CYCLODEXTRINS AS PROCOAGULANTS

Abstract

The invention relates to substituted cyclodextrins and pharmaceutically acceptable salts thereof, pharmaceutical compositions, kits of parts and their use as procoagulants. The invention further relates to methods of reversing an anticoagulant effect of an anticoagulant in a subject, methods for reducing or preventing bleeding in a subject and methods for the treatment or prevention of a blood coagulation disorder.

Claims

1. A substituted cyclodextrin of formula (I): ##STR00005## 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 thereof, for use as a procoagulant.

2. A substituted cyclodextrin or pharmaceutically acceptable salt thereof for use according to claim 1, wherein S(C.sub.nalkylene)-R is S(CH.sub.2).sub.mR, and wherein m is an integer from 3 to 7.

3. A substituted cyclodextrin or pharmaceutically acceptable salt thereof for 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 one of claims 1-3 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.

5. A substituted cyclodextrin of formula (II): ##STR00006## 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 of any of these cyclodextrins.

6. A substituted cyclodextrin according to claim 5 wherein: p is 7, q is 1, 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 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 4and RisOH; 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 4and R is OH, or a pharmaceutically acceptable salt or ester of any of these cyclodextrins.

7. A substituted cyclodextrin according to claim 5 wherein p is 0, q is 8, m is 5 and R is COOH.

8. A pharmaceutical composition comprising a substituted cyclodextrin or pharmaceutically acceptable salt or ester thereof according to any one of claims 5-7 and at least one pharmaceutically acceptable auxiliary.

9. A kit of parts comprising: a substituted cyclodextrin or pharmaceutically acceptable salt thereof as defined in any one of claims 1-3, and a recombinant or isolated coagulation factor.

10. A kit of parts according to claim 9, wherein said recombinant or isolated coagulation factor is factor VIII and said substituted cyclodextrin is a cyclodextrin wherein S(C.sub.nalkylene)-R is S(CH.sub.2).sub.mR, and wherein: p is 0, q is 8, m is 5 and R is COOH, p is 0, q is 8, m is 4and 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.

11. A kit of parts according to claim 9, wherein said recombinant or isolated coagulation factor is factor IX and said substituted cyclodextrin is a cyclodextrin wherein S(C.sub.nalkylene)-R is S(CH.sub.2).sub.mR, and 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 4and 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.

12. A kit of parts according to claim 9, wherein said recombinant or isolated coagulation factor is factor IX and said substituted cyclodextrin is a cyclodextrin wherein S(C.sub.nalkylene)-R is S(CH.sub.2).sub.mR and wherein p is 0, q is 8, m is 5 and R is COOH, or a pharmaceutically acceptable salt or ester thereof.

13. A method for inducing or stimulating coagulation in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a substituted cyclodextrin or a pharmaceutically acceptable salt thereof as defined in any one of claims 1-3.

14. A substituted cyclodextrin or a pharmaceutically acceptable salt thereof as defined in any one of claims 1-3 for use in a method for reversing an anticoagulant effect of an anticoagulant in a subject.

15. 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 cyclodextrin or a pharmaceutically acceptable salt thereof as defined in any one of claims 1-3.

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

17. 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 substituted cyclodextrin or a pharmaceutically acceptable salt thereof as defined in any one of claims 1-3.

18. A substituted cyclodextrin or a pharmaceutically acceptable salt thereof for use or method according to any one of claims 14-17, wherein 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 drug-induced thrombocytopenia.

19. A substituted cyclodextrin or a pharmaceutically acceptable salt thereof for use or method according to any one of claims 14, 15 or 18, wherein the anticoagulant is selected from the group consisting of: a direct thrombin inhibitor, such as dabigatran, hirudin, bivalirudin, lepirudin or argatroban, a direct factor Xa inhibitor, such as rivaroxaban, apixaban, edoxaban, betrixaban, darexaban, letaxaban or eribaxaban, a pentasaccharide, such as fondaparinux or idraparinux, a low molecular weight heparin, 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 inhibitors (such as aspirin or a derivative thereof or triflusal), an ADP receptor inhibitor (such as clopidogrel, prasugrel, ticagrelor, ticlopedine, cangrelor or elinogrel), a phosphodiesterase inhibitor (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 antagonist (such as terutroban or picotamide).

20. A substituted cyclodextrin or a pharmaceutically acceptable salt thereof as defined in any one of claims 1-3 for use in a method for the treatment or prevention of a blood coagulation disorder.

21. A method for the treatment or prevention of a blood coagulation disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a substituted cyclodextrin or a pharmaceutically acceptable salt thereof as defined in any one of claims 1-3.

22. A substituted cyclodextrin or a pharmaceutically acceptable salt thereof for use or method according to claim 20 or 21, wherein said disorder is selected from the group consisting of congenital or acquired hemophilia A, hemophilia B, hemophilia 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 neonatal alloimmune thrombocytopenia and post-transfusion thrombocytopenic purpura, Wiskott-Aldrich Syndrome, Glanzmann's thrombasthenia, Bernard-Soulier Syndrome, idiopathic dense-granule disorder, Hermansky-Pudlak syndrome, Chediak-Higashi syndrome, gray platelet syndrome, Paris-Trousseau/Jacobsen's syndrome, disseminated intravascular coagulation and vitamin K deficiency, including vitamin K deficiency of the newborn.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0258] FIG. 1: Basic structure of -, - and -cyclodextrins. a. chemical structure; b. 3-D structure.

[0259] FIG. 2: Structures of OKL-1108 (A), OKL-1109 (B), OKL-1110 (C) and OKL-1111 (D).

[0260] FIGS. 3-8, 12, 14-19 and 22-27: Pooled normal plasma was spiked with cyclodextrins (100 M, 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 section with 1 pM tissue factor (TF) as initiator of coagulation.

[0261] FIG. 9: Pooled normal plasma was spiked with OKL-1111 (100 M, 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 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.

[0262] FIG. 10: Pooled normal plasma was spiked with OKL-1111 (100 M) and anticoagulants (A-F). The concentrations of the anticoagulants were 2 g/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 g/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.

[0263] FIG. 11: Plasma was used from individuals taking vitamin K-antagonists (VKA plasma). Two different intensities of treatment (given as INR) were available, as depicted in (A) and (B). 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.

[0264] FIG. 13: Pooled normal plasma was spiked with OKL-1147 (100 M, 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 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.

[0265] FIG. 20: Plasma deficient in coagulation factor VIII was spiked with cyclodextrin OKL-1111 (A), OKL-1180 (B), and OKL-1187 (C) at the indicated concentrations.

[0266] FIG. 21: Effect of OKL-1111 on normal plasma spiked with inhibitory antibodies against factor VIII (A); factor IX (B), and factor XI (C).

[0267] FIG. 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.

[0268] FIG. 29: In vivo analysis of procoagulant potential of OKL-1111 and OKL-1147. Except for controls, animals were treated with rivaroxaban for 4 days and cyclodextrins were administered 5 min prior bleeding assay.

[0269] FIG. 30: Effect of OKL-1111 and OKL-1187 on coagulation in mouse hemophilia A assay.

EXAMPLES

Example 1

Materials and Methods

Synthesis of Cyclodextrins

[0270] General Procedure for the Synthesis of Decorated -Cyclodextrins with Thiols.

[0271] For the synthesis of mono-decorated -cyclodextrin derivatives, a solution of 6-monotosyl--cyclodextrin (500 mg, 0.388 mmol, 1.0 equiv.) in DMSO (3 mL) was degassed. The solution was added dropwise to a degassed solution of the appropriate thiol (HSR; 4.67 mmol, 12 equiv) and NaOH (460 mg, 11.5 mmol, 30 equiv) in DMSO/H.sub.2O (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 precipitate was dissolved in H.sub.2O (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.

[0272] The synthesis of per-decorated f-cyclodextrin derivatives with a sulfur tether was performed using commercial heptakis-(6-bromo-6-deoxy)--cyclodextrin as starting material. The reactions with the appropriate thiol (HSR) and NaOH were performed successfully with NaH as base in DMF with stirring overnight at room temperature.

[0273] Scheme 1 shows the reaction for per-decorated -cyclodextrin derivatives.

##STR00003##

General Procedure for the Synthesis of Decorated -Cyclodextrins with Thiols.

[0274] For the synthesis of per-decorated -cyclodextrin derivatives a solution of Octakis-6-bromo-6-deoxy--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/H.sub.2O (12 mL/6 mL). The suspension was stirred overnight at 50 C. The reaction 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 H.sub.2O (50 mL) and the pH was adjusted to 7 with aqueous 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 reduced pressure.

[0275] For the synthesis of mono-decorated -cyclodextrin derivatives, monotosylated -cyclodextrines were functionalized in a similar fashion as described for the mono-decorated beta-cyclodextrins.

##STR00004##

[0276] Analogous to the beta-cyclodextrins, the -cyclodextrins were functionalized (see scheme 2 for the per-substituted -cyclodextrins). The compounds were synthesized using NaOH and DMSO as solvent, providing difficult isolations but eventually addition of EtOAc led to good precipitation.

Purification

[0277] In general, the functionalized cyclodextrins were isolated by precipitation from a suitable solvent, followed by several washings with solvents to remove excess of reagents and side-products. Often this procedure provided materials that were considered pure for the application based on either .sup.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 decorated 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

[0278] Alpha-mono-S-C6-acid (OKL-1186) was prepared according to the general procedure described above using 6-Mercaptohexanoic acid (131 l, 140 mg, 0.943 mmol), NaOH (38 mg, 0.0925 mmol) and 6-monodeoxy-6-monoiodo--cyclodextrin (200 mg, 0.185 mmol). Other alpha-mono-substituted cyclodextrins according to the invention can be prepared in the same way using the appropriate starting compounds.

[0279] 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 dropwise. After 10 minutes, hexakis-(6-bromo-6-deoxy)--cyclodextrin (102 mg, 0.0741 mmol, 1.0 eq.) was added and the reaction mixture was stirred at room temperature overnight. The reaction mixture was precipitated by addition of acetone (large excess), 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 acetone, filtered, washed with acetone and dried in vacuo to give the product. Other alpha-per-substituted cyclodextrins according to the invention can be prepared in the same way using the appropriate starting compounds.

[0280] Tables 1 and 2 shows the cyclodextrins that have been prepared. FIG. 2 shows the structure of four exemplary mono- and per-substituted, beta- and gamma cyclodextrins (compounds OKL-1108, OKL-1109, OKL-1110 and OKL-111).

TABLE-US-00001 TABLE 1 Cyclodextrins with procoagulant activity. cyclodextrin substitution compound type pattern substituent OKL-1105 beta per S(CH.sub.2).sub.3COOH OKL-1106 gamma mono S(CH.sub.2).sub.3COOH OKL-1107 gamma per S(CH.sub.2).sub.3COOH OKL-1108 beta mono S(CH.sub.2).sub.5COOH OKL-1109 beta per S(CH.sub.2).sub.5COOH OKL-1110 gamma mono S(CH.sub.2).sub.5COOH OKL-1111 gamma per S(CH.sub.2).sub.5COOH OKL-1146 gamma per S(CH.sub.2).sub.3OH OKL-1171 gamma per S(CH.sub.2).sub.4COOH OKL-1172 gamma per S(CH.sub.2).sub.6COOH OKL-1174 gamma per S(CH.sub.2).sub.4OH OKL-1178 beta per S(CH.sub.2).sub.4COOH OKL-1179 beta per S(CH.sub.2).sub.6COOH OKL-1180 beta per S(CH.sub.2).sub.7COOH OKL-1181 beta per S(CH.sub.2).sub.3OH OKL-1186 alpha mono S(CH.sub.2).sub.5COOH OKL-1187 alpha per S(CH.sub.2).sub.5COOH OKL-1188 beta mono S(CH.sub.2).sub.6COOH OKL-1189 beta mono S(CH.sub.2).sub.4OH OKL-1190 gamma mono S(CH.sub.2).sub.6COOH OKL-1191 gamma mono S(CH.sub.2).sub.4OH

TABLE-US-00002 TABLE 2 Comparative cyclodextrins. cyclodextrin substitution compound type pattern substituent OKL-1100 beta mono S(CH.sub.2).sub.2COOH OKL-1101 beta per S(CH.sub.2).sub.2COOH OKL-1102 gamma mono S(CH.sub.2).sub.2COOH OKL-1103 gamma per S(CH.sub.2).sub.2COOH OKL-1147 gamma per NH.sub.2 OKL-1170 gamma per S(CH.sub.2).sub.2COOH

Coagulation Assays

[0281] 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 described by Hemker et al. (Pathofysiol. 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 (Innovin, Siemens, Marburg, Germany), 4 M phospholipids, and 417 M fluorogenic 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

[0282] All animal protocols were approved by the Institutional Animal Care and Use Committee of the University of North Carolina. C57BL6/J mice were purchased from Charles Rivers Laboratories (Willmington, Mass.). Bleeding studies were done essentially as previously described Pastoft et al. (Haemophilia 2012; 18:782-8). Mice were anesthetized with isoflurane throughout all procedures. The hair on the ventral side of both hind limbs was removed. The animals 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 occurred. 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 minutes. 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 bleeding 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

[0283] Thrombin generation analyses were performed in pooled normal plasma with and without the addition of anticoagulants. The results are summarized in table 3.

[0284] Alpha-mono-carboxyl Cyclodextrins:

[0285] OKL-1186 had a substantial procoagulant effect in normal plasma and antagonized the anticoagulant effect of dabigatran, rivaroxaban, apixaban and edoxaban (table 4 and FIG. 14).

Alpha-per-carboxyl Cyclodextrins:

[0286] OKL-1187 showed a very strong procoagulant effect in normal plasma and strongly antagonized the anticoagulant effect of dabigatran, rivaroxaban, apixaban and edoxaban (table 4 and FIG. 15).

Beta-mono-carboxyl Cyclodextrins:

[0287] OKL-1108 had a substantial procoagulant effect in plasma and antagonized the anticoagulant effect of dabigatran, rivaroxaban, apixaban and edoxaban (table 4 and FIG. 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 FIG. 16).

Beta-per-carboxyl Cyclodextrins:

[0288] Addition of OKL-1105 gave a significant procoagulant effect in plasma. In the presence of dabigatran, rivaroxaban, apixaban and edoxaban also procoagulant effects were observed. As such the anticoagulant effect of the NOACs was antagonized (table 4 and FIG. 4). OKL-1109 had small effects on thrombin generation in plasma. In the presence of dabigatran, rivaroxaban, apixaban and edoxaban there were marginal or no effects on thrombin generation (table 4 and FIG. 5). OKL-1178, OKL-1179 and OKL-1180 showed procoagulant activity in normal plasma at varying degrees (table 4 and FIGS. 25-26).

Gamma-mono-carboxylic Cyclodextrins

[0289] OKL-1106 induced a procoagulant 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 FIG. 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 effects of the direct thrombin inhibitor dabigatran (table 4 and FIG. 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 FIG. 18).

Gamma-per-carboxylic Cyclodextrins

[0290] 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 edoxaban the effects of OKL-1107 remained, and thrombin generation was completely restored or even higher than in the non-anticoagulated plasma (table 4 and FIG. 8). OKL-1171 and OKL-1172 showed significant procoagulant activity in plasma (table 4 and FIGS. 22-23). Addition of OKL-1111 gave very strong procoagulant effects in plasma (table 4 and FIG. 9). Peak thrombin was largely increased 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-1111 were still highly procoagulant with restoration of thrombin generation to levels far above that of non-anticoagulated plasma (FIG. 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 (FIG. 10). Also, in plasma of patients using vitamin K antagonists, thrombin generation could be improved by the addition of OKL-1111 (FIG. 11).

Beta-mono-hydroxylic Cyclodextrins

[0291] OKL-1189 showed strong procoagulant activity in normal plasma and strongly antagonized the anticoagulant effect of dabigatran, rivaroxaban, apixaban and edoxaban (table 4 and FIG. 17).

Beta-per-hydroxylic Cyclodextrins

[0292] OKL-1181 showed significant procoagulant activity in plasma (table 4 and FIG. 27).

Gamma-mono-hydroxylic Cyclodextrins

[0293] OKL-1191 showed strong procoagulant activity in plasma and also antagonized the anticoagulant effect of dabigatran, rivaroxaban, apixaban and edoxaban (table 4 and FIG. 19).

Gamma-per-hydroxylic Cyclodextrins

[0294] Addition of OKL-1146 gave very strong procoagulant effects in plasma (table 4 and FIG. 12). Peak thrombin was largely increased 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 (FIG. 12). Similarly, OKL-1174 gave strong procoagulant effects in plasma, also in the presence of anticoagulants (FIG. 24).

Substituted Cyclodextrins Containing a C2 Substituent

[0295] 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 anticoagulants. These cyclodextrins were therefore not tested in deficient plasma.

Gamma-per-amine-Substituted Cyclodextrins

[0296] In several experiments the amine-substituted -cyclodextrin OKL-1147 was used as a negative control for the -series cyclodextrins since it had no procoagulant effect in plasma, nor did it influence the anticoagulant effect of the NOACs (FIG. 13).

Coagulation Assays in Deficient Plasma

[0297] The procoagulant effect of the substituted cyclodextrins 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 investigated in human plasma deficient in coagulation factor XI. The results are summarized in table 4 and representative graphs for OKL-1111, OKL-1180 and OKL-1187 are shown in FIG. 20.

[0298] OKL-1111 was able to stimulate coagulation significantly in factor in factor VIII, IX and XI deficient plasma (table 4, FIG. 20). OKL-1171, OKL-1172, OKL-1174, OKL-1180, 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 activity in factor IX deficient plasma (table 4).

Coagulation Assays in Antibody Pre-Treated Plasma

[0299] Normal plasma was spiked with inhibitory antibodies against factor VIII (Sanquin, VK34, 14 g/ml), factor IX (Sanquin, 5F5, 20 g/ml) or factor XI (Sanquin, mix of #203 and #175, 75 g/ml). The effect of OKL-1111 was tested in thrombin generation 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. OKL-1111 concentration-dependently stimulated thrombin generation in normal human plasma pretreated with inhibitory antibodies against factor VIII, factor IX and factor XI (FIG. 21 A-C).

Coagulation Assays in Plasma of a Hemophilia a Patient

[0300] 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 contained high levels of anti-FVIII antibodies (50 BU). All tested cyclodextrins concentration-dependently stimulated thrombin generation in this plasma containing anti-FVIII antibodies (FIG. 28).

In Vivo Bleeding Model

[0301] 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 anticoagulated with rivaroxaban.

[0302] In a non-anticoagulated mouse a clot forms in a little over 1 minute after puncture 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 25 M in plasma, gave a normalization of the clotting times. In contrast, OKL-1147 was without any significant effect in this respect (FIG. 29).

[0303] In order to test the efficacy of OKL-1111 and OKL-1187 in hemophilia in vivo, the vena saphena 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 mol/kg, designed to give a plasma value of 25 M, 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 (FIG. 30). Similar results were obtained in the presence of 0.2 mol/kg of OKL-1187.