Antidote

Abstract

The present invention discloses a recombinant antithrombin for use in the treatment of haemorrhagic risk in a patient.

Description

[0001] ANTIDOTE

[0002] Field of technology

[0003] The present invention falls within the pharmaceutical sector and relates to an antidote against heparins and its medical use in the treatment of hemorrhagic risk.

[0004] Background

[0005] Heparin is a highly sulfated glycosaminoglycan. It has the highest density of negative charges known in a biological molecule. It is an anticoagulant drug that acts by preventing the formation and spread of clots within the blood, but is unable to break or lyse a clot already formed. It is used for the treatment of deep vein thrombosis and pulmonary embolism, acute coronary syndrome, atrial fibrillation, cardiopulmonary bypass, extracorporeal circulation, hemodialysis, and central or peripheral venous catheters. The main complications of heparin treatment are bleeding and thrombocytopenia, which involves a decrease in the number of circulating platelets in the bloodstream below normal levels. Heparin's mechanism of action is based on the binding and activation of antithrombin, the main physiological inhibitor of the coagulation cascade.This binding occurs through electrostatic interactions that cause a conformational change in antithrombin, which consequently more efficiently inhibits its targets, primarily coagulation factor Xa (FXa) and coagulation factor Ha or thrombin (PHI). In this way, heparin acts as a cofactor for antithrombin.

[0006] There are different types of heparins used clinically. Unfractionated heparin is the only physiological type. It is isolated from mast cells present in the intestinal mucosa of porcine or bovine cells. Low-molecular-weight heparins are obtained by chemical or enzymatic fragmentation from unfractionated heparins. These heparins also have different actions. Unfractionated heparins primarily have anti-Flla action, while low-molecular-weight heparins (LMWH) and pentasaccharide primarily have anti-FXa action.

[0007] In the clinic, the heparins in use are the following:

[0008] Sodium heparin 1000 U / ml and 5000 U / ml (Unit of enzyme activity per ml) Low molecular weight heparins, Table 1:

[0009] Table 1: Low molecular weight heparins used in clinical practice

[0010] Ul: international units

[0011] Se: subcutaneous

[0012] Different heparins also have different indications:

[0013] - Sodium heparin: When there is a very high risk of thrombosis and hemorrhage. Example: prosthetic valves. Treatment of deep vein thrombosis (DVT) of the lower extremities with or without pulmonary embolism, only in the acute phase.

[0014] - Low-molecular-weight heparins: Prophylaxis of venous thromboembolism (VTE) in general surgery, when there is renal failure or obesity. Treatment of lower extremity DVT with or without pulmonary embolism.

[0015] - Fondaparinux: VTE prophylaxis in hip fracture surgery and when there is renal failure.

[0016] Furthermore, dosage adjustment is necessary in patients with renal failure, the elderly, obese patients, and pregnant women. This is important because heparins are considered high-risk medications by the Institute for Safe Medication Management (ISMP). "High-risk medications" are those that, when misused, present a high probability of causing serious or even fatal harm to patients. There is only one antidote for unfractionated heparins, protamine sulfate, but it causes hypotension and bradycardia. There is no approved method for neutralizing low-molecular-weight heparins. The use of protamine sulfate is reflected in the data sheet when bleeding is severe. Protamine sulfate will only neutralize the anti-HIV fraction of LMWH and is only effective if it has been administered less than 8 hours before administration. Therefore, its effectiveness is minimal.It should be noted that excessive doses may have anticoagulant effects, so it is best not to use it. The recommended doses in the product data sheet are: Protamine sulfate: 1 mg per 100 IU of Anti-Xa LMWH and if bleeding persists: 0.5 mg per 100 IU of Anti-Xa LMWH.

[0017] There is no specific antidote for pentasaccharide Fondapañux. If there is severe bleeding, prothrombin complex can be used.

[0018] Another hepatic antidote described in the literature is based on antithrombin, since it has a hepatic-binding domain. This recombinant antithrombin lacked one of its four glycans, specifically the N135 glycan, which favored greater affinity for hepatic. Additionally, the addition of a proline at position 394 decreased antithrombin's anticoagulant capacity by three orders of magnitude. In experiments with mice, the authors demonstrate that this antithrombin administered at a concentration 2.5 times higher than the plasma antithrombin concentration, neutralized 86% of the anti-FXa activity in 5 minutes in mice treated with pentasaccharide (Bianchini EP, Fazavana J, Picard V, Borgel D. Development of a recombinant antithrombin variant as a potent antidote to fondaparinux and other heparin derivatives. Blood. 2011;117(6): 2054-60). They do not disclose in vivo experiments with the other heparins, only ex vivo.This mutated antithrombin and its use for the treatment or prevention of coagulation disorders is patented (EP2175877B1). In the patent, they describe the FXa inhibitory activity of different antithrombin mutants (AT-R393H, AT-Pro394, AT-AR393-S394, AT-AR393, AT-AS394, AT-N135Q-R393H, AT-N135Q-Pro394, AT-N135Q-AR393-S394, AT-N135Q-AR393, ATN135Q-AS394). In mice, they test the effect of all these mutants as antidotes to pentasaccharide.

[0019] The antidote based on a recombinant antithrombin of the present invention is much better than the one described in EP2175877B1 since the mutations it presents with respect to the antithrombin sequence make it express itself better and have greater affinity for heparin.

[0020] The present invention relates to an antidote based on a recombinant antithrombin that lacks the ability to inhibit coagulation target proteases, but has a higher affinity for heparin than the wild-type protein. In in vivo mouse models and ex vivo models in plasma from anticoagulated patients, we have verified that the antidote of the present invention is capable of reversing the anticoagulant effect of low molecular weight heparins.

[0021] Description

[0022] In this specification, the terms "antidote" and "recombinant antithrombin" are synonymous and are used interchangeably. In the prior art, an antidote could also be a molecule capable of binding to heparin and preventing its action, without necessarily being a recombinant antithrombin.

[0023] The term "recombinant antithrombin" refers to an antithrombin, preferably a human antithrombin, comprising at least one substitution, insertion and / or deletion of one or more amino acids within its amino acid sequence.

[0024] As used herein, the term “low molecular weight heparins” refers to heparin with a weight in the range of about 4000 to 6500 Daltons.

[0025] As used herein, the term "nucleotide" may refer to either ribonucleotide or deoxyribonucleotide, unless otherwise stated.

[0026] There are two glycoforms of antithrombin in plasma. The alpha glycoform, with four N-glycans, is the majority (90-95%), while the beta glycoform, which lacks N-glycosylation at position 135, is the minority (5-10%). The generation of the beta glycoform is explained by the presence of a serine residue at position 137 (in the mature antithrombin sequence, once the signal peptide has been removed) and is responsible for the N-glycosylation of asparagine (N) 135. The presence of serine instead of threonine at position 137 reduces the efficiency of the first process of incorporation of the carbohydrate nucleus into the protein backbone. The absence of the carbohydrate component at residue N135 has important functional consequences, as it increases the affinity for heparin and other glycosaminoglycans such as heparan sulfate by two to five times, a key process in the activation of the anticoagulant activity of antithrombin.This glycosylation at position 135 produces a spherical impediment of the alpha conformation to bind to heparin.

[0027] Surprisingly, the inventors have discovered that by introducing the mutations 1039TG>AC to give rise to the change of amino acid M to N and 1044GTG>ACA to give rise to the change of amino acid V to T. These mutations produce the addition of a new N-glycan, this glycan is very far from position 135 whose relevance has been explained in the previous paragraph, therefore the technical effect of these mutations is that the secretion of the protein is increased when it is synthesized in vitro, since the glycosylation sites are necessary for the secretion of antithrombin when it is expressed in eukaryotic cells and additionally allows an affinity for heparin of more than 2.5 times greater than that of Beta-antithrombin since the glycosylation chains are involved in the binding of antithrombin-glycosaminoglycans similar to cellular heparin.

[0028] The state of the art only discloses a mutation in the nucleotide sequence corresponding to amino acid 137 to eliminate the N-glycan at position 135 that differentiates Alpha-antithrombin from Beta-antithrombin, however, the introduction of the 2 mutations indicated above allows the introduction of a new N-glycan group in the sequence that allows the antidote of the invention to be secreted better and have greater affinity for heparin.

[0029] The present invention relates to an antidote comprising

[0030] - a recombinant antithrombin encoded by the polynucleotide sequence SEQ ID NO: 2, which has the following mutations with respect to SEQ ID NO: 1

[0031] A) 1039 TG>AC

[0032] B) 1044 GTG>ACA

[0033] C) 504 TCC> NNN, where N is any combination of three nucleotides that does not translate into a serine, threonine, or cysteine

[0034] D) Deletion of the nucleotides at position 1273, 1274 and 1275 of SEQ ID NO:1 or

[0035] - a recombinant antithrombin encoded by a variant of SEQ ID NO: 2 having at least 80% identity, more preferably 85% identity, even more preferably at least 90% identity, and even more preferably 91% or 92% or 93% or 94% or 95% or 96% or 97% or 98% or even up to 99% identity with respect to the polynucleotide sequence SEQ ID NO: 2, keeping the mutations A)-D) of SEQ ID NO: 2 fixed with respect to SEQ ID NO: 1 for use in the prevention and / or treatment of hemorrhagic risk in a subject.

[0036] SEQ ID NO: 1 is the nucleotide sequence of the gene encoding the SERPINC1 protein in Homo sapiens. This sequence is the region from nucleotides 69 to 1463, Genebank CDS (Coding Sequence), NM_000488.4 (1552 bp linear mRNA PRI 10-APR-2023).

[0037] The term “mutations” includes at least one substitution, insertion and / or deletion of one or more nucleotides that result in an amino acid change when the sequence is translated.

[0038] The recombinant antithrombin encoded by a variant of SEQ ID NO: 2 may comprise one or more mutations as indicated in the preceding paragraph.

[0039] The technical effect of the variations 1039 TG>AC and 1044 GTG>ACA has been explained in the previous paragraphs.

[0040] The technical effect of the 504 TCC> NNN variation is that N-glycosylation of the asparagine residue at position 135 is avoided, which means that antithrombin is in the beta conformation rather than the alpha glycoform.

[0041] In a particular embodiment, the variation 504 TCC> NNN is 504 T>G, this variation results in the change of amino acid S to A.

[0042] The technical effect of the deletion of 3 nucleotides in position 1273, 1274 and 1275 of SEQ ID NO:1 which results in a deletion of an arginine amino acid (AR) in said position, the advantage of this deletion is that the antidote of the invention binds with greater affinity to heparin, thus achieving a substantially reduced capacity to inhibit factor Xa (FXa) and factor II (FII)

[0043] In another particular embodiment, the antidote of the invention comprises a recombinant antithrombin encoded in SEQ ID NO: 3 that presents the following mutations with respect to SEQ ID NO: 1

[0044] A) 1039 TG>AC B) 1044 GTG>ACA

[0045] C) 504 T> G, and

[0046] D) Deletion of the nucleotides at position 1273, 1274 and 1275 of SEQ ID NO:1 or a recombinant antithrombin encoded by a variant of SEQ ID NO:3 having at least 80% identity, more preferably 85% identity, even more preferably at least 90% identity, and even more preferably 91% or 92% or 93% or 94% or 95% or 96% or 97% or 98% or even up to 99% identity with respect to the polynucleotide sequence SEQ ID NO:3, keeping mutations A)-D) of SEQ ID NO:3 fixed with respect to SEQ ID NO:1 for use in the prevention and / or treatment of hemorrhagic risk in a subject

[0047] The recombinant antithrombin encoded by a variant of SEQ ID NO: 3 may comprise one or more mutations as indicated above.

[0048] The amino acid sequence encoding SEQ ID NO:1 is found in SEQ ID NO:4. This is the native sequence of human antithrombin disclosed in GenPept NP_000479; 464 aa; linear PRI 10-APR-2023

[0049] The first 32 amino acids of SEQ ID NO: 4 correspond to a signal peptide that is expressed physiologically, but is processed inside the cell so that the protein that is secreted does not contain that peptide because it is proteolyzed, in SEQ ID NO: 5 the same sequence is disclosed but without the signal peptide.

[0050] Thus, the amino acid sequence translated from SEQ ID NO: 3 is SEQ ID NO: 6, and the amino acid variations are S169A / M347N / V349T / AR425 with respect to SEQ ID NO: 4.

[0051] SEQ ID NO: 7 is identical to SEQ ID NO: 6, but without the first 32 amino acids corresponding to the signal peptide. Therefore, the S169A / M347N / V349T / AR425 mutations of SEQ ID NO: 6 are equivalent to S137A / M315N / V317T / AR393 of SEQ ID NO: 7.

[0052] In another particular embodiment, the antidote of the invention comprises a recombinant antithrombin encoded in SEQ ID NO: 7 that presents the following mutations with respect to SEQ ID NO: 5

[0053] A) S137A

[0054] B) M315N

[0055] C) V317T, and

[0056] D) AR393 of SEQ ID NO: 5 or a recombinant antithrombin encoded by a variant of SEQ ID NO: 7 having at least 80% identity, more preferably 85% identity, even more preferably at least 90% identity, and even more preferably 91% or 92% or 93% or 94% or 95% or 96% or 97% or 98% or even up to 99% identity with respect to the polynucleotide sequence SEQ ID NO: 7, keeping variations A)-D) of SEQ ID NO: 7 fixed with respect to SEQ ID NO: 5 for use in the prevention and / or treatment of hemorrhagic risk in a subject.

[0057] The antidote that is purified once expressed does not have the signal peptide, since it is lost when it is transported outside the cell, this would be the functional form of the peptide

[0058] The term “mutations” includes at least one substitution, insertion and / or deletion of one or more amino acids within the sequence.

[0059] The recombinant antithrombin encoded by a variant of SEQ ID NO: 7 may comprise one or more mutations as indicated in the preceding paragraph.

[0060] In another particular embodiment, the antidote of the invention comprises a recombinant antithrombin encoded in SEQ ID NO: 6 that presents the following mutations with respect to SEQ ID NO: 4

[0061] A) S169A

[0062] B) M347N

[0063] C) V349T, and

[0064] D) AR425 of SEQ ID NO:4 or a recombinant antithrombin encoded by a variant of SEQ ID NO:6 having at least 80% identity, more preferably 85% identity, even more preferably at least 90% identity, and even more preferably 91% or 92% or 93% or 94% or 95% or 96% or 97% or 98% or even up to 99% identity with respect to the polynucleotide sequence SEQ ID NO:6, keeping variations A)-D) of SEQ ID NO:6 fixed with respect to SEQ ID NO:4 for use in the prevention and / or treatment of hemorrhagic risk in a subject

[0065] The recombinant antithrombin encoded by a variant of SEQ ID NO: 6 may comprise one or more mutations as indicated above.

[0066] The recombinant antithrombin of the invention has a substantially reduced anticoagulant activity relative to the anticoagulant activity of native antithrombin, or having substantially no anticoagulant activity, said recombinant antithrombin further having: a substantially reduced, or substantially lost, thrombin inhibitory activity, or a reduced or substantially lost factor Xa (FXa) inhibitory activity, or a reduced or substantially lost factor Ha (Fila) inhibitory activity, or a substantially reduced, or substantially lost thrombin inhibitory activity and factor Xa and factor Ha inhibitory activity.

[0067] The thrombin or factor Xa inhibitory activity is "substantially reduced" means that the activity of antithrombin to inhibit thrombin or factor Xa is reduced compared to said activity in wild-type antithrombin. Preferably, according to the invention, the thrombin or factor Xa inhibitory activity is considered reduced when said activity represents from about 90%, or 80%, or 70%, or 60%, or 50%, or 40%, or 30% to about 5% of the thrombin or factor Xa inhibitory activity of wild-type antithrombin. The anti-Xa and anti-ILA inhibitory activities can be measured with any system known in the state of the art.

[0068] "Substantially lost" thrombin or factor Xa inhibitory activity means that the activity of antithrombin to inhibit thrombin or factor Xa is absent compared to such activity in wild-type antithrombin.

[0069] The terms "treatment" and "treat" as used herein refer to the medical treatment of a subject with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, i.e., treatment specifically directed at ameliorating a disease, pathological condition, or disorder, and also includes causal treatment, i.e., treatment directed at eliminating the cause of the associated disease, pathological condition, or disorder.Furthermore, this term includes palliative treatment, i.e., treatment designed to alleviate symptoms rather than cure the disease, pathological condition, or disorder; preventative treatment, i.e., treatment aimed at minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, i.e., treatment used to complement another specific therapy aimed at ameliorating the associated disease, pathological condition, or disorder. It is understood that treatment, although intended to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder, need not actually result in the cure, amelioration, stabilization, or prevention. The effects of treatment may be measured or evaluated as described herein and as known in the art, as appropriate for the disease, pathological condition, or disorder in question.Such measurements and assessments may be performed qualitatively and / or quantitatively. Thus, for example, the characteristics or features of a disease, pathological condition, or disorder and / or the symptoms of a disease, pathological condition, or disorder may be reduced to any effect or in any amount. In the context of a subject suffering from an inflammatory disease, the terms "treatment" and "treat" refer to the medical management of a subject with the intent to cure, ameliorate, or stabilize said bleeding risk.

[0070] As used herein, the term "prevent" refers to the administration of the recombinant antithrombin of the invention before the onset of clinical symptoms of a disease or condition in order to prevent a physical manifestation of the aberrations associated with the disease or condition. In the context of hemorrhagic risk, the term "prevent" refers to the administration of the recombinant antithrombin of the invention before the onset of clinical symptoms of the hemorrhagic risk in order to prevent bleeding in the subject.

[0071] As used herein, the term "subject" includes, but is not limited to, animals. The subject may be a vertebrate, more specifically a mammal (e.g., a human, a horse, a pig, a rabbit, a dog, a sheep, a goat, a non-human primate, a cow, a cat, a guinea pig, or a rodent), a fish, a bird, or a reptile or amphibian. The term does not denote a particular age or sex. Therefore, adult and newborn subjects, as well as fetuses, whether male or female, are covered by the definition of subject. A patient is a subject afflicted with a disease or disorder. The term "patient" includes human and veterinary subjects. In another aspect of the invention, the antidote for use in treating the bleeding risk may be administered to a subject comprising a human or an animal, including, but not limited to, a mouse, a dog, a cat, a horse, a bovine or ovine animal, and the like.

[0072] In another particular embodiment, the "vahante" of any of the sequences SEQ_ID NO: 2 or SEQJD NO: 3 that code for the recombinant antithrombin of the invention could have the codon sequence optimized for expression in any host cell (a particular organism or cell type, for example, a human cell line,) in at least 10% of the codons, preferably at least 20% of the codons, more preferably at least 30% of the codons, even more preferably at least 40% of the codons, even more preferably at least 50% of the codons, even more preferably at least 60% of the codons, even more preferably at least 70% of the codons, even more preferably at least 80% of the codons, and even more preferably at least 90% of the codons. The codon optimization does not comprise a change in the amino acid sequence.

[0073] In a particular embodiment, the hemorrhagic risk is caused by the administration of heparin to the subject, said heparin may be one or more of the following: low molecular weight heparin, unfractionated heparin and pentasaccharide, preferably low molecular weight heparins (LMWH) and / or unfractionated heparin (UFH), since as demonstrated later in the embodiment examples, the antidote of the invention comprises a reduced or substantially lost inhibitory activity of factors Ha and Xa.

[0074] In another particular embodiment, heparin has been administered, previously, simultaneously with or with the recombinant antithrombin of the invention.

[0075] It is important that the antidote of the invention be administered within 24 hours after the subject has been administered LMWH or UFH. A physician would be able to identify the optimal time period in which to administer the antidote of the invention to achieve the best antihemorrhagic effect, given the risk of hemorrhage. This specialist would monitor the condition of the patient who has been administered LMWH or UFH, and if bleeding or symptoms of hemorrhage occur, they would administer the antidote of the invention to the subject.

[0076] In another particular embodiment, the hemorrhagic risk is associated with one or more of the following processes, for which treatment with heparin is indicated: infections, inflammations or hypoxic injuries, in particular sepsis and ischemia / reperfusion related to stroke, ischemia / reperfusion related to surgery and ischemia / reperfusion related to organ transplantation, deep vein thrombosis, pulmonary embolism, acute coronary syndrome, cardiopulmonary bypass, extracorporeal circulation, hemodialysis, central or peripheral venous catheters, venous thromboembolism (VTE) in general surgery when there is renal failure or obesity, treatment of DVT of lower extremities with or without pulmonary embolism, hip fracture surgery, renal failure, pulmonary thromboembolism and atrial fibrillation.

[0077] In a preferred embodiment, the hemorrhagic risk is associated with pulmonary thromboembolism or atrial fibrillation.

[0078] In the present invention, the hemorrhagic risk also includes thrombocytopenia.

[0079] The recombinant antithrombin of the invention can be administered to a subject suffering from, or at risk of suffering from, a hemorrhagic risk an effective amount of said recombinant antithrombin that exhibits reduced or substantially lost factor Ia and Xa inhibitory activity. The amount can be an amount from a minimum of about 0.1 mg of the recombinant antithrombin per kg of the subject, about 0.2 mg / kg, or about 0.5 mg / kg, to a maximum of about 100 mg / kg, about 200 mg / kg, or about up to 500 mg / kg. For example, the amount of recombinant antithrombin that exhibits reduced or substantially lost factor Ia and Xa inhibitory activity can be from about 0.1 mg / kg to about 100 mg / kg, from about 0.2 mg / kg to about 200 mg / kg, from about 0.5 mg / kg to about 500 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 20 mg / kg, about 0.1 mg / kg to about 50 mg / kg, about 0. 5 mg / kg to about 20 mg / kg, about 0.5 mg / kg to about 50 mg / kg, about 0.5 mg / kg to about 100 mg / kg, about 0.5 to about 200 mg / kg, about 0. 5 mg / kg to about 500 mg / kg, about 15 mg / kg to about 25 mg / kg, about 15 mg / kg to about 50 mg / kg, about 18 mg / kg to about 30 mg / kg, about 18 mg / Kg to about 50 mg / Kg.

[0080] A person skilled in the art would understand that the recombinant antithrombin of the present invention is for occasional use, when there is a risk of bleeding and / or heparin has been administered to a subject, not for sustained use over time unless the subject is being administered one or more doses of heparin continuously over time.

[0081] In a further embodiment, the antidote of the invention is administered to a subject at a dose of about 20 IU / kg to about 600 IU / kg, preferably about 40 IU / kg to about 300 IU / kg.

[0082] The antidote of the invention may be administered to humans and other animals intravenously, orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as powders, ointments or drops), orally, as an oral or nasal spray, or the like, preferably intravenously.

[0083] Another additional object of the invention relates to a pharmaceutical composition comprising the antidote of the invention and one or more additional active compounds, and / or one or more pharmaceutically acceptable carriers, adjuvants or vehicles.

[0084] The additional active compounds are all those commonly used in clinical practice to prevent the risk of bleeding.

[0085] By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, that is, the material can be administered to a subject together with the selected compound without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.

[0086] Each of the terms "comprising," "consisting essentially of," and "consisting of" may be substituted for either of the other two terms. The terms "a" or "an" may refer to one or a plurality of the elements they modify (e.g., "a reactant" may mean one or more reactants), unless it is contextually clear that one or more of the elements is being described. The term "about," as used herein, refers to a value within 10% of the underlying parameter (i.e., plus or minus 10%; e.g., a weight of "about 100 grams" may include a weight between 90 grams and 110 grams). The use of the term "about" at the beginning of a listing of values ​​modifies each of the values ​​(e.g., "about 1 , 2 , and 3" refers to "about 1 , about 2 , and about 3").When describing a list of securities, the list includes all intermediate securities and all fractional values ​​thereof (for example, the list of securities "80%, 85%, or 90%" includes the intermediate value 86% and the fractional value 86.4%). When a list of securities is followed by the term "or more," the term "or more" applies to each of the listed securities (for example, the list of "80%, 90%, 95%, or more" or "80%, 90%, or 95% or more" refers to "80% or more, 90% or more, or 95% or more"). When describing a list of values, the list includes all ranges between two of the listed values ​​(for example, the list of "80%, 90%, or 95%" includes ranges of "80% to 90%," "80% to 95%," and "90% to 95%"). Some examples of how the technology is used are given below.

[0087] Brief description of the figures

[0088] Figure 1: Western blot showing the increased secretion of recombinant antithrombin (antidote) of the invention compared to alpha antithrombin (alpha-AT) and beta antithrombin (beta-AT). R-SDS means that the electrophoresis is performed with sodium dodecyl sulfate (SDS) under reducing conditions (with beta-mercaptoethanol or dithiothreitol).

[0089] Figure 2: Silver staining of the recombinant antithrombin (antidote) of the invention.

[0090] Figure 3: Graphs showing the activity of the antidote of the invention (recombinant antithrombin) on Fila and FXa. PBS is the control and phosphate-buffered saline is the control.

[0091] Figure 4: Graph showing FXa activity in mice treated with PBS (control), heparin (HPBM), the antidote of the invention (recombinant antithrombin), and the antidote of the invention (recombinant antithrombin) together with heparin (HPBM). Vmax denotes maximum velocity.

[0092] Examples of implementation

[0093] Generation of the invention's antidote

[0094] To introduce the desired mutations in the antithrombin sequence we used the plasmid pCEP4 with the cDNA that codes for the expression of human antithrombin NG_012462.1 RefSeqGene, specifically SEQ ID NO: 1.

[0095] Directed mutagenesis was performed on this plasmid using the primers indicated in Table 1. The mutations were performed sequentially in the order indicated in the table from top to bottom. A person skilled in the art would know how to perform these mutations using common general knowledge in the field and standard laboratory protocols.

[0096] Table 1. Sequences of the primers used to introduce the desired mutations into the gene encoding antithrombin by directed mutagenesis.

[0097] PCR reactions were performed according to the steps described in Table 2.

[0098] Table 2. Temperature and time conditions for the POR reaction of site-directed mutagenesis.

[0099] The reagents for the PCR reaction are described in Table 3.

[0100] Table 3. Reagents and quantities required for the PCR reaction with polymerase

[0101] Roche Pfu Turbo.

[0102] Once the PCR reactions were performed and the correct introduction of mutations was verified by Sanger sequencing, HEK-EBNA eukaryotic cells were transfected for recombinant protein expression. Cells were grown to 60% confluence at 37°C, 5% CO2, in DMEM with GlutaMAX-l medium (Invitrogen), supplemented with 5% FBS (Sigma-Aldrich). 200 µg / mL of plasmid were transfected for 30 minutes in OptiMEM with LX (Invitrogen), as suggested by the manufacturer. After 24 hours, cells were washed with PBS and switched to CD-CHO medium (Invitrogen) supplemented with 4 mM L-glutamine and 0.25 mg / mL of Geneticin (Invitrogen). Cells were grown at 37°C for 10 days. The medium was collected and replaced with fresh medium every 2 days.The protein was purified by heparin affinity chromatography on HiTrap Heparin columns (GE Healthcare) using an ÁKTA Purifier (GE Healthcare) in 100 mM Tris-HCl and 10 mM citric acid, in a gradient from 0.15 to 2 M NaCl. Finally, the protein was eluted and desalted in 5 mL HiTrap Desalting columns (GE Healthcare) and stored at 70°C. The protein was obtained with high efficiency, due to its high secretion rate into the medium and its high affinity for heparin. A homogeneity greater than 90% was obtained with a single purification step. This means that the protein is at least 90% pure in the supernatant and that if there are contaminating proteins, they do not exceed 10% of the total. For a single purification step, this is a very high efficiency, which saves time and resources for the production of the antidote of the invention.These results demonstrate that the 1039TG>AC and 1044GTG>ACA mutations promote the secretion of the recombinant antithrombin of the invention.

[0103] In vitro expression of the antidote of the invention and evaluation of secretion levels

[0104] A protocol similar to that described in https: / / doi.org / 10.3390 / iims22020516 was used. Human embryonic kidney cells expressing Epstein-Barr nuclear antigen 1 (HEK-EBNA) were grown to 60% confluence at 37°C, 5% CO2, in DMEM with GlutaMAX-l medium (Invitrogen, Barcelona, ​​Spain) supplemented with 5% fetal bovine serum (Sigma-Aldrich, Madrid, Spain). 200 pg / mL of control plasmids or those expressing the antidote of the invention were transfected for 30 minutes in OptiMEM with LTX (Invitrogen, Barcelona, ​​Spain), as suggested by the manufacturer. Two control plasmids have been used, one expressing AT-alpha and the other AT-beta, with AT-alpha being SEQ ID NO: 1, and AT-beta being SEQ ID NO:1 +S137A.

[0105] After 24 hours, cells were washed with PBS and changed to CD-CHO medium (Invitrogen, Barcelona, ​​Spain) supplemented with 4 mM L-glutamine and 0.25 mg / mL Geneticin (Invitrogen, Barcelona, ​​Spain). Cells were grown at 37°C for 10 days. The medium was collected and replaced with fresh medium every 2 days. Purification of the antidote of the invention was performed from the medium on days 8 to 10 (48 hours).

[0106] The antidote of the invention was purified from the culture medium collections by heparin affinity chromatography on HiTrap Heparin columns (GE Healthcare), using an AKTA purifier (GE Healthcare) in 100 mMTr¡s-HCI pH 7.4 and 10 mM citric acid, in a gradient of 0.15 to 3 M NaCl. The fractions with antithrombin were applied to a HiTrap Q column (GE Healthcare) in 50 mMThs-HCI pH 7.4, in a gradient of 0.15 to 2 M NaCl. Finally, the proteins were desalted in 5 ml HiTrap Desalting columns (GE Healthcare).

[0107] Polyacrylamide gel electrophoresis was performed at 8% under denaturing conditions. After separation, proteins were transferred by Western blot onto a polyvinylidene difluoride membrane. Antithrombin was detected by immunostaining with a rabbit polyclonal antibody against human antithrombin (Sigma-Aldrich, Madrid, Spain), followed by horseradish peroxidase-conjugated donkey anti-rabbit IgG (GE Healthcare, Barcelona, ​​Spain), with detection using an ECL kit (Amersham Biosciences, Piscataway, NJ, USA). The secretion rate at 24 h posttransfection relative to the wild-type protein was assessed by densitometry using ImageJ software.

[0108] As can be seen in Figure 1, the antidote of the invention has the same electrophoretic mobility as the alpha form (since both have 4 N-glycans) and is present in the secreted medium more than the normal alpha and beta forms, specifically 2.7 times more. Figure 2 shows the silver staining of the antidote of the invention on a polyacrylamide gel in which electrophoresis with SDS has been performed under reducing conditions, which shows the purity of the recombinant antithrombin (antidote) at different concentrations in micrograms.

[0109] Hepahnal affinity by determining intrinsic fluorescence emission

[0110] The dissociation constant for the interaction of antithrombin with heparin was determined as previously described (Langdown J, Belzar KJ, Savory WJ, Baglin TP, Huntington JA. The critical role of hinge-region expulsion in the induced-fit heparin binding mechanism of antithrombin. J. Mol. Biol. 2009;386:1278-1289). Briefly, the change in intrinsic antithrombin fluorescence at 25-50 nM was monitored at 340 nM during titration of low molecular weight heparin (Enoxaparin, Rovi, Spain) in a Gary Eclipse spectrofluorometer (Agilent technologies, Barcelona), exciting at 280 nm and with an aperture of 2.5 nm for both excitation and emission. All titrations were carried out at room temperature with a physiological ionic strength (I = 0.15) in 20 mM Na2HPO4, 100 mM NaCl, 0.1 mM EDTA, 0.1% polyethylene glycol 8000, pH 7.4 buffer.The fluorescence emission intensity was taken as the average of 100 measurements recorded at 1-second intervals for each addition of hepahn. The data were adjusted using the following formula: Table 4: Affinity constant for heparin

[0111] The data in Table 4 demonstrate that the recombinant antithrombin of the invention (antidote) has a higher affinity for heparin than Beta-antithrombin, which means that it is capable of binding to heparin more efficiently than physiological antithrombin with a higher affinity for heparin. Likewise, none of the recombinant antithrombins described in EP2379101 B1 comprise the mutations of the present invention at positions 1039 and 1044 of the nucleotide chain that translate into the amino acid changes of M315N and V317N, therefore, the recombinant antithrombin of the invention has a higher affinity for heparin than those described in EP2379101 B1, which translates into greater anticoagulant activity.

[0112] Ex vivo assay: measurements of anti-FXa activity of plasma from anticoagulated patients in the presence of the antidote

[0113] Anti-FXa and anti-FIla activities were assayed by incubating antithrombin with low molecular weight heparin (0.096 nM) or unfractionated heparin (0.03 nM) with FXa (2 pM) or Fila (2 pM), respectively. Hydrolysis of the chromogenic substrates (S-2765 for FXa or S-2238 for Fila) (Chromogenix, Izasa, Spain) was recorded at 405 nm for 10 min in a plate reader. (Fig 3a and 3b)

[0114] Using the ACL-TOP equipment from Werfen (Madrid, Spain), we measured anti-FXa activity by adding different concentrations of the antidote of the invention to 500 pl of plasma from patients anticoagulated with low-molecular-weight heparins. The equipment used is automated, so an expert in the field would be able to reproduce this experiment with the information provided.

[0115] In Examples 1-4 below, subjects were treated with the various drugs, and a blood sample was subsequently collected from the patients, from which the plasma was separated. Once the sample was observed to be anticoagulated, the antidote of the invention was administered, and it was observed that it could reverse the anticoagulation of the sample. The objective of the experiment was to determine whether the antidote of the invention is capable of reversing the binding of heparin to antithrombin. The patients in the experiments did not show bleeding.

[0116] Blood samples were obtained after drug administration for a period of up to 24 hours. The initial concentration of the antidote of the invention is 600 pg / mL, and starting from this solution, 0, 1, 5, 10, 25, or 50 pL of said solution were administered to 500 pL of serum sample.

[0117] Example 1. Patient anticoagulated with Enoxaparin (60 mg every 12 hours) due to pulmonary thromboembolism, Table 5:

[0118] Table 5: Patient results from Example 1

[0119] Example 2. Patient anticoagulated with a type of low molecular weight heparin (Bemiparin) (7500 IU / day) due to atrial fibrillation, Table 6.

[0120] Table 6: Patient results from Example 2

[0121] Example 3. Patient anticoagulated with Bemiparin (5000 lll / day) due to atrial fibrillation Table 7.

[0122] Table 7: Patient results from Example 3

[0123] Example 4. Patient anticoagulated with Bemiparin (5000 lll / day) due to atrial fibrillation Table 8.

[0124] Table 8: Patient results from Example 4

[0125] These results in patients show that the recombinant antithrombin of the invention is capable of reversing the anticoagulant effect of Bemiparin and Enoxaparin.

[0126] In vivo tests: Injection into mice

[0127] To test the effect of our antidote in vivo, we performed experiments with C57BL / 6J mice. The animal experiments were approved by the Animal Experimentation Committee of the University of Murcia and were carried out in accordance with European guidelines for animal experimentation. We grouped the mice into 4 groups. Each group contained 5 males and 5 females. Each mouse was first anesthetized with isoflurane administered by inhalation and received a subcutaneous injection of 100 l and 10 minutes later a retroorbital sinus injection of 100 l. In group 1, both injections contained PBS. In group 2, we injected subcutaneously with low molecular weight heparin (Clexane) and PBS into the retroorbital sinus. In group 3, we injected subcutaneously with PBS and antidote into the retroorbital sinus. In group 4, we injected subcutaneous low molecular weight heparin (Clexane) and antidote into the retroorbital sinus.Five minutes after the last injection, blood was drawn from the vena cava using syringes containing sodium citrate, and then euthanasia was performed. Low-molecular-weight heparin (Clexane) was injected at a dose of 3 mg / kg, and the antidote at a concentration of 20 mg / kg. Once the blood was drawn, plasma was isolated by centrifugation at 2300g for 15 minutes, and anti-FXa activity was measured.

[0128] The results demonstrate that the antidote or recombinant antithrombin of the invention is capable of reversing the anticoagulant effect of heparin, Figure 4.

[0129] Sequence listing.

[0130] SEQ ID NO: 1 : Nucleotide sequence of the gene encoding the SERPINC1 protein in Homo sapiens. This sequence is the region from nucleotides 69 to 1463, CDS (Coding Sequence) of Genebank, NM_000488.4 (1552 bp linear mRNA PRI 10-APR-2023)

[0131] SEQ ID NO: 2: Nucleotide sequence encoding the antidote or recombinant protein of the invention, including, with respect to SEQ ID NO: 1, the mutations 504TCC>NNN, 1039TG>AC and 1044GTG>ACA and the deletion of 1272CGT

[0132] SEQ ID NO: 3: Nucleotide sequence encoding the antidote or recombinant protein of the invention, including, with respect to SEQ ID NO: 1, the mutations 504T>G, 1039TG>AC and 1044GTG>ACA and the deletion of 1272CGT

[0133] SEQ ID NO: 4 Amino acid sequence of SERPINC1, see also GenPept NP_000479; 464 aa; linear PRI 10-APR-2023

[0134] SEQ ID NO: 5 Amino acid sequence of SERPINC1 without the signal peptide

[0135] SEQ ID NO: 6 Amino acid sequence of the recombinant antithrombin of the invention with all mutations with the signal peptide: S169A / M347N / V349T / AR425

[0136] SEQ ID NO: 7 Amino acid sequence of the recombinant antithrombin of the invention with all mutations without the signal peptide S137A / M315N / V317T / AR393 SEQ ID NO: 8-15 primers used

Claims

Claims 1. An antidote that includes - a recombinant antithrombin encoded in the polynucleotide sequence SEQ ID NO: 2, which has the following mutations with respect to SEQ ID NO: 1 A) 1039 TG>AC B) 1044 GTG>ACA C) 504 TCC> NNN, where N is any combination of three nucleotides that does not translate into a serine, threonine, or cysteine D) Deletion of the nucleotides at position 1273, 1274 and 1275 of SEQ ID NO:1 or - a recombinant antithrombin encoded by a variant of SEQ ID NO: 2 having at least 80% identity, more preferably 85% identity, even more preferably at least 90% identity, and even more preferably 91% or 92% or 93% or 94% or 95% or 96% or 97% or 98% or even up to 99% identity with respect to the polynucleotide sequence SEQ ID NO: 2, keeping the mutations A)-D) of SEQ ID NO: 2 fixed with respect to SEQ ID NO: 1 for use in the prevention and / or treatment of hemorrhagic risk in a subject 2. The antidote according to the preceding claim, comprising a recombinant antithrombin encoded in SEQ ID NO: 3 having the following mutations with respect to SEQ ID NO: 1 A) 1039 TG>AC B) 1044 GTG>ACA C) 504 T> G, and D) deletion of the nucleotides at position 1273, 1274 and 1275 of SEQ ID NO:1 or a recombinant antithrombin encoded by a variant of SEQ ID NO: 3 having at least 80% identity, more preferably 85% identity, even more preferably at least 90% identity, and even more preferably 91% or 92% or 93% or 94% or 95% or 96% or 97% or 98% or even up to 99% identity with respect to the polynucleotide sequence SEQ ID NO: 3, keeping the mutations A)-D) of SEQ ID NO: 3 fixed with respect to SEQ ID NO: 1 for use in the prevention and / or treatment of hemorrhagic risk in a subject 3. The antidote according to any one of the preceding claims comprises a recombinant antithrombin encoded in SEQ ID NO: 6 that has the following mutations with respect to SEQ ID NO: 4 A) S169A B) M347N C) V349T, and D) AR425 of SEQ ID NO:4 or a recombinant antithrombin encoded by a variant of SEQ ID NO:6 having at least 80% identity, more preferably 85% identity, even more preferably at least 90% identity, and even more preferably 91% or 92% or 93% or 94% or 95% or 96% or 97% or 98% or even up to 99% identity with respect to the polynucleotide sequence SEQ ID NO:6, keeping variations A)-D) of SEQ ID NO:6 fixed with respect to SEQ ID NO:4 for use in the prevention and / or treatment of hemorrhagic risk in a subject 4. An antidote comprising a recombinant antithrombin encoded in SEQ ID NO: 7 that has the following mutations with respect to SEQ ID NO: 5 A) S137A B) M315N C) V317T, and D) AR393 of SEQ ID NO:5 or a recombinant antithrombin encoded by a variant of SEQ ID NO: 7 having at least 80% identity, more preferably 85% identity, even more preferably at least 90% identity, and even more preferably 91% or 92% or 93% or 94% or 95% or 96% or 97% or 98% or even up to 99% identity with respect to the polynucleotide sequence SEQ ID NO: 7, keeping variations A)-D) of SEQ ID NO: 7 fixed with respect to SEQ ID NO: 5 for use in the prevention and / or treatment of hemorrhagic risk in a subject.

5. The antidote according to any one of the preceding claims, wherein the subject is selected from a human, a horse, a pig, a rabbit, a dog, a sheep, a goat, a non-human primate, a cow, a cat, a guinea pig or a rodent.

6. The antidote according to any one of the preceding claims, wherein the hemorrhagic risk is caused by the administration of heparin to the subject, said heparin may be one or more of the following: low molecular weight heparin, unfractionated heparin and pentasaccharide.

7. The antidote according to the preceding claim wherein the hepahn is low molecular weight hepahn and / or unfractionated hepahn.

8. The antidote according to any one of the preceding claims, wherein the hemorrhagic risk is associated with one or more of the following processes: infections, inflammations or hypoxic injuries, in particular sepsis and ischemia / reperfusion related to stroke, ischemia / reperfusion related to surgery and ischemia / reperfusion related to organ transplantation, deep vein thrombosis, pulmonary embolism, acute coronary syndrome, cardiopulmonary bypass, extracorporeal circulation, hemodialysis, central or peripheral venous catheters, pulmonary thromboembolism and atrial fibrillation.

9. The antidote according to any one of the preceding claims, wherein an amount of between 0.1 mg / kg to 500 mg / kg is administered to a subject.

10. The antidote according to any one of the preceding claims, which is administered intravenously, orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically, buccally, or nasally.

11. A pharmaceutical composition comprising the antidote according to any one of claims 1 to 10 and one or more additional active compounds, and / or one or more pharmaceutically acceptable carriers, adjuvants or vehicles.

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