pharmaceutically acceptable salt thereof.

Further preferred substituted cyclodextrins described herein or for use described herein are cyclodextrins of formula (II) wherein: - p + q is 7, y p is 0 and q is 7; - the -(CH2)m- moiety is unsubstituted; - m is 3 or 4, preferably 3; and - R is selected from the group consisting of -COOH and -OH, preferably R is COOH, or a pharmaceutically acceptable salt thereof.

Further preferred substituted cyclodextrins described herein or for use described herein are cyclodextrins of formula (II) wherein: - p + q is 7, whereby p is 6 and q is 1; - the -(CH2)m- moiety is unsubstituted; - m is an r from 3 to 7, preferably an integer from 3 to 5, more preferably an integer from 3 to 5, more preferably m is 5; and - R is selected from the group consisting of -COOH and -OH, preferably R is COOH, or a pharmaceutically acceptable salt thereof.

Further preferred substituted cyclodextrins described herein or for use described herein are cyclodextrins of formula (II) wherein: - p + q is 8, y p is 0 and q is 8; - the -(CH2)m- moiety is unsubstituted; - m is an integer from 3 to 7, preferably from 3 to 5, more preferably 3 or 5; and - R is selected from the group consisting of -COOH and -OH, preferably R is COOH, or a pharmaceutically acceptable salt or ester thereof.

Further preferred substituted cyclodextrins bed herein or for use described herein are cyclodextrins of formula (II) wherein: - p + q is 8, whereby p is 7 and q is 1; - the -(CH2)m- moiety is unsubstituted; - m is an r from 3 to 7, ably from 3 to 5, more ably 3 or 5; and - R is selected from the group consisting of -COOH and -OH, preferably R is COOH, or a pharmaceutically acceptable salt or ester thereof.

Particularly preferred substituted cyclodextrins described herein and/or used in accordance with the present disclosure are cyclodextrins of formula (II) wherein: - p is 0, q is 7, m is 3 and R is COOH; - p is 7, q is 1, m is 3 and R is COOH; - p is 0, q is 8, m is 3 and R is COOH; - p is 6, q is 1, m is 5 and R is COOH; - p is 0, q is 7, m is 5 and R is COOH; - p is 7, q is 1, m is 5 and R is COOH; - p is 0, q is 8, m is 5 and R is COOH; - p is 0, q is 8, m is 3 and R is OH; - p is 0, q is 8, m is 4 and R is COOH; - p is 0, q is 8, m is 6 and R is COOH; - p is 0, q is 8, m is 4 and R is OH; - p is 0, q is 7, m is 4 and R is COOH; - p is 0, q is 7, m is 6 and R is COOH; - p is 0, q is 7, m is 7 and R is COOH; - p is 0, q is 7, m is 3 and R is OH; - p is 5, q is 1, m is 5 and R is COOH; - p is 0, q is 6, m is 5 and R is COOH; - p is 6, q is 1, m is 6 and R is COOH; - p is 6, q is 1, m is 4 and R is OH; - p is 7, q is 1, m is 6 and R is COOH; or - p is 7, q is 1, m is 4 and R is OH, or pharmaceutically acceptable salts or esters of any of these substituted extrins. In a preferred embodiment, said substituted cyclodextrin is not 6-Per-deoxyper-(5-carboxypentyl)thio-γ-cyclodextrin sodium salt, 6-Per-deoxypercarboxypropyl)thio-γ-cyclodextrin sodium salt or 6-Perdeoxyper- (3-carboxypropyl)thio-β-cyclodextrin sodium salt. In another further preferred embodiment said pharmaceutically acceptable salt is not a sodium salt.

More preferably, a substituted extrin described herein or for use described herein is a cyclodextrins of formula (II) wherein: - p is 0, q is 8, m is 5 and R is COOH, - p is 0, q is 8, m is 4 and R is COOH, - p is 0, q is 8, m is 6 and R is COOH, - p is 0, q is 8, m is 4 and R is OH, - p is 0, q is 8, m is 3 and R is COOH - p is 0, q is 7, m is 7 and R is COOH, - p is 0, q is 7, m is 3 and R is OH, - p is 0, q is 6, m is 5 and R is COOH, - p is 6, q is 1, m is 6 and R is COOH, - p is 6, q is 1 m is 4 and R is OH, or - p is 7, q is 1, m is 4 and R is OH, or a ceutically acceptable salt or ester of any of these substituted cyclodextrins. In a red embodiment, said substituted cyclodextrin is not 6- Per-deoxyper-(5-carboxypentyl)thio-γ-cyclodextrin sodium salt or 6-Per-deoxy percarboxypropyl)thio-γ-cyclodextrin sodium salt. In another further preferred embodiment said pharmaceutically acceptable salt is not a sodium salt.

In a further preferred embodiment, a substituted cyclodextrin described herein or for use described herein is a cyclodextrin wherein: - p is 0, q is 8, m is 5 and R is COOH, - p is 0, q is 6, m is 5 and R is COOH, - p is 6, q is 1, m is 6 and R is COOH, - p is 7, q is 1, m is 3 and R is COOH, - p is 6, q is 1, m is 6 and R is COOH, - p is 6, q is 1, m is 4 and R is OH, or - p is 7, q is 1, m is 4 and R is OH, or a pharmaceutically acceptable salt or ester of any of these substituted cyclodextrins. In a preferred ment, said substituted cyclodextrin is not 6- Per-deoxyper-(5-carboxypentyl)thio-γ-cyclodextrin sodium salt or 6-Per-deoxy per- 3-carboxypropyl)thio-γ-cyclodextrin sodium salt. In another further preferred ment said pharmaceutically acceptable salt is not a sodium salt.

In another further preferred embodiment, a substituted cyclodextrin described herein or for use described herein is a cyclodextrin wherein: - p is 0, q is 6, m is 5 and R is COOH, - p is 6, q is 1, m is 6 and R is COOH, - p is 7, q is 1, m is 3 and R is COOH, - p is 6, q is 1, m is 6 and R is COOH, - p is 6, q is 1, m is 4 and R is OH, or - p is 7, q is 1, m is 4 and R is OH, or a pharmaceutically acceptable salt or ester of any of these substituted cyclodextrins.

A particularly preferred substituted cyclodextrin described herein or for use described herein is a cyclodextrin of formula (II) wherein p is 0, q is 8, m is 5 and R is COOH or an ester thereof, ably a cyclodextrin wherein of a (II) p is 0, q is 8, m is 5 and R is COOH. Another particularly preferred cyclodextrin described herein or for use described herein is a cyclodextrin of formula (II), wherein p is 0, q is 6, m is 5 and R is COOH or a pharmaceutically acceptable salt or ester thereof, preferably a cyclodextrin of formula (II) n p is 0, q is 6, m is and R is COOH.

Salts of substituted cyclodextrins described herein are also described.

Such salts can be used as procoagulants in the methods and uses described .

Such salts include, but are not d to, acid addition salts and base addition salts. The term "pharmaceutically acceptable salt" as used herein refers to those salts retain the pharmacological activity of the substituted cyclodextrins and that are, within the scope of sound medical judgment, suitable for use in humans or animals without undue toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically able salts are well-known in the art. They can be prepared in situ when isolating and purifying the substituted cyclodextrins described herein, or separately by reacting them with pharmaceutically acceptable non-toxic bases or acids, including inorganic or c bases and inorganic or organic acids, for ce by reacting the free acid or free base forms of the product with one or more equivalents of the appropriate acid or base in a solvent or medium in which the salt is insoluble, or in a solvent such as water or an organic solvent which is then d in vacuo or by freeze-drying, or by exchanging the s of an existing salt for another cation on a suitable ion exchange resin. es of ceutically acceptable acids and bases include organic and inorganic acids such as acetic acid, propionic acid, lactic acid, glycolic acid, oxalic acid, pyruvic acid, succinic acid, maleic acid, malonic acid, trifluoroacetic acid, cinnamic acid, sulfuric acid, hydrochloric acid, hydrobromic acid, nitric acid, perchloric acid and phosphoric acid, and bases such as ethylamine, methylamine, dimethylamine, triethylamine, isopropylamine, ropylamine, and other mono-, di-and trialkylamines, and arylamines, and sodium salts, potassium salts and m salts.

Esters of substituted cyclodextrins described herein are also described.

Such esters can be used as procoagulants in the methods and uses described herein. Compounds containing an ester group or bond are a well known as prodrugs for a compound containing a carboxylic acid. Such esters are activated by an esterase in vivo after stration to a patient. Such ester is preferably a cyclodextrin as defined herein wherein R is -COO(1-4C)alkyl, preferably -COO(1- 2C)alkyl.

Also described is a pharmaceutical composition comprising a substituted cyclodextrin described herein or pharmaceutically acceptable salt or ester thereof and at least one pharmaceutically acceptable auxiliary. Examples of a pharmaceutically able auxiliary include a pharmaceutically acceptable carrier, diluent and/or excipient. By "pharmaceutically acceptable" it is meant that the auxiliary, carrier, t or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. In general, any pharmaceutically suitable additive which does not interfere with the function of the active nds can be used. A ceutical composition described herein is preferably le for human use. Examples of suitable carriers comprise a on, lactose, starch, cellulose derivatives and the like, or mixtures thereof. In a preferred embodiment said le carrier is a solution, for example saline. For making dosage units, e.g. tablets, the use of conventional additives such as fillers, colorants, polymeric binders and the like, is contemplated.

Examples of excipients which can be incorporated in tablets, capsules and the like are the following: a binder such as gum tragacanth, acacia, corn starch or gelatin; an excipient such as microcrystalline cellulose; a egrating agent such as corn starch, pregelatinized starch, alginic acid and the like; a lubricant such as magnesium stearate; a sweetening agent such as sucrose, lactose or saccharin; a flavoring agent such as peppermint, oil of wintergreen or cherry. itions for intravenous administration may for example be solutions comprising the antibodies described herein in sterile isotonic aqueous buffer. The intravenous itions may include for ce solubilizing agents, stabilizing agents and/or a local anesthetic to ease the pain at the site of the ion.

In one embodiment, a pharmaceutical composition described herein is ated from systemic administration, preferably for parenteral administration, including but not limited to intravenous, intramuscular and aneous administration, or for oral administration, including but not limited to tablets, capsules, liquids, emulsions, suspensions.

A ceutical composition described herein is preferably suitable for topical stration, for topical (local) treatment of bleeding. A pharmaceutical composition is therefore preferably formulated for topical administration, preferably as a gel, cream, ointment, spray, mouth wash, eye drops, ng, compress, plaster, band-aid or patch. Such topical ations are particularly suitable for use in treatment of a wound and/or local (major) bleeding.

Further described is a kit of parts comprising: - a substituted cyclodextrin or pharmaceutically acceptable salt or ester thereof described herein, and - a recombinant or ed coagulation factor.

The term “isolated recombinant ation factor” refers to a coagulation that is recombinantly produced or isolated from blood or plasma. red, but not limiting, coagulation factors are factor V, factor VII, factor VIII, factor IX, factor X, factor XI, factor XIII, alpha2-antiplasmin, von Willebrand factor. Particularly preferred are factor VIII and factor IX. Preferably, a coagulation factor present in a kit of parts described herein is recombinant coagulation factor.

Such kit of parts is particularly suitable to provide a combination therapy for treatment of patients that are deficient in a coagulation factor, such as patients suffering from hemophilia A, hemophilia B, Von Willebrand disease or hemophilia C. The use of such kit of parts has the advantage that less ed or recombinant coagulation factor is needed for treatment of such patients.

In a preferred embodiment, a kit of parts described herein comprises factor VIII as the recombinant or ed ation factor and a substituted cyclodextrin selected from the group of cyclodextrins of formula (II) wherein: - p is 0, q is 8, m is 5 and R is COOH, - p is 0, q is 8, m is 4 and R is COOH, - p is 0, q is 8, m is 6 and R is COOH, - p is 0, q is 8, m is 4 and R is OH, - p is 0, q is 7, m is 7 and R is COOH, - p is 0, q is 7, m is 3 and R is OH, - p is 0, q is 6, m is 5 and R is COOH, - p is 6, q is 1, m is 6 and R is COOH, - p is 6, q is 1 m is 4 and R is OH or - p is 7, q is 1, m is 4 and R is OH, or a pharmaceutically acceptable salt or ester thereof.

In another preferred embodiment, a kit of parts described herein comprises factor IX as the recombinant or isolated coagulation factor and a substituted cyclodextrin selected from the group of cyclodextrins of a (II) wherein: - p is 0, q is 8, m is 5 and R is COOH, - p is 0, q is 8, m is 6 and R is COOH, - p is 0, q is 8, m is 4 and R is OH, - p is 0, q is 7, m is 7 and R is COOH, - p is 0, q is 7, m is 3 and R is OH, - p is 6, q is 1, m is 6 and R is COOH, - p is 6, q is 1 m is 4 and R is OH or - p is 7, q is 1, m is 4 and R is OH, or a ceutically acceptable salt or ester thereof.

In r preferred embodiment, a kit of parts described herein comprises factor XI as the recombinant or isolated coagulation factor and a tuted cyclodextrin is a cyclodextrin of formula (II) wherein p is 0, q is 8, m is and R is COOH or a pharmaceutically acceptable salt or ester thereof.

In one embodiment described herein, a kit of parts is described comprising one or more containers filled with a substituted cyclodextrin or ceutically acceptable salt or ester f described herein and a recombinant or isolated coagulation factor. Associated with such containers can be various written materials such as instructions for use, or a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals products, which notice reflects approval by the agency of cture, use, or sale for human or veterinary administration. Preferably, a kit of parts described herein comprises instructions for use.

As used herein, the term lation” refers to the process of polymerization of fibrin monomers, resulting in the formation of a blood clot, whereby blood or plasma changes from a liquid to a gel phase. As used herein, the term “use as procoagulant” refers to initiating or accelerating the process of blood clot formation. Any methods known in the art can be used for determining procoagulant effect of a substituted cyclodextrin as bed herein, e.g. measuring thrombin generation and/or the length of time before blood clot formation in plasma or blood samples. A particularly suitable method is described in the Examples herein for measuring thrombin generation. In brief, normal plasma is spiked with cyclodextrin and ally with an anticoagulant.

Coagulation is red by ification in the presence of, e.g. recombinant human, tissue factor and fluorogenic substrate Z-Gly-Gly-Arg-AMC. Fluorescence can be monitored and followed by calculation of lag time for thrombin formation, peak thrombin, velocity index and area under the curve.

Also described is the use of a substituted cyclodextrin sing at least one substituent -S-(Cnalkylene)-R, wherein n is an integer from 3 to 10, preferably 3 to 7, and R is selected from the group consisting of -COOH, -OH,and - COO(1-4C)alkyl, or a pharmaceutically acceptable salt thereof as a procoagulant.

Also described is a method for procoagulation comprising administering to a subject in need thereof, a therapeutically effective amount of a substituted cyclodextrin comprising at least one substituent -S-(Cnalkylene)-R, wherein n is an integer from 3 to 10 and R is ed from the group consisting of -COOH, -OH, and -COO(1-4C)alkyl, or a pharmaceutically acceptable salt thereof. Also described is a method for ng or stimulating coagulation in a subject in need thereof comprising administering to the subject a therapeutically ive amount of a substituted cyclodextrin comprising at least one substituent -S-(Cnalkylene)-R, wherein n is an integer from 3 to 10 and R is selected from the group consisting of - COOH, -OH, and -COO(1-4C)alkyl, or a pharmaceutically acceptable salt thereof.

The cyclodextrins described herein are particularly suitable for reversing the agulant effect of an agulant, i.e. as an antidote for agulants. As demonstrated in the es, the substituted cyclodextrins were capable of reversing the anticoagulant activity of all anticoagulants tested.

Described is therefore a substituted cyclodextrin described herein or pharmaceutically able salt or ester thereof for use in a method for reversing an anticoagulant effect of an anticoagulant in a subject. Also described is a method for reversing an anticoagulant effect of an anticoagulant in a subject in need thereof, the method comprising administering to the subject, which subject has been administered said anticoagulant, a therapeutically effective amount of a substituted extrin described herein or pharmaceutically able salt or ester thereof. Further described is substituted cyclodextrin described herein or pharmaceutically acceptable salt or ester thereof for the ation of a medicament for reversing an anticoagulant effect of an anticoagulant in a subject.

The term “anticoagulant” as used herein refers to an agent or nd capable of preventing or delaying blood clot formation in vitro and/or in vivo.

As used herein, the term “reversing an anticoagulation effect of an anticoagulant” refers to decreasing the ability of the anticoagulant to prevent or delay blood clot formation. Hence, the agulation effect of the anticoagulant is at least partially reversed. In particular, the term refers to a shortening of the time to initiation of blood clot formation or to an increase in strength of the blood clot in the ce of a tuted cyclodextrin as described herein and an anticoagulant as compared to the time to tion to blood clot formation or strength of blood clot in the presence of the anticoagulant but in the absence of the substituted cyclodextrin. Any s known in the art can be used for determining procoagulant effect of a substituted cyclodextrin as described herein, e.g. measuring thrombin generation, blood clot strength and/or the length of time before clot ion in plasma or blood samples or in an in vivo bleeding model. A suitable thrombin formation assay is described in the Examples and above. A suitable in vivo bleeding assay is also described in the Examples. In brief, the saphenous veins in the hind limb of anesthetized mice are transected by piercing with a needle followed by an incision. Blood is gently wicked away until haemostasis occurs. The clot is then d and blood is again wicked away until haemostasis, which is repeated for 30 minutes. Parameters that can be assessed are the number of times that haemostasis occurs in 30 minutes and the time required for each haemostasis.

The anticoagulant can be any anticoagulant known in the art, since the extrins described herein have demonstrable activity against all tested anticoagulants. In a preferred embodiment, the anticoagulant is selected from the group consisting of: - a direct thrombin inhibitor, such as dabigatran, n, bivalirudin, lepirudin or oban, - a direct factor Xa tor, such as xaban, apixaban, edoxaban, aban, darexaban, ban or eribaxaban, - a pentasaccharide, such as fondaparinux or idraparinux, - a low molecular weight heparins, such as nadroparin, tinzaparin, dalteparin, enoxaparin, bemiparin, reviparin, parnaparin or certoparin, - unfractionated heparin, - a vitamin K antagonist, such as acenocoumarol, phenprocoumon, warfarin, atromentin or phenindione, and - an antiplatelet drug, such as an irreversible cyclooxygenase tors (such as aspirin or a derivative thereof or triflusal), an ADP receptor tor (such as clopidogrel, prasugrel, ticagrelor, ticlopedine, cangrelor or rel), a phosphodiesterase tor (such as cilostazol), a PAR-1 antagonist (such as voraxapar), a GPIIB/IIIa inhibitor (such as abciximab, eptifibatide, tirofiban, roxifiban or orbofiban), an adenosine reuptake inhibitor (such as dipyridamole), a thromboxane inhibitor (such as ifetroban or picotamide) or a thromboxane receptor nist (such as terutroban or picotamide).

It is noted that this list is non-exhaustive, many other anticoagulants ing to the listed categories of anticoagulants are known to a person skilled in the art.

Their anticoagulant effects can also be reversed using the substituted cyclodextrins described herein. In a specific embodiment described herein, the anticoagulant is selected from the group consisting of dabigatran, rivaroxaban, apixaban, edoxaban, fondaparinux, nadroparin, tinzaparin, unfractionated heparin, hirudin, rudin and a vitamin K antagonist. In one preferred embodiment, the anticoagulant is selected from the group ting of dabigatran, rivaroxaban, apixaban and edoxaban.

In a preferred embodiment, the substituted cyclodextrin used described herein for reversing an anticoagulant effect of an anticoagulant in a subject is selected from the group consisting of OKL-1105, OKL-1106, OKL-1107, OKL-1108, OKL-1109, OKL-1110, OKL-1111, OKL-1146, OKL-1171, OKL-1172, OKL-1174, OKL-1178, OKL-1180, OKL-1181, OKL-1186, OKL-1187, OKL-1188, OKL-1189, OKL-1190, OKL-1191, the structures of which are indicated in table 1, and combinations thereof, more preferably selected from the group consisting of OKL- 1105, OKL-1106, OKL-1107, OKL-1108, OKL-1110, OKL-1111, OKL-1146, OKL- 1171, OKL-1172, OKL-1174, OKL-1178, OKL-1180, 81, OKL-1186, OKL- 1187, OKL-1188, OKL-1189, OKL-1190, OKL-1191 and ations thereof. In a particularly preferred embodiment, the substituted cyclodextrin used described herein for reversing an anticoagulant effect of an agulant in a subject is OKL-1111, a per-substituted γ-cyclodextrin of formula (II), wherein the substituent is 2)5-COOH or a ceutically acceptable salt or ester thereof, or OKL- 1187, a per-substituted α-cyclodextrin of formula (II), wherein the substituent is -S- (CH2)5-COOH or a pharmaceutically acceptable salt or ester thereof.

Whether or not the effect of a particular anticoagulant can be reversed with a particular substituted cyclodextrin described herein can be readily assessed by a d person, for ce by performing a coagulation assay as described in the Examples herein. In such assay, normal human plasma ning the anticoagulant is incubated with and without the substituted cyclodextrin and one or more of the parameters as described herein (lag time for thrombin formation, peak thrombin, velocity index and area under the curve) are determined to assess whether the substituted cyclodextrin is able to reverse the anticoagulant effect in the specific plasma sample.

The Examples further show that the cyclodextrins described herein have a procoagulant effect in plasma of patients that are deficient in one of the coagulation s. The cyclodextrins described herein are ore further particularly suitable for antagonizing blood coagulation disorders, i.e. as prohemostatic agents. bed is therefore a substituted cyclodextrin described herein or a pharmaceutically acceptable salt or ester thereof for use in a method for the treatment or prevention of a blood coagulation disorder. Also decribed is a method for the treatment or prevention of a blood ation disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a substituted cyclodextrin described herein or a pharmaceutically acceptable salt or ester f. Also decribed is a tuted cyclodextrin described herein or a pharmaceutically acceptable salt or ester thereof for the preparation of a medicament for the treatment or prevention of a blood coagulation disorder.

As used herein, the term “blood coagulation disorder” refers to a disease which causes an anomaly in the hemostatic and/or coagulation system. Such disorder is typically characterized by a tendency to bleeding. Well known examples of blood coagulation disorders include hemophilia A, which is characterized by a deficiency of coagulation factor VIII (FVIII), hemophilia B, which is characterized by a deficiency of coagulation factor IX (FIX) and hemophilia C, which is characterized by a deficiency of coagulation factor XI (FXI). Whether or not a particular blood coagulation er is ble with a particular substituted cyclodextrin described herein is can be readily assessed by a skilled person, for instance by performing a coagulation assay as described in the Examples herein. In such assay, plasma of one or more ts suffering from the blood coagulation disorder is incubated with and t the substituted cyclodextrin and one or more of the parameters as bed herein (lag time for thrombin formation, peak thrombin, velocity index and area under the curve) are determined to assess whether the substituted cyclodextrin has a procoagulant effect in the ic plasma . Preferred, but non-limiting, examples of blood ation disorders are congenital or acquired hemophilia A, ilia B, hemophilia C, von Willebrand disease, coagulation factor deficiency, such as factor V, factor VII, and/or factor X deficiency, factor XIII or alpha2-antiplasmin deficiency, hereditary or drug-induced thrombocytopenia, including immune thrombocytopenia purpura, thrombotic thrombocytopenic purpura, fetal or al alloimmune thrombocytopenia and post-transfusion thrombocytopenic purpura, Wiskott- Aldrich Syndrome, Glanzmann's thrombasthenia, d-Soulier Syndrome, idiopathic dense-granule er, Hermansky-Pudlak syndrome, Chediak-Higashi syndrome, gray platelet syndrome, Paris-Trousseau/Jacobsen's syndrome, disseminated intravascular coagulation and vitamin K deficiency, including n K deficiency of the newborn. In a red ment described herein, the blood coagulation disorder is selected from the group consisting of said disorders. In a further preferred embodiment, said disorder is deficiency of a coagulation factor deficiency, in particular deficiency of a coagulation factor selected from the group consisting of factor V, factor VII, factor VIII, factor IX, factor X, factor XI, factor XIII and -antiplasmin. Particularly preferred disorders that is treated or prevented in accordance with the present disclosure is selected from the group consisting of hemophilia A, hemophilia B and ilia In a preferred ment, the substituted cyclodextrin used described herein for treatment or prevention of a blood coagulation disorder, preferably ilia A, hemophilia B or hemophilia C, is selected from the group consisting of OKL-1111, OKL-1171, OKL-1172, OKL-1174, OKL-1180, OKL-1181, OKL-1187, OKL-1188, OKL-1189, OKL-1191, preferably selected from the group consisting of OKL-1111, OKL-1172, OKL-1180, OKL-1187, OKL-1188, OKL-1189 and OKL- 1191, preferably selected from the group consisting of OKL-1111, OKL-1180 and OKL-1187, the structures of which are indicated in table 1, and combinations thereof. In a further preferred embodiment, the substituted cyclodextrin used described herein for treatment or tion of a blood coagulation disorder, preferably hemophilia A, hemophilia B or hemophilia C, is a substituted cyclodextrin of formula (II), wherein m is an integer from 5 to 10, more preferably from 5 to 7, more preferably 5, p is 0 and q is 7 or 8. In a particularly preferred embodiment, the substituted cyclodextrin used described herein for treatment or tion of a blood coagulation disorder, preferably hemophilia A, hemophilia B or hemophilia C, is selected from the group consisting of OKL-1111, OKL-1172, OKL-1180, 87, 88, OKL-1189 en OKL1191, or mixtures thereof, preferably 11, a per-substituted odextrin of formula (II), wherein the substituent is -S-(CH2)5-COOH or a pharmaceutically acceptable salt or ester thereof.

In a preferred ment the blood coagulation disorder is ilia A and the substituted cyclodextrin is a cyclodextrin of formula (II) wherein: - p is 0, q is 8, m is 5 and R is COOH, - p is 0, q is 8, m is 4 and R is COOH, - p is 0, q is 8, m is 6 and R is COOH, - p is 0, q is 8, m is 4 and R is OH, - p is 0, q is 7, m is 7 and R is COOH, - p is 0, q is 7, m is 3 and R is OH, - p is 0, q is 6, m is 5 and R is COOH, - p is 6, q is 1, m is 6 and R is COOH, - p is 6, q is 1 m is 4 and R is OH or - p is 7, q is 1, m is 4 and R is OH, or a pharmaceutically acceptable salt or ester of any of these substituted cyclodextrins.

In another preferred embodiment the blood coagulation disorder is ilia B and the substituted cyclodextrin is a cyclodextrin of formula (II) wherein: - p is 0, q is 8, m is 5 and R is COOH, - p is 0, q is 8, m is 6 and R is COOH, - p is 0, q is 8, m is 4 and R is OH, - p is 0, q is 7, m is 7 and R is COOH, - p is 0, q is 7, m is 3 and R is OH, - p is 6, q is 1, m is 6 and R is COOH, - p is 6, q is 1 m is 4 and R is OH or - p is 7, q is 1, m is 4 and R is OH, or a pharmaceutically acceptable salt or ester of any of these substituted cyclodextrins.

In another preferred embodiment the blood coagulation disorder is hemophilia C and the substituted cyclodextrin is a cyclodextrin of formula (II) wherein p is 0, q is 8, m is 5 and R is COOH or a pharmaceutically able salt or ester thereof.

The es further show that the cyclodextrins described herein have a procoagulant effect in normal plasma both in the presence and absence of an anticoagulant. Hence, the cyclodextrins described herein are also particularly suitable for reducing or preventing bleeding in a subject, i.e. as prohemostatic agents in a bleeding situation, irrespective of the cause of bleeding. Further described is therefore a substituted cyclodextrin described herein or a pharmaceutically acceptable salt or ester thereof for use in a method for reducing or preventing ng in a subject. Also bed is a method for reducing or preventing ng in a subject in need thereof, the method comprising administering to the subject a eutically effective amount of a substituted cyclodextrin bed herein or a pharmaceutically acceptable salt or ester thereof.

Also described is a substituted cyclodextrin bed herein or a ceutically acceptable salt or ester thereof for the preparation of a medicament for reducing or preventing bleeding in a subject.

Preferably, said subject has been treated with an anticoagulant, is undergoing surgery, is undergoing dental treatment, is suffering from trauma, is suffering from induced or spontaneous major bleeding, such as intracranial or gastro-intestinal bleeding, and/or is suffering from or at risk of hereditary or druginduced thrombocytopenia.

The anticoagulant can be any anticoagulant known in the art, since the cyclodextrins bed herein have demonstrable activity against all tested anticoagulants. In a preferred ment, the anticoagulant is selected from the group consisting of: - a direct thrombin inhibitor, such as tran, hirudin, rudin, lepirudin or argatroban, - a direct factor Xa inhibitor, such as rivaroxaban, apixaban, edoxaban, betrixaban, darexaban, letaxaban or eribaxaban, - a pentasaccharide, such as fondaparinux or rinux, - a low molecular weight n, such as nadroparin, tinzaparin, dalteparin, enoxaparin, bemiparin, reviparin, parnaparin or certoparin, - unfractionated heparin, - a vitamin K nist, such as acenocoumarol, phenprocoumon, warfarin, atromentin or phenindione, and - an antiplatelet drug, such as an irreversible cyclooxygenase inhibitors (such as aspirin or a derivative thereof or triflusal), an ADP or inhibitor (such as clopidogrel, prasugrel, ticagrelor, ticlopedine, cangrelor or elinogrel), a phosphodiesterase inhibitor (such as cilostazol), a PAR-1 antagonist (such as voraxapar), a IIIa tor (such as abciximab, eptifibatide, tirofiban, roxifiban or orbofiban), an adenosine reuptake inhibitor (such as dipyridamole), a thromboxane inhibitor (such as ifetroban or picotamide) or a thromboxane receptor antagonist (such as terutroban or picotamide).

It is noted that this list is non-exhaustive, many other anticoagulants belonging to the listed categories of anticoagulants of which the effects can be reversed with the methods or used described herein are known to a person skilled in the art. In a specific embodiment described herein, the anticoagulant is ed from the group ting of tran, rivaroxaban, apixaban, edoxaban, fondaparinux, nadroparin, tinzaparin, unfractionated heparin, hirudin, bivalirudin and a vitamin K antagonist.

In a preferred embodiment, the substituted extrin used described herein for reducing or preventing bleeding in a subject is selected from the group consisting of a cyclodextrin of formula (II) wherein: - p is 0, q is 7, m is 3 and R is COOH; - p is 7, q is 1, m is 3 and R is COOH; - p is 0, q is 8, m is 3 and R is COOH; - p is 6, q is 1, m is 5 and R is COOH; - p is 0, q is 7, m is 5 and R is COOH; - p is 7, q is 1, m is 5 and R is COOH; - p is 0, q is 8, m is 5 and R is COOH; - p is 0, q is 8, m is 3 and R is OH; - p is 0, q is 8, m is 4 and R is COOH; - p is 0, q is 8, m is 6 and R is COOH; - p is 0, q is 8, m is 4 and R is OH; - p is 0, q is 7, m is 4 and R is COOH; - p is 0, q is 7, m is 6 and R is COOH; - p is 0, q is 7, m is 7 and R is COOH; - p is 0, q is 7, m is 3 and R is OH; - p is 5, q is 1, m is 5 and R is COOH; - p is 0, q is 6, m is 5 and R is COOH; - p is 6, q is 1, m is 6 and R is COOH; - p is 6, q is 1, m is 4 and R is OH; - p is 7, q is 1, m is 6 and R is COOH; or - p is 7, q is 1, m is 4 and R is OH, or a pharmaceutically acceptable salt or ester thereof, more preferably selected from the group consisting of OKL-1105, OKL-1106, OKL-1107, OKL-1108, OKL- 1109, OKL-1110, 11, OKL-1146, OKL-1171, OKL-1172, OKL-1174, OKL- 1178, OKL-1180, 81, OKL-1186, OKL-1187, OKL-1188, OKL-1189, OKL- 1190, OKL-1191 or a pharmaceutically able salt or ester thereof and combinations thereof, more preferably selected from the group consisting of OKL- 1105, OKL-1106, OKL-1107, OKL-1110, OKL-1111, OKL-1146, OKL-1172, OKL- 1174, OKL-1180, 81, OKL-1187, OKL-1188, OKL-1189, 91 or a pharmaceutically acceptable salt or ester thereof and combinations thereof, more preferably selected from the group consisting of OKL-1106, OKL-1107, OKL-1111, OKL-1146, OKL-1172, OKL-1174, 80, OKL-1187, OKL-1188, OKL-1189, OKL-1191 or a pharmaceutically able salt or ester thereof and combinations thereof, more preferably selected from the group consisting of OKL-1106, OKL- 1111, 74, OKL-1187, OKL-1188, OKL-1189, OKL-1191 or a ceutically acceptable salt or ester thereof and combinations thereof. In a further preferred embodiment, the substituted cyclodextrin used described herein for reducing or preventing bleeding in a subject is a substituted cyclodextrin of formula (II), wherein m is an integer from 5 to 10, more preferably from 5 to 7, more preferably 5, p is 0 and q is 7 or 8. In a particularly preferred embodiment, the substituted cyclodextrin used described herein for reducing or ting bleeding in a subject is OKL-1111, a per-substituted γ-cyclodextrin of a (II), wherein the substituent is -S-(CH2)5-COOH or a pharmaceutically acceptable salt or ester thereof, or OKL-1187, a per-substituted α-cyclodextrin of a (II), wherein the substituent is -S-(CH2)5-COOH or a pharmaceutically acceptable salt or ester f.

The term "therapeutically effective amount" as used herein refers to the amount of the pharmaceutical composition, which provides a therapeutic benefit in the prevention, ent, or management, of the disease being treated.

As used , the term ct” asses humans and animals, preferably mammals. Preferably, a subject is a mammal, more preferably a human.

In a particular embodiment, a subject is a patient that has been treated with an anticoagulant, is suffering from a blood coagulation disorder, is undergoing surgery, is undergoing dental treatment, is suffering from trauma, is suffering from induced or spontaneous major bleeding, such as intracranial or gastrointestinal bleeding, and/or is suffering from or at risk of hereditary or drug-induced thrombocytopenia.

As used herein, the term “prevention” refers to preventing or delaying the onset of a disease and/or the appearance of clinical ms of the disease in a subject that does not yet experience clinical symptoms of the disease. The term “treatment” refers to ting the disease, i.e., halting or reducing its development or at least one clinical symptom f, and to relieving symptoms of the disease.

The substituted cyclodextrins described herein can be prepared using any method known in the art for the preparation of extrins. Particularly suitable methods for the preparation of substituted cyclodextrins, in particular substituted α-cyclodextrins, substituted β-cyclodextrins and substituted γcyclodextrins , starting from cially available intermediate cyclodextrins and their purification are described in the Examples.

Features may be described herein as part of the same or separate aspects or embodiments of the present invention for the purpose of clarity and a concise ption. It will be appreciated by the skilled person that the scope of the invention may include embodiments having combinations of all or some of the features described herein as part of the same or separate embodiments.

The invention will be explained in more detail in the following, nonlimiting examples.

Brief description of the drawings Figure 1: Basic structure of α-, β- and γ-cyclodextrins. a. al structure; b. 3-D structure.

Figure 2: Structures of OKL-1108 (A), OKL-1109 (B), OKL-1110 (C) and OKL-1111 (D).

Figures 3-8, 12, 14-19 and 22-27: Pooled normal plasma was spiked with cyclodextrins (100 µM, unless otherwise indicated) and anticoagulants. The concentrations of the anticoagulants were 100 ng/ml for dabigatran (A), 100 ng/ml for rivaroxaban (B), 60 ng/ml for apixaban (C), 60 ng/ml for edoxaban (D). The plasmas were subjected to thrombin generation analysis as described in the Materials and Methods n with 1 pM tissue factor (TF) as initiator of coagulation.

Figure 9: Pooled normal plasma was spiked with OKL-1111 (100 µM, unless otherwise indicated (A)) and anticoagulants (B-E). The concentrations of the anticoagulants were 100 ng/ml for dabigatran (B), 100 ng/ml for rivaroxaban (C), 60 ng/ml for apixaban (D), 60 ng/ml for edoxaban (E). The s were subjected to thrombin generation analysis as described in the Materials and Methods n with 1 pM tissue factor (TF) as initiator of coagulation.

Figure 10: Pooled normal plasma was spiked with OKL-1111 (100 µM) and anticoagulants (A-F). The concentrations of the anticoagulants were 2 µg/ml for fondaparinux (A), 0.4 U/ml for nadroparin (B), 0.1 U/ml for tinzaparin (C), 0.03 U/ml for unfractionated heparin (UFH) (D), 0.5 U/ml for hirudin (E) and 10 µg/ml for bivalirudin (F). The plasmas were subjected to thrombin generation analysis as described in the Materials and Methods section with 1 pM tissue factor (TF) as initiator of coagulation.

Figure 11: Plasma was used from individuals taking vitamin K-antagonists (VKA plasma). Two different ities of treatment (given as INR) were available, as depicted in (A) and (B). The s were subjected to thrombin generation is as bed in the Materials and Methods section with 1 pM tissue factor (TF) as initiator of coagulation.

Figure 13: Pooled normal plasma was spiked with OKL-1147 (100 µM, unless ise indicated (A)) and anticoagulants (B-E). The concentrations of the anticoagulants were 100 ng/ml for dabigatran (B), 100 ng/ml for rivaroxaban (C), 60 ng/ml for apixaban (D), 60 ng/ml for edoxaban (E). The plasmas were ted to in generation analysis as described in the Materials and Methods section with 1 pM tissue factor (TF) as initiator of coagulation.

Figure 20: Plasma ent in coagulation factor VIII was spiked with cyclodextrin OKL-1111 (A), OKL-1180 (B), and OKL-1187 (C) at the indicated concentrations.

Figure 21: Effect of OKL-1111 on normal plasma spiked with inhibitory antibodies against factor VIII (A); factor IX (B), and factor XI (C).

Figure 28: Effect of OKL-1111 (A), OKL-1180 (B) and OKL-1187 (C) on coagulation in plasma of a hemophilia A patient with anti-factor VIII antibodies. BU = Bethesda Units.

Figure 29: In vivo analysis of procoagulant ial of OKL-1111 and OKL-1147.

Except for controls, s were treated with rivaroxaban for 4 days and cyclodextrins were administered 5 min prior bleeding assay.

Figure 30: Effect of OKL-1111 and OKL-1187 on coagulation in mouse hemophilia A assay.

Examples Example 1 Materials and s Synthesis of extrins General Procedure for the synthesis of decorated β-cyclodextrins with thiols.

For the synthesis of mono-decorated β -cyclodextrin derivatives, a solution of 6- monotosyl-β-cyclodextrin (500 mg, 0.388 mmol, 1.0 equiv.) in DMSO (3 mL) was degassed. The on was added dropwise to a degassed solution of the riate thiol (H-S-R; 4.67 mmol, 12 equiv) and NaOH (460 mg, 11.5 mmol, 30 equiv) in DMSO/H2O (4 mL/2 mL). The suspension was stirred overnight at 50 °C.

The reaction mixture was allowed to cool to room temperature. Methanol (8 mL) was added. The white precipitate was filtered and washed with methanol. The itate was dissolved in H2O (5 mL) and the pH was adjusted to 7 with aqueous 3 M HCl. The solution was poured into MeOH (8-16 mL) or acetone. The precipitate was filtered, washed with methanol or acetone and dried under reduced pressure.

The synthesis of per-decorated β-cyclodextrin derivatives with a sulfur tether was performed using commercial heptakis-(6-bromodeoxy)-β-cyclodextrin as starting material. The reactions with the appropriate thiol (H-S-R) and NaOH were performed successfully with NaH as base in DMF with ng overnight at room temperature.

Scheme 1 shows the on for per-decorated β-cyclodextrin derivatives.

Scheme 1: Procedure for the sulphur tethering of beta-cyclodextrins General Procedure for the synthesis of decorated γ-cyclodextrins with thiols.

For the synthesis of per-decorated γ -cyclodextrin derivatives a solution of Octakis- 6-bromodeoxy-γ-cyclodextrin (1.8 g, 1 mmol, 1.0 equiv.) in DMSO (9 mL) was degassed. The solution was added dropwise to a degassed solution of the appropriate thiol (12.5 mmol, 12.5 equiv) and NaOH (1.1 g, 27.5 mmol, 27 equiv) in DMSO/H2O (12 mL/6 mL). The suspension was stirred overnight at 50 °C. The on mixture was allowed to cool to room temperature. Methanol (80 mL) was added. The white precipitate was filtered and washed with methanol. The precipitate was dissolved in H2O (50 mL) and the pH was adjusted to 7 with s 3 M HCl. The solution was poured into EtOH (100 mL) or acetone. The precipitate was filtered, washed with methanol or acetone and dried under d pressure.

For the synthesis of ecorated γ-cyclodextrin derivatives, monotosylated γcyclodextrines were functionalized in a r fashion as described for the mono- decorated beta-cyclodextrins.

Scheme 2: Procedure for the r tethering of gamma-cyclodextrins Analogous to the beta-cyclodextrins, the γ-cyclodextrins were functionalized (see scheme 2 for the per-substituted γ-cyclodextrins). The compounds were synthesized using NaOH and DMSO as solvent, providing ult isolations but eventually addition of EtOAc led to good precipitation.

Purification In general, the onalized cyclodextrins were isolated by precipitation from a suitable t, followed by several washings with solvents to remove excess of ts and side-products. Often this procedure provided materials that were considered pure for the ation based on either 1H NMR (often broad peaks or especially in the case of mono-substitution rather complex spectra were observed) or HPLC-MS or the combination of both. In a number of cases the reaction towards the ted cyclodextrin had to be repeated to prepare a new batch in order to isolate pure product. In addition, other methods to purify cyclodextrins were made, including normal phase chromatography, reversed-phase chromatography and preparative-HPLC.

Synthesis of decorated alpha cyclodextrins Alpha-mono-S-C6-acid (OKL-1186) was prepared according to the general procedure described above using 6-Mercaptohexanoic acid (131 µl, 140 mg, 0.943 mmol), NaOH (38 mg, 0.0925 mmol) and 6-monodeoxymonoiodo-α-cyclodextrin (200 mg, 0.185 mmol). Other alpha-mono-substituted cyclodextrins described herein can be prepared in the same way using the appropriate starting compounds.

Alpha-per-S-C6-acid (OKL-1187): Under a N2 atmosphere, NaH (70 mg, 1.70 mmol, 23.0 eq.) was suspended in DMF (5 mL). A solution of 6-mercaptohexanoic acid (134 mg, 0.897 mmol, 12.1 eq.) in DMF (2 mL) was added se. After 10 minutes, hexakis-(6-bromodeoxy)-α-cyclodextrin (102 mg, 0.0741 mmol, 1.0 eq.) was added and the on mixture was stirred at room temperature overnight.

The reaction mixture was precipitated by addition of acetone (large ), filtered and washed with acetone. The precipitate was dissolved in demi-water (5 mL) and the pH was adjusted to just below 7 with a 3 M HCl solution in demi-water. The resulting suspension was diluted with e, filtered, washed with acetone and dried in vacuo to give the product. Other alpha-per-substituted extrins described herein can be prepared in the same way using the appropriate starting compounds.

Tables 1 and 2 shows the cyclodextrins that have been prepared. Figure 2 shows the structure of four ary mono- and per-substituted, beta- and gamma cyclodextrins (compounds OKL-1108, 09, OKL-1110 and OKL-1111).

Table 1. Cyclodextrins with procoagulant ty. compound cyclodextrin substitution substituent type pattern OKL-1105 beta per -S-(CH2)3-COOH OKL-1106 gamma mono -S-(CH2)3-COOH OKL-1107 gamma per -S-(CH2)3-COOH OKL-1108 beta mono -S-(CH2)5-COOH OKL-1109 beta per -S-(CH2)5-COOH OKL-1110 gamma mono -S-(CH2)5-COOH OKL-1111 gamma per -S-(CH2)5-COOH OKL-1146 gamma per -S-(CH2)3-OH OKL-1171 gamma per -S-(CH2)4-COOH OKL-1172 gamma per -S-(CH2)6-COOH OKL-1174 gamma per -S-(CH2)4-OH OKL-1178 beta per -S-(CH2)4-COOH OKL-1179 beta per -S-(CH2)6-COOH OKL-1180 beta per 2)7-COOH OKL-1181 beta per 2)3-OH OKL-1186 alpha mono -S-(CH2)5-COOH OKL-1187 alpha per -S-(CH2)5-COOH 88 beta mono -S-(CH2)6-COOH OKL-1189 beta mono -S-(CH2)4-OH OKL-1190 gamma mono -S-(CH2)6-COOH OKL-1191 gamma mono -S-(CH2)4-OH Table 2. Comparative cyclodextrins. compound cyclodextrin substitution substituent type n OKL-1100 beta mono -S-(CH2)2-COOH OKL-1101 beta per -S-(CH2)2-COOH OKL-1102 gamma mono -S-(CH2)2-COOH OKL-1103 gamma per -S-(CH2)2-COOH OKL-1147 gamma per -NH2 OKL-1170 gamma per -S-(CH2)2-COOH Coagulation assays The Calibrated Automated Thrombogram® assays the generation of thrombin in clotting plasma using a microtiter plate reading fluorometer (Fluoroskan Ascent, ThermoLab systems, Helsinki, Finland) and Thrombinoscope® software (Thrombinoscope BV, Maastricht, The Netherlands). The assay was carried out as bed by Hemker et al. fysiol. Haemost. Thromb. 2003, 33, 4-15), and the Thrombinoscope® manual. Coagulation was triggered by recalcification in the presence of 1 or 5 pM recombinant human tissue factor in®, Siemens, Marburg, Germany), 4 μM phospholipids, and 417 μM genic substrate Z-Gly- Gly-Arg-AMC (Bachem, Bubendorf, Switzerland). Fluorescence was monitored using the Fluoroskan Ascent fluorometer (ThermoLabsystems, Helsinki, Finland), and the lag time, peak thrombin, velocity index and area under the curve (ETP) were calculated using the Thrombinoscope® software (Thrombinoscope BV).

In vivo bleeding model All animal protocols were approved by the Institutional Animal Care and Use Committee of the sity of North Carolina. C57BL6/J mice were purchased from Charles Rivers Laboratories (Willmington, MA). Bleeding studies were done essentially as previously bed Pastoft et al. (Haemophilia 2012; 18:782–8).

Mice were anesthetized with rane throughout all procedures. The hair on the ventral side of both hind limbs was removed. The s were placed supine on a temperature and ECG monitoring board. The paws were gently restrained by looping soft polyethylene tubing around them and attaching the tubing to the ECG board. The skin on the left and right ventral hind limb was incised which exposes a length of the saphenous neurovascular bundle; the bundle was covered with normal saline to prevent drying. To assess haemostasis, the right saphenous vein was transected by piercing it with a 23-G needle followed by a longitudinal incision made in the distal portion of the vessel. Blood was gently wicked away until haemostasis ed. The clot was then removed to restart bleeding and the blood was again wicked away until haemostasis occurs again. Clot disruption was repeated after every incidence of haemostasis for 30 s. Mice were fed chow that contained 0.1 mg rivaroxaban per g of chow. The mice were on this diet for 4 days to allow them to reach a steady state. Cyclodextrins were administered by a tail vein injection 5 minutes before the start of the ng assay. Two parameters were measured: 1) the number of times that haemostasis occurs in a 30 minute period, and 2) the time required for each haemostasis.

Results Coagulation assays in normal plasma Thrombin generation analyses were med in pooled normal plasma with and without the addition of anticoagulants. The results are summarized in table 3.

Table 3. Overview of procoagulant activity of tested cyclodextrins.

Modification α-mono α-per β-mono β-per γ-mono γ-per S-C2-COOH 1100 1101 1102 1103/1170 S-C3-COOH 1105 1106 1107 OOH 1178 1171 S-C5-COOH 1186 1187 1108 1109 1110 1111 OOH 1188 1179 1190 1172 S-C7-COOH 1180 S-C2-OH S-C3-OH 1181 1146 S-C4-OH 1189 1191 1174 Shading shows the strength of procoagulant activity. Darker shading indicates stronger procoagulant activity.

White boxes with number indicate no effect in coagulation assay.

White boxes without number: not ined.

Alpha-mono-carboxyl cyclodextrins: 86 had a substantial procoagulant effect in normal plasma and antagonized the anticoagulant effect of dabigatran, xaban, apixaban and edoxaban (table 4 and Figure 14).

Alpha-per-carboxyl cyclodextrins: OKL-1187 showed a very strong procoagulant effect in normal plasma and ly antagonized the anticoagulant effect of dabigatran, rivaroxaban, apixaban and edoxaban (table 4 and Figure 15) .

Beta-mono-carboxyl cyclodextrins: 08 had a substantial procoagulant effect in plasma and antagonized the anticoagulant effect of dabigatran, rivaroxaban, apixaban and edoxaban (table 4 and Figure 3). OKL-1188 showed a strong procoagulant effect in normal plasma and antagonized the anticoagulant effect of dabigatran, rivaroxaban, apixaban and edoxaban (table 4 and Figure 16).

Beta-per-carboxyl cyclodextrins: Addition of OKL-1105 gave a significant procoagulant effect in plasma. In the presence of dabigatran, rivaroxaban, an and edoxaban also procoagulant effects were ed. As such the anticoagulant effect of the NOACs was antagonized (table 4 and Figure 4). OKL-1109 had small effects on thrombin generation in plasma. In the presence of dabigatran, rivaroxaban, apixaban and an there were marginal or no effects on thrombin tion (table 4 and Figure 5). OKL-1178, OKL-1179 and OKL-1180 showed procoagulant ty in normal plasma at varying s (table 4 and s . mono-carboxylic cyclodextrins OKL-1106 induced a gulant effect in normal plasma and significantly antagonized the anticoagulant actions of dabigatran and rivaroxaban. Its effect on the anticoagulant actions of apixaban and edoxaban were less pronounced (table 4 and Figure 6). OKL-1110 induced a very potent procoagulant effect in normal plasma and fully counteracted the anticoagulant effect of the Xa-antagonists rivaroxaban, edoxaban and apixaban. OKL-1110 also strongly antagonized the anticoagulant s of the direct thrombin inhibitor dabigatran (table 4 and Figure 7). OKL-1190 had a procoagulant effect in normal plasma and also antagonized the anticoagulant effect of dabigatran, rivaroxaban, apixaban and edoxaban (table 4 and figure 18).

Gamma-per-carboxylic cyclodextrins Addition of OKL-1107 gave strong procoagulant effects in plasma. Peak thrombin was largely increased and the lag time was considerably shorter. After addition of dabigatran, rivaroxaban, apixaban or an the effects of OKL-1107 remained, and thrombin generation was completely restored or even higher than in the nonanticoagulated plasma (table 4 and Figure 8). OKL-1171 and OKL-1172 showed significant procoagulant activity in plasma (table 4 and figures 22-23).

Addition of OKL-1111 gave very strong procoagulant effects in plasma (table 4 and Figure 9). Peak thrombin was largely increased and the lag time was considerably shorter than in the absence of CD. After addition of dabigatran, rivaroxaban, an or edoxaban the effects of OKL-1111 were still highly procoagulant with ation of thrombin generation to levels far above that of non-anticoagulated plasma (Figure 10). OKL-1111 was also capable of restoring thrombin generation in plasma anticoagulated with unfractionated and low molecular weight heparin, pentasaccharide (arixtra), hirudin and bivalirudin (Figure 10). Also, in plasma of patients using n K antagonists, thrombin generation could be improved by the addition of OKL-1111 (Figure 11).

Beta-mono-hydroxylic cyclodextrins OKL-1189 showed strong procoagulant ty in normal plasma and strongly antagonized the anticoagulant effect of tran, rivaroxaban, apixaban and edoxaban (table 4 and Figure 17).

Beta-per-hydroxylic cyclodextrins OKL-1181 showed significant procoagulant activity in plasma (table 4 and figure 27).

Gamma-mono-hydroxylic extrins OKL-1191 showed strong procoagulant activity in plasma and also antagonized the agulant effect of dabigatran, rivaroxaban, apixaban and edoxaban (table 4 and figure 19).

Gamma-per-hydroxylic cyclodextrins Addition of OKL-1146 gave very strong procoagulant effects in plasma (table 4 and figure 12). Peak thrombin was largely sed and the lag time was considerably shorter than in the absence of CD. After addition of dabigatran, rivaroxaban, apixaban or edoxaban the effects of OKL-1146 were still highly procoagulant with restoration of thrombin generation to levels far above that of non-anticoagulated plasma (Figure 12). Similarly, OKL-1174 gave strong procoagulant effects in plasma, also in the presence of anticoagulants e 24).

Substituted extrins containing a C2 substituent Beta-mono-, beta-per-, gamma-mono- and gamma-per-hydroxylic substituted cyclodextrins OKL-1100, OKL-1101, OKL-1102 and OKL-1103 / OKL-1170 did not show procoagulant activity in normal plasma, nor in plasma containing agulants. These cyclodextrins were therefore not tested in deficient plasma.

Gamma-per-amine-substituted extrins In several experiments the amine-substituted γ-cyclodextrin OKL-1147 was used as a negative control for the γ-series cyclodextrins since it had no procoagulant effect in plasma, nor did it influence the anticoagulant effect of the NOACs (Figure 13).

Coagulation assays in deficient plasma The procoagulant effect of the substituted extrins was also tested in plasma deficient in coagulation factor VIII and IX. The procoagulant effect of the cyclodextrins OKL-1107, OKL-1110, OKL-1111 has further been igated in human plasma deficient in coagulation factor XI. The results are summarized in table 4 and representative graphs for 11, OKL-1180 and OKL-1187 are shown in figure 20.

OKL-1111 was able to stimulate coagulation significantly in factor in factor VIII, IX and XI deficient plasma (table 4, figure 20). OKL-1171, OKL-1172, OKL-1174, 80, OKL-1181, OKL-1187, OKL-1188, OKL-1189 and OKL-1191 also showed procoagulant effects in factor VIII deficient plasma and OKL-1172, OKL- 1174, OKL-1180, OKL-1181, OKL-1188, OKL-1189 and OKL-1191 showed procoagulant ty in factor IX deficient plasma (table 4).

Table 4. Effect of procoagulant CDs in Factor VIII or Factor IX depleted plasma.

Shading and number of * shows the th of gulant activity. Darker shading and sing number of * indicates stronger procoagulant activity. - indicates no procoagulant activity in coagulation assay.

Compound Normal plasma FVIII dp FIX dp * OKL-1105 ** - - *** OKL-1106 **** - - **** OKL-1107 *** - - ***** OKL-1108 * - - ****** OKL-1109 * - - OKL-1110 ** - - OKL-1111 **** ** * OKL-1146 *** - - OKL-1171 * * - OKL-1172 *** *** *** OKL-1174 **** * * OKL-1178 * - - OKL-1180 *** *** *** OKL-1181 ** * * OKL-1186 * - - OKL-1187 ****** ***** - OKL-1188 **** ** *** OKL-1189 **** ** ** OKL-1190 * - - OKL-1191 **** * ** Coagulation assays in antibody pre-treated plasma Normal plasma was spiked with inhibitory antibodies against factor VIII in, VK34, 14 µg/ml), factor IX (Sanquin, 5F5, 20 µg/ml) or factor XI (Sanquin, mix of #203 and #175, 75 µg/ml). The effect of OKL-1111 was tested in thrombin tion assay using 1 pM tissue factor (TF). This model is representative for hemophilia A, B and C patients that have developed inhibitory antibodies against plasma-derived or recombinant factor VIII or IX or XI they are treated with. 11 concentration-dependently ated thrombin generation in normal human plasma pretreated with inhibitory antibodies against factor VIII, factor IX and factor XI (Figure 21 A-C).

Coagulation assays in plasma of a hemophilia A patient The procoagulant effect of OKL-1111, OKL-1180 and OKL-1187 was also tested in plasma of a hemophilia A patient (George King Bio-Medical, USA). The patient had developed antibodies against factor VIII prior to plasma withdrawal. The plasma ned high levels of VIII antibodies (50 BU). All tested cyclodextrins concentration-dependently stimulated thrombin generation in this plasma containing anti-FVIII antibodies (figure 28).

In vivo bleeding model In order to investigate whether the procoagulant effect observed in the in vitro assays was also observed in vivo, OKL-1111 was administered to mice that were agulated with rivaroxaban.

In a non-anticoagulated mouse a clot forms in a little over 1 minute after re of the blood vessel. As such, in a 30 minute time period about 20-25 clots will form.

In mice fed with rivaroxaban, bleeding time was roughly doubled, so that animals only formed about 10-13 clots in 30 minutes. A dose of OKL-1111 expected to give μM in , gave a normalization of the clotting times. In contrast, OKL-1147 was without any significant effect in this respect (Figure 29).

In order to test the efficacy of OKL-1111 and OKL-1187 in hemophilia in vivo, the vena a bleeding model was used as well. Hemophilia A mice were injected with a very low dose of factor VIII (2.5 IU/kg which is designed to give plasma levels of about 0.0625 IU/dL) with or without OKL-1111 or OKL-1187.

Hemophilic mice (that completely lack factor VIII) do not or only form one clot in 30 minutes after puncture of the vena saphena, whereas in wild type mice this amounts to about 20 clots. In the presence of a low dose of factor VIII, the number of clots in hemophilic mice is increased to approximately 2-3 clots. At a dose of 1 µmol/kg, designed to give a plasma value of 25 µM, OKL-1111 increased hemostasis significantly higher compared to factor VIII alone. In the presence of OKL-1111, 7-9 clots are formed over a 30 minute period (figure 30). Similar s were obtained in the presence of 0.2 µmol/kg of OKL-1187.

The term “comprising” as used in this specification and claims means “consisting at least in part of”. When reting statements in this specification, and claims which include the term “comprising”, it is to be understood that other features that are additional to the features prefaced by this term in each statement or claim may also be t. Related terms such as “comprise” and “comprised” are to be interpreted in similar manner.

In this ication where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, nce to such external documents is not to be construed as an ion that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

In the description in this specification reference may be made to subject matter that is not within the scope of the claims of the current application. That t matter should be readily identifiable by a person skilled in the art and may assist in putting into practice the invention as defined in the claims of this application.

Claims (21)

Claims

1. A use of a substituted cyclodextrin of formula (I): Formula (I), 5 wherein n is an integer from 3 to 7 and R is selected from the group consisting of - COOH, -OH and -COO(1-4C)alkyl, and wherein p + q is 6, 7 or 8, whereby p is 5 and q, is 1 or p is 6 and q is 1, or p is 7 and q is 1, or p is 0 and q is 6, or p is 0 and q is 7, or p is 0 and q is 8, or a pharmaceutically acceptable salt or ester thereof, in the manufacture of a medicament for initiating or rating blood clot formation 10 in a subject in need thereof.

2. A use according to claim 1, wherein S-(Cnalkylene)-R is -S-(CH2)m-R, and wherein m is an integer from 3 to 7. 15

3. A use according to claim 1 or 2 wherein R is selected from the group consisting of -COOH and -OH.

4. A pharmaceutical composition comprising at least one substituted cyclodextrin or pharmaceutically acceptable salt or ester thereof as defined in any 20 one of claims 1-3 and at least one pharmaceutically acceptable auxiliary, n said pharmaceutical composition is ated for topical administration as a gel, cream, ointment, dressing, ss, plaster, band-aid or patch.

5. A substituted cyclodextrin of formula (II): Formula (II), wherein: - p is 6, q is 1, m is 5 and R is COOH; 5 - p is 0, q is 7, m is 5 and R is COOH; - p is 7, q is 1, m is 5 and R is COOH; - p is 0, q is 7, m is 4 and R is COOH; - p is 0, q is 7, m is 6 and R is COOH; - p is 0, q is 7, m is 7 and R is COOH; 10 - p is 5, q is 1, m is 5 and R is COOH; - p is 0, q is 6, m is 5 and R is COOH; - p is 6, q is 1, m is 6 and R is COOH; - p is 6, q is 1, m is 4 and R is OH; - p is 7, q is 1, m is 6 and R is COOH; or 15 - p is 7, q is 1, m is 4 and R is OH, or a ceutically acceptable salt or ester of any of these cyclodextrins, or a substituted cyclodextrin of formula (II) wherein p is 0, q is 8, m is 5 and R is COOH. 20

6. A substituted cyclodextrin according to claim 5 wherein: - p is 5, q is 1, m is 5 and R is COOH; or - p is 0, q is 6, m is 5 and R is COOH; or a pharmaceutically acceptable salt or ester of any of these cyclodextrins.

7. A pharmaceutical composition comprising a substituted cyclodextrin or pharmaceutically acceptable salt or ester thereof according to claim 5 or 6 and at least one pharmaceutically acceptable auxiliary. 5

8. A kit of parts comprising: - a substituted cyclodextrin or pharmaceutically acceptable salt thereof according to claim 5 or 6, and - a recombinant or isolated coagulation factor. 10

9. A kit of parts according to claim 8, wherein said recombinant or isolated coagulation factor is factor VIII and said substituted cyclodextrin is a cyclodextrin wherein S-(Cnalkylene)-R is -S-(CH2)m-R, and wherein: - p is 0, q is 7, m is 7 and R is COOH, - p is 0, q is 6, m is 5 and R is COOH, 15 - p is 6, q is 1, m is 6 and R is COOH, - p is 6, q is 1 m is 4 and R is OH or - p is 7, q is 1, m is 4 and R is OH, or a pharmaceutically acceptable salt or ester thereof, or a substituted extrin of formula (II) wherein p is 0, q is 8, m is 5 and R is 20 COOH.

10. A kit of parts according to claim 8, wherein said recombinant or isolated coagulation factor is factor IX and said substituted cyclodextrin is a cyclodextrin wherein S-(Cnalkylene)-R is -S-(CH2)m-R, and n: 25 - p is 0, q is 7, m is 7 and R is COOH, - p is 6, q is 1, m is 6 and R is COOH, - p is 6, q is 1 m is 4 and R is OH or - p is 7, q is 1, m is 4 and R is OH, or a pharmaceutically able salt or ester thereof.

11. A kit of parts ing to claim 8, wherein said recombinant or isolated coagulation factor is factor IX and said substituted extrin is a cyclodextrin wherein S-(Cnalkylene)-R is -S-(CH2)m-R and wherein p is 0, q is 8, m is 5 and R is COOH.

12. A use of a substituted cyclodextrin or a pharmaceutically able salt thereof as defined in any one of claims 1-3 for the manufacture of a medicament for 5 reversing an anticoagulant effect of an anticoagulant in a t.

13. A use of a substituted cyclodextrin or a pharmaceutically acceptable salt thereof as defined in any one of claims 1-3 for the manufacture of a medicament for reducing or preventing bleeding in a subject.

14. A use according to claim 12 or 13, wherein said subject has been treated with an anticoagulant, is undergoing surgery, is undergoing dental treatment, is suffering from trauma, is ing from induced or spontaneous major bleeding, such as intracranial or gastro-intestinal ng, and/or is suffering from or at risk 15 of hereditary or drug-induced thrombocytopenia.

15. A use according to claim 12 or 14, wherein the anticoagulant is selected from the group consisting of: - a direct thrombin inhibitor, such as dabigatran, hirudin, rudin, lepirudin or 20 argatroban, - a direct factor Xa inhibitor, such as rivaroxaban, apixaban, edoxaban, aban, darexaban, ban or eribaxaban, - a pentasaccharide, such as fondaparinux or idraparinux, - a low molecular weight heparin, such as nadroparin, tinzaparin, dalteparin, 25 enoxaparin, bemiparin, reviparin, parnaparin or certoparin, - unfractionated heparin, - a n K antagonist, such as acenocoumarol, phenprocoumon, warfarin, atromentin or dione, and - an antiplatelet drug, such as an irreversible cyclooxygenase inhibitors (such as 30 aspirin or a derivative thereof or triflusal), an ADP receptor inhibitor (such as clopidogrel, prasugrel, ticagrelor, ticlopedine, cangrelor or elinogrel), a phosphodiesterase tor (such as cilostazol), a PAR-1 antagonist (such as voraxapar), a GPIIB/IIIa inhibitor (such as abciximab, eptifibatide, ban, roxifiban or orbofiban), an adenosine reuptake inhibitor (such as dipyridamole), a thromboxane inhibitor (such as ifetroban or picotamide) or a thromboxane receptor antagonist (such as terutroban or picotamide).

16. A use of a substituted cyclodextrin or a ceutically able salt 5 thereof as defined in any one of claims 1-3 in the manufacture of a medicament for the treatment or prevention of a blood coagulation disorder.

17. A use according to claim 16, wherein said disorder is selected from the group ting of congenital or acquired hemophilia A, hemophilia B, ilia 10 C, von Willebrand disease, factor V, factor VII, factor X and/or factor XI deficiency, factor XIII or alpha2-antiplasmin deficiency, hereditary or drug-induced thrombocytopenia, including immune thrombocytopenia purpura, thrombotic thrombocytopenic purpura, fetal or al alloimmune thrombocytopenia and post-transfusion thrombocytopenic a, Wiskott-Aldrich Syndrome, 15 Glanzmann's thrombasthenia, Bernard-Soulier Syndrome, idiopathic densegranule disorder, Hermansky-Pudlak syndrome, Chediak-Higashi syndrome, gray platelet me, Paris-Trousseau/Jacobsen's syndrome, disseminated intravascular coagulation and vitamin K deficiency, including vitamin K deficiency of the newborn.

18. A use as claimed in any one of claims 1-3 and 12-17 substantially as herein described and with reference to any example thereof.

19. A pharmaceutical composition as claimed in claim 4 or 7 substantially as 25 herein described and with reference to any example thereof.

20. A substituted cyclodextrin of Formula (II) as claimed in claim 5 or 6 substantially as herein described and with reference to any example thereof. 30

21. A kit of parts as claimed in any one of claims 8-11 substantially as herein described and with reference to any example thereof.