Antithrombotic molecules with APAC activity for preventing and / or treating thrombocytopenia - Patent Application 20070122999

Abstract

The present invention relates to an antithrombotic molecule having both antiplatelet and anticoagulant (APAC) activity, in particular its use as a pharmaceutical for preventing and / or treating heparin-induced thrombocytopenia (HIT) type I or type II; and / or heparin-induced thrombocytopenia and thrombosis (HITT); and / or heparin-independent thrombocytopenia autoimmune HIT (aHIT); and / or vaccine-induced thrombocytopenia and thrombosis (VITT). The present invention is used in both the medical and veterinary industries.

Description

[Technical Field]

[0001] The present invention relates to an antithrombotic molecule having both antiplatelet and anticoagulant (APAC) activity, in particular its use as a pharmaceutical for preventing and / or treating heparin-induced thrombocytopenia (HIT) type I or type II; and / or heparin-induced thrombocytopenia and thrombosis (HITT); and / or heparin-independent thrombocytopenia autoimmune HIT (aHIT); and / or vaccine-induced thrombocytopenia and thrombosis (VITT). The present invention is used in both the medical and veterinary industries. [Background technology]

[0002] Thrombocytopenia can result from conditions that result in increased platelet destruction or decreased platelet production. Heparin can cause thrombocytopenia through immune and non-immune-mediated mechanisms. Two types of heparin-induced thrombocytopenia (HIT), non-immune-mediated type I and immune-mediated type II, can result from heparin administration. The term "non-immune heparin-associated thrombocytopenia" is used to refer to type I, a benign condition in which heparin-dependent antibodies are absent. The term "immune-mediated heparin-induced thrombocytopenia" (HIT) is used to refer to thrombocytopenia in which pathogenic heparin-dependent antibodies are detectable, and this term is the most widely accepted designation for HIT type II.

[0003] It has also been recognized that some patients exhibit clinical and laboratory features of HIT despite having received heparin in the recent past or never (spontaneous HIT syndrome). The sera of these patients contain antibodies that potently activate platelets even in the absence of heparin. However, such "heparin-independent" platelet activation properties are not unique to spontaneous HIT syndrome; they are also found in the sera of a small number of (heparin-dependent) typical HIT patients. Furthermore, patients with this in vitro reactivity profile are more likely to have atypical HIT syndromes, such as delayed-onset HIT, persistent HIT, fondaparinux-associated HIT, and HIT induced by exposure to heparin "flush." ​​This form of HIT is referred to as aHIT. More recently, aHIT-like PF4-dominant antibodies have also been recognized in vaccine-induced thrombocytopenia and thrombosis (VITT).

[0004] Additionally, any type of thrombocytopenia (HIT I, HIT II, ​​aHIT, or VITT) can lead to thrombosis, and thus patients can present with thrombocytopenia and thrombosis (HITT). HITT specifically can lead to either arterial or venous thrombosis, which can occur at multiple sites.

[0005] In summary, HIT I is heparin-related and transient, whereas HIT II is immunological, long-term, and more pathological, as additional HIT II antibodies can predispose to HITT.

[0006] Heparin-induced thrombocytopenia (HIT type II) is a dangerous and potentially fatal immunological response to unfractionated heparin (UFH) or, less commonly, low molecular weight heparins (LMWH).

[0007] The prevalence of HIT II ranges from 0.1 to 5% of patients receiving heparin, with 35 to 50% of these patients developing thrombosis and thus exhibiting HITT. The risk of developing HITT increases with the duration of heparin therapy (>5 days), the type of heparin (UFH / LMWH) and dose, the treatment application (surgery and trauma pose a higher risk, exposing tissues to platelets and coagulation activity), and the patient's gender (women are at higher risk). Thus, HITT is a potentially fatal immunological complication of heparin therapy. The cardinal clinical manifestations are a decrease in platelet count and an increased propensity for thromboembolism in clots proximal to heparin exposure, or some other pathogenicity.

[0008] The diagnosis of HIT II requires both laboratory and clinical testing. The clinical 4T score assesses the degree of thrombocytopenia, the timing of thrombocytopenia after heparin administration, the presence of thrombus, and the possibility of other causes of thrombocytopenia. This scoring system has a high negative predictive value, making it useful for ruling out HITT. Furthermore, if an asymptomatic patient with a 4T score indicating HIT II subsequently undergoes ultrasound to detect thrombosis, this 4T scoring is important as a prerequisite for ordering laboratory testing; once this is established, the patient requires a curative rather than preventative approach.

[0009] If HIT II or HITT is diagnosed or suspected, all heparin therapy must be discontinued, and warfarin therapy must be tapered to prevent venous limb gangrene. All patients with HIT II require 4 weeks of anticoagulation therapy, which may extend to 3 months if complicated by thrombotic HITT. In some indications, intravenous immunoglobulin can be used.

[0010] Thus, HIT II is a potent prothrombotic disorder with the unfortunate paradox that thromboprophylaxis and / or antithrombotic treatments are transformed into inducers of new thrombosis. It is caused by ultra-large immune complexes (ULCs), which can reach micron size. ULICs are composed of polyanions, such as unfractionated heparin (UFH), other glycosaminoglycans (GAGs), polyphosphates, or DNA, bound to platelet factor 4 (PF4), which is released upon platelet activation, e.g., after cardiopulmonary bypass or other forms of contact activation of coagulation. PF4 tetramers oligomerize along the UFH backbone, incorporating additional UFH molecules, additional PF4 molecules, and so on. This structural modification creates a large antigen array that stabilizes the epitope on PF4 recognized by several HIT antibodies.

[0011] These antigenic complexes are capable of binding multiple anti-PF4 / UFH antibodies, some of which then promote oligomerization.

[0012] Antigenic complexes are also formed between PF4 and glycosaminoglycans expressed by hematopoietic and vascular cells, which remain targets for HITT antibodies long after heparin has been resolved and metabolized.

[0013] ULIC also activates platelets via the IgG receptor FcγRIIA, releasing PF4, which sustains the formation of neoantigens and leads to uncontrolled thrombin formation. PF4 neutralizes heparin and other GAGs upon binding, thus preventing anticoagulation. ULIC also activates neutrophils to generate DNA NETs, ​​monocytes to express tissue factor, and induces endothelium to express tissue factor, activate complement, release von Willebrand factor, etc. Expression of tissue factor leads to the generation of thrombin, which amplifies the activation of these cell types and exacerbates the risk of thrombosis.

[0014] Current therapies involve administering the maximum tolerated dose of thrombin inhibitors, factor Xa inhibitors, or danaparoid therapy. However, a significant proportion of patients develop new thromboembolic complications, and a reported risk of major bleeding of up to 40% has been reported. Therefore, there is a need for disease-specific interventions that address the stage of the pathogenic process proximal to thrombin formation, allowing for safe and effective use of lower doses of antithrombotic agents. Specifically, drugs that prevent or disrupt antigen formation or immune complex formation, preferably all of which act at the most proximal stage of the pathogenic process, not only attenuate thrombin generation but also block other deleterious effects of IgG-Fc receptor activation or complement activation.

[0015] Accordingly, the inventors herein describe the use of heparin-based compositions that are surprisingly effective in preventing or disrupting the PF4 / UFH complex and / or preventing or interrupting the formation of ULICs. [Prior art documents] [Patent documents]

[0016] [Patent Document 1] International Publication No. 2016 / 030316 Summary of the Invention

[0017] According to a first aspect of the present invention, there is provided an antithrombotic molecule having both antiplatelet and anticoagulant (APAC) activity for use in the prevention and / or treatment of thrombocytopenia, the antithrombotic molecule comprising a human plasma protein to which are attached via a plurality of linker molecules a plurality of heparin chains selected from the group consisting of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16, each chain having a MW of 10 to 21 KDa.

[0018] In a preferred embodiment of the present invention, said thrombocytopenia is selected from the group comprising heparin-induced thrombocytopenia (HIT) type I; heparin-induced thrombocytopenia (HIT) type II; thrombocytopenia and thrombosis (HITT); heparin-independent thrombocytopenia aHIT; and vaccine-induced thrombocytopenia and thrombosis (VITT).

[0019] Most ideally, said thrombocytopenia is selected from the group comprising heparin-induced thrombocytopenia (HIT) type II; and thrombocytopenia and thrombosis (HITT).

[0020] Most ideally, said thrombocytopenia is further immunologically based and selected from the group comprising heparin-induced thrombocytopenia (HIT) type II; thrombocytopenia and thrombosis (HITT); heparin-independent thrombocytopenia aHIT; and vaccine-induced thrombocytopenia and thrombosis (VITT).

[0021] Even more ideally, said thrombocytopenia is further not immunologically based and is selected from the group comprising heparin-induced thrombocytopenia (HIT) type I; thrombocytopenia and thrombosis (HITT); and vaccine-induced thrombocytopenia and thrombosis (VITT).

[0022] Even more ideally, said thrombocytopenia is caused by heparin and is selected from the group comprising heparin-induced thrombocytopenia (HIT) type I; heparin-induced thrombocytopenia (HIT) type II; thrombocytopenia and thrombosis (HITT); and vaccine-induced thrombocytopenia and thrombosis (VITT).

[0023] Without wishing to be bound by theory, the inventors believe that APAC has properties that make it effective for use in the treatment of thrombocytopenia. APAC not only has desirable dual antiplatelet / anticoagulant activity, but is also a relatively small, anionic molecule that competes with UFH for HIT antigen and / or ULIC formation / stability, making it a rational intervention in HIT type II or HITT and / or aHIT and / or VITT.

[0024] Furthermore, the inventors believe it is surprising that heparin-based compositions, i.e., APACs, can be used to prevent or treat HIT type I, HIT type II or HITT, and / or VITT, diseases caused by the presence of heparin (typically UFH / LMWH).

[0025] The manufacture and use of APAC to treat thrombosis caused by factors other than heparin administration and the immunological response thereto is described in PCT / EP2015 / 069327 (WO2016 / 030316).

[0026] In particular, HIT type II or HITT can cause either arterial or venous thrombosis, and therefore references throughout this specification to the prevention or treatment of thrombosis refer to the treatment of either arterial or venous thrombosis.

[0027] In yet a further aspect of the present invention, there is provided the use of an antithrombotic molecule having both antiplatelet and anticoagulant (APAC) activity in the manufacture of a medicament for treating heparin-induced thrombocytopenia, the antithrombotic molecule comprising a human plasma protein having attached thereto a plurality of heparin chains selected from the group comprising 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16 via a plurality of linker molecules, each chain having a MW of 10 to 21 KDa.

[0028] In a preferred embodiment of the invention, the APAC molecule has up to 6 heparin chains attached to the plasma protein, for example between 4, 5 or 6, ideally 5 heparin chains. Even more preferably, the APAC molecule has a heparin concentration of 1.1 mg / ml and the human plasma protein, ideally serum albumin (HSA), has a concentration of 0.87 mg / ml.

[0029] In a preferred embodiment of the invention, the APAC is formulated for administration at a dose ranging from 0.15 μg / ml to 10 μg / ml, inclusive, with near-complete inhibition of HIT type I or HITT type II at the upper end of the range. More preferably, HITT is inhibited at 0.3 μg / ml, with significant inhibitory effects seen at 1 μg / ml or greater. Thus, the invention includes APAC formulated for administration at a dose ranging from 0.15 μg / ml to 3 μg / ml in blood or plasma, inclusive, including all 0.1 μg / ml doses therebetween. Formulations containing 1 to 3 μg / ml are particularly preferred. Additionally or alternatively, formulations in the range of 0.1 to 0.3 mg / kg are preferred.

[0030] In a preferred embodiment of the present invention, the heparin-conjugated human plasma protein is albumin, globulin, or fibrinogen, ideally serum albumin or α2-macroglobulin, and more ideally human serum albumin (HSA) or human α2-macroglobulin. As is generally known, serum albumin is produced by the liver, dissolved in plasma, and is the most abundant blood protein in mammals. Serum albumin is a globular, water-soluble protein with a molecular weight of approximately 66,000 daltons. As is also known, α2-macroglobulin (α2M and A2M) is a large plasma protein, in fact, the largest major non-immunoglobulin protein in plasma, and is primarily produced by the liver. α2-macroglobulin acts as an antiprotease and can inactivate a wide variety of proteinases.

[0031] In a still further preferred embodiment of the invention, said plasma protein is recombinant.

[0032] In yet a further preferred embodiment of the present invention, the heparin is unfractionated heparin. Even more ideally, the heparin is of mammalian origin, ideally human or porcine origin. In the case where the plasma protein is human and the heparin is porcine or bovine heparin, the APAC molecule represents a chimeric molecule.

[0033] Preferably the heparin has a MW selected from the group including 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 KDa, ideally 15 or 16 or 17 KDa.

[0034] In a still further preferred embodiment of the invention, said heparin is recombinant.

[0035] In an even more preferred embodiment of the present invention, the linker molecule is a single linker molecule that binds one molecule of heparin, at least when the binding of the heparin to the plasma protein is complete, and thus the binding of one linker molecule to the plasma protein results in the binding of one molecule of heparin to the plasma protein. Thus, the stoichiometry of the linker to the heparin is 1:1. Preferably, the linker is an amine linker, so that it binds to amino groups on the heparin and plasma protein, and ideally, but not exclusively, the linker conjugates with serines located on the heparin chain, ideally at or near the end of the chain, and ideally, but not exclusively, with lysines on the plasma protein. Even more ideally, the linker conjugates the heparin and plasma protein by using disulfide bridges. Even more preferably, the linker is a heterobifunctional crosslinker, such as a 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP) linker, or a homobifunctional crosslinker, such as a 3,3′-dithiodipropionic acid di(N-hydroxysuccinimide (NHS)-ester (DTSP) linker.

[0036] SPDP (commercially available, for example, from Sigma-Aldrich or Thermo Scientific Pierce) is a short-chain crosslinker used to conjugate amines with sulfhydryls via N-hydroxysuccinimide (NHS)-ester and pyridyldithiol-reactive groups, which form cleavable (reducible) disulfide bonds with cysteine ​​sulfhydryls. It is available in short- and long-chain versions. The long-chain version is available in sulfonated form and is water-soluble. We prefer to use 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester. All SPDPs contain an amine-reactive N-hydroxysuccinimide (NHS) ester that reacts with lysine residues to form stable amide bonds, while the other end of the linker has a pyridyl disulfide group that reacts with sulfhydryls to form reversible disulfide bonds.

[0037] DTSP (3,3'-dithiodipropionic acid di(N-hydroxysuccinimide (NHS)-ester), commercially available from, for example, Sigma-Aldrich or Thermo Scientific Pierce, is a short-chain crosslinker used for amine-to-amine conjugation via an N-hydroxysuccinimide (NHS) ester group. It is available in short- and long-chain versions. The long-chain version is available in a sulfonated form (N-hydroxysulfosuccinimide (sulfo-NHS) ester) and is water-soluble. DTSP contains two amine-reactive N-hydroxysuccinimide (NHS) ester groups and a disulfide bridge in the spacer arm. The N-hydroxysuccinimide ester reacts with primary amine-containing residues to form stable amide bonds, including cleavable disulfide bonds, in the linker molecule.

[0038] In a preferred embodiment, the APAC has a coupling level (CL) of 5 heparin per human serum albumin (HSA) and the linker used for coupling is SPDP.

[0039] When the APAC molecule has six or fewer heparin chains attached to the plasma protein, e.g., four to six, the molecule functions predominantly or to a greater extent as an anticoagulant, given that the antithrombotic molecule / APAC has both antiplatelet and anticoagulant (APAC) activity.

[0040] When the APAC molecule has 8 to 16 heparin chains attached to the plasma protein, the molecule functions to a greater extent as an antiplatelet / platelet inhibitor, given that the antithrombotic molecule / APAC has both antiplatelet and anticoagulant (APAC) activity.

[0041] According to a further aspect of the present invention there is provided a method for the prevention and / or treatment of thrombocytopenia, the method comprising: An effective amount of an antithrombotic molecule is administered to the individual to be treated, the antithrombotic molecule having both antiplatelet and anticoagulant (APAC) activity, comprising a plasma protein to which multiple heparin chains, each having a MW of 10-21 KDa, are attached via multiple linker molecules, and further wherein the number of heparin chains attached to the plasma protein is selected from the group consisting of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16.

[0042] In a further preferred method of the invention, said APAC is a replacement for heparin, ie LMW heparin or unfractionated UFH.

[0043] In a further preferred embodiment of the invention, the APAC is administered at a dose ranging from 0.15 μg / ml to 10 μg / ml in blood or plasma, inclusive, with near-complete inhibition of HIT or HITT at the upper end of the range. More preferably, HITT is inhibited at 0.3 μg / ml, with significant inhibitory effects seen at 1 μg / ml or greater. Thus, the invention includes APAC formulated for administration at a dose ranging from 0.15 μg / ml to 3 μg / ml, inclusive, including all 0.1 μg / ml doses therebetween. Additionally or alternatively, formulations in the range of 0.1 to 3 mg / kg are preferred.

[0044] In the following claims and the foregoing description of the invention, unless the context, express language, or necessary implication dictates otherwise, the word "comprise" or variations such as "comprises" or "comprising" are used in the inclusive sense, i.e., specifying the presence of stated features but not excluding the presence or addition of further features in various embodiments of the invention.

[0045] All references, including any patents or patent applications, cited herein are hereby incorporated by reference. No admission is made that any reference constitutes prior art. Further, no admission is made that any of the prior art constitutes part of the common general knowledge in the art.

[0046] Preferred features of each aspect of the invention may be similar to those described in relation to any of the other aspects.

[0047] Other features of the present invention will become apparent from the following examples. Generally speaking, the present invention extends to any novel or novel combination of features disclosed in this specification (including the accompanying claims and drawings). Accordingly, it should be understood that any property, integer, feature, compound, or chemical moiety described in connection with a particular aspect, embodiment, or example of the present invention is applicable to any other aspect, embodiment, or example described herein, unless incompatible therewith.

[0048] Moreover, unless stated otherwise, any feature disclosed herein may be replaced by an alternative feature serving the same or a similar purpose.

[0049] The invention will now be described, by way of example only, with particular reference to the following figures: [Brief explanation of the drawings]

[0050] [Figure 1A] Figure 1A shows the binding of the HIT-like monoclonal Ab KKO (Arepally et al. Blood. 2000;95:1533-1540 https: / / www.ncbi.nlm.nih.gov / pubmed / 10688805) to PF4 in the presence and absence of UFH and APAC. Figure 1A shows the binding of the HIT-like monoclonal Ab KKO (a mouse monoclonal IgG[2bkappa] antibody against the complex of human PF4 and heparin) to 1) immobilized PF4 (50 μl of 5 μg / ml / well = 0.25 μg), 2) immobilized PF4 in the presence of UFH (0.1 IU / ml), and 3) immobilized PF4 in the presence of APAC (0.5, 1, 3, 10, 30, 100, 200, and 300 μg / ml; heparin-equivalent concentrations). Results are shown as the mean±standard error of the mean (SEM) of three independent experiments. [Figure 1B]Figure 1B shows the binding of Ab KKO to 1) immobilized PF4 (0.25 μg), 2) immobilized PF4 in the presence of UFH (0.1 IU / ml), 3) immobilized PF4 in the presence of both UFH (0.1 IU / ml) and APAC (3, 10, 30, and 100 μg / ml), and 4) immobilized PF4 in the presence of APAC (3, 10, 30, and 100 μg / ml). Results are shown as the mean ± standard error of the mean (SEM) of three independent experiments. [Figure 2] The inhibitory effect of APAC on the formation of large antigenic PF4 / UFH complexes is shown. The size of particle formation between PF4 (10 μg / ml) and UFH (0.1 IU / ml) in the presence and absence of APAC (0.15, 0.3, 1, 2, 3, 5, or 10 μg / ml; reported as heparin equivalent concentrations) is shown after 0, 1, 2, 3, 4, 5, 6, and 24 hours of incubation. Particle formation was detected by dynamic light scattering (DLS). Results are shown as the mean ± standard deviation (SD) of three independent experiments. [Figure 3] This figure shows the dissociation effect of APAC on preformed large antigenic PF4 / UFH complexes. PF4 / UFH complexes were initially formed between 10 μg / ml PF4 and 0.2 IU / ml UFH. The particle size of this preformed PF4 / UFH complex in the presence and absence of APAC (0.15, 0.3, 1, 2, 3, 5, or 10 μg / ml; heparin-equivalent concentrations) is shown at 0, 1, 2, 3, 4, 5, 6, and 24 hours of incubation. Particle formation was detected by DLS. Results are shown as the mean ± SD of three independent experiments. [Figure 4]The competitive effect of APAC on the formation of ultra-large immune complexes (ULCs) of KKO / PF4 / UFH is shown. The size of ULIC particles formed between PF4 (10 μg / ml), UFH (0.2 IU / ml), and the HIT-like monoclonal antibody KKO (30 μg) in the presence and absence of APAC (0.15, 0.3, 1, 2, 3, 5, or 10 μg / ml; reported as heparin equivalent concentrations) at 0, 1, 2, 3, 4, 5, 6, and 24 hours of incubation. Particle formation was detected by DLS. Results are shown as the mean ± SD of three independent experiments. [Figure 5] This figure shows the dissociation effect of APAC on preformed ultra-large immune complexes (ULCs) of KKO / PF4 / UFH. PF4 (10 μg / ml), UFH (0.2 IU / ml), and the HIT-like monoclonal antibody KKO (30 μg) were used to first form ULICs. The particle size of preformed KKO / PF4 / UFH complexes in the presence and absence of APAC (0.15, 0.3, 1, 2, 3, or 5 μg / ml; heparin-equivalent concentration) is shown at 0, 1, 2, 3, 4, 5, 6, and 24 hours of incubation. Particle formation was detected by DLS. Results are shown as the mean ± SD of three independent experiments. [Figure 6] The effect of APAC on the induction of tissue factor (TF) activity in a human monocyte-like cell line (THP-1) is shown. THP-1 cells were first incubated with PF4 (10 μg / ml) and then supplemented with APAC (10, 50, or 100 μg / ml; heparin-equivalent concentration). Control THP-1 cells were not treated with APAC. The HIT-like monoclonal antibody KKO (50 μg / ml) was included to induce immune complex (IC) formation. The generation of FXa activity reflected active TF expression in the cell suspension. Data are presented as the mean fold increase in the initial rate of FXa generation compared to THP-1 cells alone. Results are presented as the mean ± SEM of four experiments. DETAILED DESCRIPTION OF THE INVENTION

[0051] method Conjugates Unfractionated heparin, Hep (UFH chains), was conjugated to human serum albumin (HSA) via disulfide bridges created by two alternative crosslinkers and reaction routes using the following: i) Heterobifunctional crosslinker 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP). For conjugation, a free amine on Ser in the Hep linker region and a free amine on Lys on HSA were utilized. Hep and HSA were modified to sulfhydryl (-SH) and pyridyldithiol (-PDP) derivatives, respectively, in separate reactions. In the final conjugation reaction, the pyridyldithiol group of HSA reacted with the sulfhydryl group of Hep, resulting in the formation of a disulfide-bonded complex and the release of pyridine 2-thione.

[0052] ii) The homobifunctional crosslinker 3,3'-dithiodipropionic acid di(N-hydroxysuccinimide (NHS)-ester) (DTSP). For conjugation, the free amine on Ser in the Hep linker region and Lys on HSA were utilized. Hep was first modified to an N-hydroxysuccinimide (NHS) ester derivative, releasing the first NHS group. In the final conjugation reaction, Lys on HSA reacted with the N-hydroxysuccinimide (NHS)-ester group of the derivatized Hep, resulting in the formation of a complex with a cleavable disulfide bond in the linker region and the release of the second N-hydroxysuccinimide group.

[0053] The ratio of the intermediate derivatives of HSA and Hep defined above in the final conjugation reaction to produce the Hep-HSA conjugate is selected to obtain a particular average conjugate level (CL) in the final purified Hep-HSA conjugate.

[0054] The Hep-HSA complex was purified by anion exchange chromatography using ultrafiltration / diafiltration and Q Sepharose medium (GE Healthcare, USA) or ultrafiltration / diafiltration. Finally, the Hep-HSA complex was eluted in phosphate-buffered saline (PBS) at pH 7.4-7.5. The complex was designated APAC-, a suffix indicating the average conjugation level of Hep chains to HSA. Thus, references herein to a plurality of heparin chains conjugated to human plasma proteins selected from the group including 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16 refer to the average conjugation level.

[0055] The general formula for an APAC complex exemplifying the present invention is: (Hep-NH-CO-CH2-CH2-SS-CH2-CH2-CO-NH) n -HSA where the average number of unfractionated heparin chains bound to HSA is defined as n.

[0056] The concentrations of Hep and HSA and their average molecular weights were used to determine the average conjugate level (CL) of Hep to HSA using the following formula: Molar Hep = Hep[C] / Average Hep MW moles of HSA = HSA[C] / HSA MW CL = moles Hep / moles HSA Hep MW=15800 or 17000 HSA MW=66472 Binding of HIT-like monoclonal Ab KKO to PF4 in the presence and absence of UFH and APAC Immulon 4 HBX plates (Thermo Scientific, Waltham, MA, USA) were first coated with PF4 (50 μl of 5 μg / ml in phosphate-buffered saline [PBS]). In experiment A, wells were supplemented with APAC at final concentrations of 0.5, 1, 3, 10, 30, 100, 200, and 300 μg / ml. In experiment B, APAC was supplemented alone or with a fixed concentration of UFH (0.1 IU / ml) at final concentrations of 3, 10, 30, 100, 200, and 300 μg / ml. PF4 alone and PF4 supplemented with UFH (0.1 IU / ml) were used as controls. The plates were incubated overnight at room temperature (RT). The next day, wells were washed four times with 180 μl of PBS, and nonspecific binding was blocked with 1% bovine serum albumin (BSA) in PBS for 1 hour at room temperature. Next, the wells were supplemented with 100 μg / ml of the HIT-like monoclonal antibody KKO (in 1% BSA / PBS) for 30 min at 37°C, after which the wells were washed four times with 180 μl of PBS / 0.1% Tween-20. The wells were incubated for 30 min with goat anti-mouse IgG (Fc) conjugated with horseradish peroxidase (HRP) 1:3000 in 1% BSA / PBS, which was used as the secondary Ab (Bethyl Laboratories, Montgomery, TX, USA). Wells were washed four more times with 180 μl of PBS / 0.1% Tween-20 and HRP substrate, and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (Roche diagnostics, Mannheim, Germany) was added at 100 μl / well at room temperature, and secondary Abs were detected at 405 nm and 490 nm in a BioTek Synergy 2 plate reader (BioTek Instruments Inc., Winooski, VT, USA). Results are calculated as the mean ± SEM of three independent experiments.

[0057] Effect of APAC on the formation of large antigenic PF4 / UFH complexes PF4 (10 μg / ml) was supplemented with UFH (Hep; 0.2 IU / ml) alone or with both UFH (Hep; 0.2 IU / ml) and increasing concentrations of APAC (0.15, 0.3, 1, 2, 3, 5, or 10 μg / ml). Particle size was measured by dynamic light scattering (DLS) immediately after addition of UFH and / or APAC and after 1, 2, 3, 4, 6, and 24 hours of incubation. Results are calculated as the mean ± SD of three independent experiments.

[0058] Effect of APAC on the dissociation of preformed large antigenic complexes PF4 (10 μg / ml) was supplemented with UFH (0.2 IU / ml) and pre-incubated at room temperature for 30 minutes, after which APAC was added at increasing concentrations of 0.15, 0.3, 0.5, 1, 2, 3, and 5 μg / ml. The size of the formed particles was measured by DLS immediately and after 1, 2, 3, 4, 6, and 24 hours of incubation. Results are calculated as the mean ± SD of three independent experiments.

[0059] Effect of APAC on the formation of ultra-large immune complexes APAC (0.15, 0.3, 1, 2, 3, or 5 μg / ml), PF4 (10 μg / ml), UFH (0.2 IU / ml), and the HITT-like monoclonal antibody KKO (30 μg) were incubated together, and the size of the formed ultra-large immune complexes (ULCs) was measured by DLS immediately and after 1, 2, 3, 4, 6, and 24 hours of incubation. Results are calculated as the mean ± SD of three independent experiments.

[0060] Effect of APAC on the disruption of preformed ultra-large immune complexes PF4 (10 μg / ml) was first incubated with the HIT-like monoclonal Ab KKO for 5 minutes at room temperature, after which UFH (0.2 IU / ml) was added for another 5 minutes to form PF4 / KKO / UFH complexes. These preformed PF4 / KKO / UFH complexes were then supplemented with 0.15, 0.3, 1, 2, 3, or 5 μg / ml of APAC. The size of the formed particles was measured by DLS immediately and after 1, 2, 3, 4, 6, and 24 hours of incubation. Results are calculated as the mean ± SD of three independent experiments.

[0061] Effect of APAC on the induction of FXa activity by monocytic cell lines Tissue factor, TF production by THP-1 cells incubated with PF4 / KKO ± APAC. This experiment was designed to determine whether APAC prevented the generation of tissue factor (TF) activity on human monocyte-like cells by PF4 and the HIT-like monoclonal antibody KKO.

[0062] THP-1 cells (human acute leukemia monocytic cell line) were cultured at 37°C and 5% CO2 in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS), 4.5 mg / ml glucose, 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U / mL penicillin, 100 μg / ml streptomycin, and 0.25 μg / ml amphotericin B. THP-1 cells were cultured at 10°C in 100 μl of RPMI-1640 supplemented with 5% FBS. 5THP-1 cells were plated in 96-well plates at 100 cells / well. THP-1 cells were first incubated with PF4 (10 μg / ml) for 5 min at 37°C, then with APAC at final concentrations of 10, 50, or 100 μg / ml for an additional 30 min. Control THP-1 cells contained no APAC. In a third step, all cells were supplemented with the HIT-like monoclonal Ab KKO (50 μg / ml) and further incubated overnight. Binding of KKO to cell-associated glycosaminoglycans was replaced by exogenous UFH. The next day, cells were washed to remove unbound ligand. FXa activity was measured using a chromogenic assay in flat-bottom 96-well plates in which an aliquot (10 μl) of the cell suspension was added to a mixture of factor VIIa (0.5 nM) and factor X (160 nM) in 20 mM Tris buffer, pH 7.4, containing 100 mM NaCl and 10 mM CaCl, for 30 minutes at 37°C under 5% CO. Activated coagulation factor FXa chromogenic substrate (0.4 mM) was added, and the optical density at 405 nm was read in kinetic mode (one reading per minute) for 30 minutes at 37°C. The amount of FXa produced over the first 10 minutes was calculated against a standard curve using purified reagents.

[0063] The average MW of Hep polymers is based on information obtained from the heparin manufacturer. HSA MW is based on ALBU_HUMAN, P02768 from isoform 1 of UniProtKB / Swiss-Prot without signal and propeptides.

[0064] Statistics: All data are means ± SD or SEM and were analyzed using SPSS for Windows, version 15.0 (SPSS Inc, Chicago, IL). For two-group comparisons, the nonparametric Mann-Whitney U test and the parametric Student's t test were applied. For multiple-group comparisons, the nonparametric Kruskal-Wallis test with Dunn's post-hoc test and the parametric ANOVA with Dunnett's correction were applied. P < 0.05 was considered statistically significant.

[0065] Experimental procedure Our first objective was to determine whether APAC forms an antigenic complex with PF4 and / or whether it reduces the antigenicity of the PF4 / UFH complex defined by the binding of HIT-like antibodies. Having identified the concentration of APAC that interferes with antigenicity, our second objective was to examine its effect on the size of the large pathogenic complexes that cause HIT or HITT. Because we found that UICs are stable over 24 hours, we hypothesized that antigenic and immune complexes undergo a series of changes over time, making them progressively more difficult to effect. Therefore, our objective was to conduct a series of experiments that address the putative sequence of events in the pathogenesis of HIT by asking the following four questions, each addressed in turn: 1) Can APAC prevent the formation of the PF4 / UFH complex? 2) Can APAC disrupt the PF4 / UFH complex? 3) Can APAC prevent the formation of ULICs? and 4) Can APAC destroy preformed ULIC? To do so, the inventors used a well-described mouse monoclonal anti-PF4 / UFH antibody called KKO and measured the size of the complexes in solution using dynamic light scattering (DLS).

[0066] Finally, we asked whether APAC inhibits the ability of ULIC to generate the active clotting factor, FXa activity, on a monocytic cell line.

[0067] result NOTE: In all experiments, data are presented as the mean ± SD of at least three independent experiments (Figure 2–Figure 5) or the mean ± SEM of three experiments (Figure 1) or the mean ± SEM of four experiments (Figure 6).

[0068] A. Effect of APAC on the binding of HIT-like monoclonal antibody KKO.

[0069] The data shown in Figure 1A are from an ELISA measuring the binding of a mouse monoclonal HIT-like antibody to PF4 / APAC. The results show that KKO binds to PF4 / APAC starting at the lowest concentration tested (0.5 μg / ml) (see Figure 1A). However, binding of KKO decreases in a dose-dependent manner at higher, potentially therapeutic, concentrations of APAC. This is the same pattern observed at the upper optimal concentration of heparin, due to the formation of a smaller complex with PF4.

[0070] The effect of APAC added to PF4 / UFH is shown on the left side of Figure 1B.

[0071] At a concentration of 1 μg / ml of APAC, there was a slight increase in KKO binding to PF4 / UFH, comparable to the binding of PF4 / APAC alone, i.e., in the absence of UFH (data not shown). However, the most significant effect was a dose-dependent decrease in KKO binding at all increasing concentrations of APAC. Comparing the left and right slides in Figure 1B, it is likely that APAC dissociates the large PF4 / UFH complex, allowing it to bind to what we propose is a smaller PF4 / APAC complex. These results led to the question of whether PF4 binding to APAC generates a large, "pathogenic" complex. We hypothesized that the smaller size of APAC would be less likely to promote PF4 oligomerization in the manner seen with UFH, and this assumption was confirmed in the experiments described below.

[0072] B. Effect of APAC on the formation of large antigenic PF4 / UFH complexes In these experiments and subsequent DLS experiments, PF4 (10 μg / ml) was incubated with the indicated concentrations of APAC and UFH (0.2 IU / ml) as the standard starting condition. Particle size (vertical axis) was measured by DLS immediately and after 1, 2, 3, 4, 6, and 24 hours (horizontal axis). The red line in Figure 2 indicates the absence of APAC; this is the size of the complex formed between PF4 and UFH, which continues to increase over time over the 24-hour incubation. In general, there is an inverse dose-dependent relationship between APAC concentration and particle size. The apparent abnormal initial results at 1 μg / ml APAC are consistent with the ELISA results and may indicate a combination of PF4 / APAC and PF4 / UFH complexes or the incorporation of APAC into the PF4 / UFH complex. The results show that complex growth during the 24-hour incubation is inhibited by APAC at concentrations as low as 0.15 μg / ml, with inhibition nearly complete at 3 μg / ml.

[0073] C. Effect of APAC on the dissociation of preformed large antigenic complexes In the set of experiments shown in Figure 3, PF4 was preincubated with unfractionated heparin (UFH) for 30 minutes at room temperature. APAC was then added at the indicated concentrations. The results show that the lowest concentration of APAC has no effect on the size of the antigenic complexes. A decrease in size begins at 0.3 μg / ml, and at higher concentrations, no complexes larger than 20 nm in size are evident. A significant inhibitory effect is seen at 1 μg / ml.

[0074] D. Effect of APAC on ULIC formation.

[0075] In the experiment shown in Figure 4, APAC was added with PF4 (10 μg / ml), UFH (0.2 IU / ml), and KKO (30 μg), and the size of the complexes (vertical axis) was measured over time (horizontal axis). The data show that low doses of APAC enhance immune complex formation. This is consistent with data from previous studies showing enhanced antibody binding in the formation of UFH-PF4 antigenic complexes at these concentrations. At higher concentrations, APAC totally prevented the formation of ultra-large immune complexes (ULCs), again consistent with its ability to prevent and disrupt antigen formation. A significant inhibitory effect is seen at 3 μg / ml.

[0076] E. Destruction of preformed ULICs.

[0077] This is the most stringent test: the degradation of a large, stable, preformed PF4 / KKO / heparin complex. Here, PF4 (10 μg / ml) was incubated with KKO for 5 minutes at room temperature. UFH (0.2 IU / ml) was added for 5 minutes at room temperature. APAC was then added at increasing concentrations (0–5 μg / ml). The results are shown in Figure 5. As observed in all previous experimental conformations, low doses of APAC increased the size of the complex. However, the preformed complex was totally disrupted at higher concentrations of APAC. A significant inhibitory effect was observed at 3 μg / ml.

[0078] F. Effect of APAC on the induction of FXa activity by monocytic cell lines This experiment was designed to determine whether APAC interferes with the induction of tissue factor activity in THP-1 monocytic cells by PF4 and KKO. THP-1 cells were incubated with PF4, with or without APAC, as follows: PF4 (10 μg / ml, 5-minute incubation), followed by APAC (30 minutes), and then KKO (50 μg / ml), all at 37°C. Binding of KKO to cell-associated glycosaminoglycans was substituted for exogenous UFH. After further incubation, cells were washed to remove unbound ligand. As a measure of TF expression, aliquots of the cell suspension were assayed for the amount of FXa produced. Results shown in Figure 6 are the mean ± SEM of four experiments, each performed in quadruplicate wells. The results indicate that higher concentrations of APAC inhibited FXa production by this monocytic cell line stimulated with HITT immune complexes. These results are consistent with all previous sets of experiments looking at the formation / dissolution of PF4 / UFH and PF4 / UFH / KKO complexes.

[0079] conclusion These experiments support the concept that APAC offers a new approach to the treatment of HIT type II and / or HITT by combining antithrombotic activity with the ability to interfere with the formation and stability of ULICs, one of the most proximal steps in the pathogenic process.

[0080] The required APAC concentration increases continuously: prevention of antigen formation < antigen dissociation < prevention = dissociation of immune complexes. In fact, administering APAC instead of UFH could theoretically prevent the development of HIT, while using higher concentrations could interrupt the cycle of ULIC formation, cell activation, release of PF4 and thrombin, and the feed-forward prothrombin loop that develops in these patients.

[0081] In other words, APAC should be used instead of heparin.

[0082] 1) Preventively, when there is a high suspicion of developing HIT, such as a past medical history, or when there is an increased risk of HIT, for example, related to trauma or surgery, e.g., cardiovascular, intervention, etc. and / or 2) If HIT type II or HITT occurs, then heparin should be stopped and APAC should replace heparin.

Claims

1. 1. An antithrombotic molecule having both antiplatelet and anticoagulant (APAC) activity for use in the manufacture of a medicament for preventing and / or treating thrombocytopenia, comprising a human plasma protein to which are attached via a plurality of linker molecules a plurality of heparin chains selected from the group consisting of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16, each chain having a MW in the range of 10-21 KDa; The thrombocytopenia is selected from the group consisting of heparin-induced thrombocytopenia (HIT) type I; heparin-induced thrombocytopenia (HIT) type II; heparin-induced thrombocytopenia and thrombosis (HITT); heparin-independent thrombocytopenia aHIT; and vaccine-induced thrombocytopenia and thrombosis (VITT). Antithrombotic molecules.

2. 2. The antithrombotic molecule for use according to claim 1, wherein the thrombocytopenia is selected from the group consisting of heparin-induced thrombocytopenia (HIT) type II; and heparin-induced thrombocytopenia and thrombosis (HITT).

3. 2. The antithrombotic molecule for use according to claim 1, wherein the thrombocytopenia is immunologically based and is selected from the group consisting of heparin-induced thrombocytopenia (HIT) type II; heparin-induced thrombocytopenia and thrombosis (HITT); heparin-independent thrombocytopenia aHIT; and vaccine-induced thrombocytopenia and thrombosis (VITT).

4. 2. The antithrombotic molecule for use according to claim 1, wherein the thrombocytopenia is not immunologically based and is selected from the group consisting of heparin-induced thrombocytopenia (HIT) type I; heparin-induced thrombocytopenia and thrombosis (HITT); and vaccine-induced thrombocytopenia and thrombosis (VITT).

5. 2. The antithrombotic molecule for use according to claim 1, wherein the thrombocytopenia is caused by heparin and is selected from the group consisting of heparin-induced thrombocytopenia (HIT) type I; heparin-induced thrombocytopenia (HIT) type II; thrombocytopenia and thrombosis (HITT); and vaccine-induced thrombocytopenia and thrombosis (VITT).

6. The antithrombotic molecule for use according to any one of claims 1 to 5, wherein said antithrombotic molecule has 4, 5 or 6 heparin chains attached to said human plasma protein.

7. The antithrombotic molecule for use according to any one of claims 1 to 6, wherein said antithrombotic molecule has five heparin chains bound to said human plasma protein.

8. 8. The antithrombotic molecule for use according to any one of claims 1 to 7, wherein the antithrombotic molecule is formulated for administration at a dose in the range of 0.15 μg / ml to 10 μg / ml in blood or plasma.

9. The antithrombotic molecule for use according to claim 8, wherein the antithrombotic molecule is formulated for administration at a dose in the range of 1 μg / ml to 3 μg / ml.

10. The antithrombotic molecule for use according to any one of claims 1 to 7, wherein the antithrombotic molecule is formulated for administration at a dose in the range of 0.1 to 0.3 mg / kg.

11. The antithrombotic molecule for use according to any one of claims 1 to 10, wherein said human plasma protein is selected from the group consisting of albumin, globulin or fibrinogen.

12. The antithrombotic molecule for use according to claim 11, wherein said human plasma protein is serum albumin or α2-macroglobulin.

13. The antithrombotic molecule for use according to any one of claims 1 to 12, wherein said human plasma protein is recombinant.

14. An antithrombotic molecule for use in thrombocytopenia according to any one of claims 1 to 13, wherein said heparin chains are unfractionated heparin.

15. An antithrombotic molecule for use according to any one of claims 1 to 14, wherein the heparin chains have a MW of 15 or 16 or 17 KDa.

16. The antithrombotic molecule for use according to any one of claims 1 to 15, wherein said heparin chains are recombinant.

17. An antithrombotic molecule for use according to any one of claims 1 to 16, wherein each linker molecule binds one heparin molecule to said human plasma protein.

18. The antithrombotic molecule for use according to any one of claims 1 to 17, wherein said linker molecule is an amine linker and connects amino groups on said heparin chains and said human plasma proteins.

19. An antithrombotic molecule for use according to any one of claims 1 to 18, wherein said linker molecule is conjugated to a serine on said heparin chain and a lysine on said human plasma protein.

20. 20. The antithrombotic molecule for use according to any one of claims 1 to 19, wherein the linker molecule is a heterobifunctional cross-linking group such as a 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP) linker, or a homobifunctional cross-linking group such as a 3,3'-dithiodipropionic acid di(N-hydroxysuccinimide (NHS)-ester) (DTSP) linker.

21. 21. The antithrombotic molecule for use according to any one of claims 1 to 20, wherein the antithrombotic molecule has a coupling level (CL) of 5 heparin per human serum albumin (HSA), and the linker molecule used for the coupling is SPDP.

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