Tafoxiparin for the treatment of preeclampsia

Abstract

The present invention relates to the use of the heparin derivative tapoxiparin in the treatment of pre-eclampsia (PE). The treatment may be a monotherapy or a combination therapy in which tapoxiparin is used in combination with an agent (drug) used as a standard treatment in pre-eclampsia (PE).

Description

[Technical field]

[0001] The present invention relates to the use of the heparin derivative tafoxiparin in the treatment of pre-eclampsia (PE). The treatment may be a monotherapy or a combination therapy in which tafoxiparin is used in combination with an agent (drug) used as standard treatment in pre-eclampsia (PE). [Background technology]

[0002] Pre-eclampsia is a pregnancy-specific vascular disease characterized by de novo maternal hypertension and organ damage. Pre-eclampsia affects at least 8.5 million women worldwide each year and is a leading cause of maternal and perinatal morbidity and mortality (Steegers, EA et al., Pre-eclampsia. Lancet, 2010. 376(9741):631-644). Pre-eclampsia (PE), often referred to as the iconic representative of "ischemic placental disease" or "placental vascular complications," is a significant cause of maternal and perinatal morbidity and mortality. PE occurs twice as often in primiparas as in women who have previously given birth. Maternal morbidity includes severe hypertension with or without seizures (eclampsia), cerebrovascular accidents, placental abruption, pulmonary edema, and hepatic and renal insufficiency. Risks to the fetus include growth restriction in approximately 25% of cases, premature birth, and death either in utero or as a result of prematurity (approximately 2 per 100 cases) (Jean-Christophe Gris et al., Addition of enoxaparin to aspirin for the secondary prevention of placental vascular complications in women with severe pre-eclampsia. Thrombosis and Haemostasis 106.6 / 2011).

[0003] The acute manifestations of PE resolve after delivery, but women are left with permanent vascular damage (Melchiorre, K. et al., Preeclampsia is associated with persistent postpartum cardiovascular impairment. Hypertension, 2011. 58(4):709-715).

[0004] Pre-eclampsia is associated with an increased risk of future chronic hypertension, cardiovascular disease (CVD), cerebrovascular disease, and death (Bellamy, L. et al., Pre-eclampsia and risk of cardiovascular disease and cancer in later life: systematic review and meta-analysis. BMJ, 2007. 335(7627): 974). The risk of dying from ischemic heart disease after pre-eclampsia is estimated to be 2.5 times higher than that of women without pre-eclampsia (Bellamy, L. et al., Pre-eclampsia and risk of cardiovascular disease and cancer in later life: systematic review and meta-analysis. BMJ, 2007. 335(7627): 974). Furthermore, several epidemiological studies suggest that children of mothers with preeclampsia and gestational hypertension are also at increased risk for long-term hypertension and CVD (Geelhoed, JJ, et al. Preeclampsia and gestational hypertension are associated with childhood blood pressure independently of family adiposity measures: the Avon Longitudinal Study of Parents and Children. Circulation, 2010, 122(12):1192-1199). The exact mechanism by which preeclampsia and other gestational hypertensive disorders confer long-term cardiovascular risk is unclear. Maternal endothelial dysfunction has been found to persist for at least one year after delivery (Chen, CW, IZJaffe, and SA Karumanchi, Pre-eclampsia and cardiovascular disease. Cardiovasc Res, 2014.101(4):579-586).

[0005] The available treatment options for preeclampsia are symptomatic treatment by administering antihypertensive drugs, complemented by magnesium infusion in severe cases. Current clinical practice is to administer low doses of acetylsalicylic acid (ASA) as prophylaxis to high-risk subjects. The most important treatment goal is to avoid preterm birth.

[0006] Various LMWHs (low molecular weight heparins), such as dalteparin and enoxaparin, have been investigated for the treatment of preeclampsia, but to date have not proven useful in clinical practice for the treatment of preeclampsia due to safety concerns regarding anticoagulant activity and inconsistent results regarding efficacy.

[0007] WO 2003 / 055499 describes the use of certain sulfated glycosaminoglycans for the prevention and treatment of delayed progression of normal labor. WO 2013 / 147689 describes the use of chemically modified heparin or heparan sulfate in labor induction. WO 2013 / 147690 describes the use of chemically modified heparin or heparan sulfate in labor arrest. WO 2013 / 169194 describes the use of chemically modified heparin or heparan sulfate in the treatment of postpartum hemorrhage (PPH). WO 2013 / 095279 describes novel chemically modified glycosaminoglycans, which are described as useful for the prevention and treatment of delayed labor (dystocia), protein leakage such as Gorham's disease, sepsis, and protein-losing enteropathy. WO 2014 / 202982 describes a novel process for preparing chemically modified glycosaminoglycans.

[0008] Pre-eclampsia (PE) is associated with many short-term and long-term perinatal and neonatal complications for the baby, including death. Short-term complications can be fetal growth restriction (FGR), oligohydramnios, intrauterine fetal death (IUFD), preterm birth, low Apgar scores, abnormal FHR at delivery, or the need for NICU admission. Long-term complications can be cerebral palsy, low IQ, hearing loss, visual impairment, insulin resistance, diabetes, coronary artery disease, or hypertension (Poon LC et al.: The International Federation of Gynecology and Obstetrics (FIGO) initiative on pre-eclampsia: A pragmatic guide for first-trimester screening and prevention; Int J Gynecol Obstet 2019; 145(Suppl.1): 1-33 DOI: 10.1002 / ijgo.12802).

[0009] Issues with using LMWH to treat pre-eclampsia are dose-limiting factors and safety concerns: women with pre-eclampsia (PE) who need to undergo emergency cesarean section cannot be administered low molecular weight heparin due to bleeding risks, and general anesthesia is also a risk factor for these women.

[0010] Currently, there are no available treatments or preventatives for pre-eclampsia (PE) and therefore the short-term and long-term consequences of PE. As a result, there is a great medical need for new, safe and effective preventative and therapeutic (interventional) treatment options for pre-eclampsia. Summary of the Invention

[0011] The object of the present invention is to find a new treatment for pre-eclampsia (PE). More particularly, the object of the present invention is the use of the heparin derivative tafoxiparin for the preventive and therapeutic (interventional) treatment of pre-eclampsia (PE).

[0012] One aspect of the present invention is (i) having anti-factor IIa activity of less than 10 IU / mg; (ii) having anti-factor Xa activity of less than 10 IU / mg; (iii) having a weight average molecular weight (Mw) of 4.6 to 6.9 kDa; (iv) (Formula I): [ka] (In the formula, [ka] and n is an integer between 2 and 20 such that the polysaccharide chain has 2 to 20 disaccharide units corresponding to a molecular weight between 1.2 and 12 kDa. and having sugars predominantly present in (v) the polysaccharide chain is essentially free of chemically intact non-sulfated iduronic acid and / or glucuronic acid from the pentasaccharide sequence that mediates the anticoagulant effect; (vi) The chemically modified glycosaminoglycan is selected from the group consisting of: [Table 1] and its corresponding molecular weight expressed as cumulative percentage by weight, Tafoxiparin, a heparin derivative for use in the treatment of pre-eclampsia.

[0013] In one embodiment of the invention, the predominant polysaccharide chains in the heparin derivative tafoxiparin used according to the invention have 6 to 12 disaccharide units with a molecular weight of 3.6 to 7.2 kDa.

[0014] In an embodiment of the invention, the heparin derivative tafoxiparin described herein produces signals at 5.95 ppm and 6.15 ppm in the 1H-NMR spectrum.

[0015] In one embodiment of the invention, the heparin derivative tafoxiparin used according to the invention contains a non-reducing terminal unsaturated glucosamine, which is represented in the 1H-NMR spectrum as a signal in the interval 5.0 to 6.5 ppm, with an intensity (% ratio) of less than 4% relative to the signal at 5.42 ppm from native heparin.

[0016] One aspect of the invention is the heparin derivative tafoxiparin, as described herein, for use in combination therapy with standard treatments for pre-eclampsia (PE).

[0017] Examples of pharmaceutical agents (drugs) that may be useful as standard of care agents (medicines) in combination therapy according to the present invention are acetylsalicylic acid (ASA), antihypertensive agents, corticosteroids, or magnesium agents such as magnesium sulfate.

[0018] Examples of antihypertensive agents that may be useful as standard of care agents (medicines) in the combination therapy according to the invention are diuretics, calcium channel blockers, adrenergic receptor antagonists, renin inhibitors, or endothelial receptor blockers. Examples of diuretics that may be useful as antihypertensive agents in the combination therapy according to the invention are loop diuretics such as bumetanide, ethacrynic acid, furosemide, or torsemide, thiazide diuretics such as epitizide, hydrochlorothiazide, chlorothiazide, bendroflumethiazide, methyclothiazide, or polythiazide, thiazide diuretics such as indapamide, chlorthalidone, methalozone, xipamide, or clopamide, potassium-sparing diuretics such as amiloride, triamterene, spironolactone, or eplerenone.

[0019] Examples of calcium channel blockers which may be useful as antihypertensive agents in the combination therapy according to the invention are amlodipine, cilnidipine, clevidipine, felodipine, isradipine, lercanidipine, levamlodipine, nicardipine, nifedipine, nimodipine, nisoldipine, nitrendipine, diltiazem, or verapamil.

[0020] Examples of ACE inhibitors (angiotensin converting enzyme inhibitors) which may be useful as antihypertensive agents in combination therapy according to the invention are captopril, enalapril, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, trandolapril and benazepril.

[0021] Examples of angiotensin II receptor antagonists that may be useful as antihypertensive agents in the combination therapy according to the invention are azilsartan, candesartan, candesartan cilexetil, eprosartan, irbesartan, losartan, olmesartan, telmisartan, valsartan or fimasartan. Examples of adrenoceptor antagonists that may be useful as antihypertensive agents in the combination therapy according to the invention are beta blockers such as acebutolol, atenolol, bisoprolol, betaxolol, carteolol, carvedilol, labetalol, metoprolol, nadolol, nebivolol, oxprenolol, penbutolol, pindolol, propranolol or timolol, and alpha blockers such as doxazosin, phentolamine, indoramin, phenoxybenzamine, prazosin, terazosin or tolazoline.

[0022] Still further examples of agents which may be useful as antihypertensive agents in combination therapy according to the invention are renin inhibitors such as aliskiren, or endothelial receptor blockers such as bosentan.

[0023] One aspect of the invention is the heparin derivative tafoxiparin for use in the treatment of pre-eclampsia (PE).

[0024] One aspect of the invention is the heparin derivative tafoxiparin for use as a preventative treatment for pre-eclampsia (PE).

[0025] One aspect of the invention is the heparin derivative tafoxiparin for use in the therapeutic (interventional) treatment of pre-eclampsia (PE).

[0026] An embodiment of the invention is the heparin derivative tafoxiparin for use as monotherapy in the treatment of pre-eclampsia (PE).

[0027] An aspect of the present invention is the heparin derivative tafoxiparin for use in combination with standard of care (SOC) regimens for pre-eclampsia (PE).

[0028] One aspect of the invention is the heparin derivative tapoxiparin for uses as described herein, wherein said tapoxiparin is administered as add-on therapy to a subject already undergoing standard treatment for pre-eclampsia (PE).

[0029] An aspect of the invention is the heparin derivative tapoxiparin for uses as described herein, said tapoxiparin being administered prior to administration of standard treatment for pre-eclampsia (PE).

[0030] An aspect of the invention is the heparin derivative tapoxiparin for uses as described herein, said tapoxiparin being administered concomitantly with the administration of standard treatments for pre-eclampsia (PE).

[0031] In an embodiment of the invention, a pregnant woman at risk of developing pre-eclampsia (PE) is initiated into a pre-eclampsia (PE) treatment as described herein.

[0032] In an embodiment of the invention, the pre-eclampsia (PE) treatment described herein is initiated at the time a pregnant woman is diagnosed with pre-eclampsia.

[0033] In an embodiment of the invention, the pre-eclampsia (PE) treatment described herein is initiated upon a woman having a positive pregnancy test.

[0034] In an embodiment of the invention, the pre-eclampsia (PE) treatment described herein is initiated in a woman diagnosed with HELLP syndrome.

[0035] In an embodiment of the invention, the pre-eclampsia (PE) treatments described herein are initiated in women who have been diagnosed with eclampsia.

[0036] In an embodiment of the invention, the pre-eclampsia (PE) treatment described herein is initiated in an obese woman.

[0037] In an embodiment of the invention, the pre-eclampsia (PE) treatment described herein is initiated in a woman who has been diagnosed with pre-diabetes.

[0038] In an embodiment of the invention, the pre-eclampsia (PE) treatments described herein are initiated in women who have been diagnosed with diabetes.

[0039] In one aspect of the invention, the pre-eclampsia (PE) treatment described herein is terminated at the time of birth.

[0040] An aspect of the present invention is postpartum treatment (treatment after delivery) of pre-eclampsia (PE). Such treatment may commence any time after delivery and may involve up to 1 week, up to 2 weeks, up to 3 weeks, up to 4 weeks, up to 5 weeks, up to 6 weeks, up to 7 weeks, up to 8 weeks, up to 9 weeks, up to 10 weeks of duration of such treatment, or the duration of such postpartum treatment of pre-eclampsia (PE) as determined by the woman's responsible physician.

[0041] An aspect of the invention is the heparin derivative tafoxiparin for use in improving exchange of the placental circulation with the maternal (spiral artery) circulation in women diagnosed with or at risk of developing pre-eclampsia (PE).

[0042] An embodiment of the invention is a method for the treatment of pre-eclampsia (PE), in which a therapeutically effective amount of the heparin derivative tafoxiparin is administered to a woman in need of such treatment, the heparin derivative tafoxiparin comprising: (i) having anti-factor IIa activity of less than 10 IU / mg; (ii) having anti-factor Xa activity of less than 10 IU / mg; (iii) having a weight average molecular weight (Mw) of 4.6 to 6.9 kDa; (iv) (Formula I): [ka] (In the formula, [ka] and n is an integer between 2 and 20 such that the polysaccharide chain has 2 to 20 disaccharide units corresponding to a molecular weight between 1.2 and 12 kDa. and having sugars predominantly present in (v) the polysaccharide chain is essentially free of chemically intact non-sulfated iduronic acid and / or glucuronic acid from the pentasaccharide sequence that mediates the anticoagulant effect; (vi) The chemically modified glycosaminoglycan is selected from the group consisting of: [Table 2] and its corresponding molecular weight expressed as cumulative % by weight.

[0043] An embodiment of the invention is the use of the heparin derivative tafoxiparin for the manufacture of a medicament for the treatment of pre-eclampsia (PE), comprising (i) having anti-factor IIa activity of less than 10 IU / mg; (ii) having anti-factor Xa activity of less than 10 IU / mg; (iii) having a weight average molecular weight (Mw) of 4.6 to 6.9 kDa; (iv) (Formula I): [ka] (In the formula, [ka] and n is an integer between 2 and 20 such that the polysaccharide chain has 2 to 20 disaccharide units corresponding to a molecular weight between 1.2 and 12 kDa. and having sugars predominantly present in (v) the polysaccharide chain is essentially free of chemically intact non-sulfated iduronic acid and / or glucuronic acid from the pentasaccharide sequence that mediates the anticoagulant effect; (vi) The chemically modified glycosaminoglycan is selected from the group consisting of: [Table 3] and its corresponding molecular weight expressed as cumulative % by weight.

[0044] According to the present invention, tafoxiparin may be used as monotherapy or in combination therapy with agents used as standard of care (SOC) for pre-eclampsia. [Brief description of the drawings]

[0045] [Figure 1A] Figure 1A (Example 1) shows concentration-contraction graphs for one patient (Patient No. 13) in the presence of increasing concentrations of angiotensin II. The X-axis reflects the concentration of the vasoconstrictor angiotensin II, and the Y-axis reflects the contractile response. [Figure 1B] Figure 1B (Example 1) shows concentration-contraction graphs for one patient (patient no. 13) in the presence of increasing concentrations of endothelin 1. The X-axis reflects the concentration of the vasoconstrictor endothelin 1 and the Y-axis reflects the contractile response. [Figure 2A] Figure 2A (Example 2) shows the concentration-contraction graphs of one patient (patient number 14) induced by the addition of increasing concentrations of angiotensin II. The X-axis reflects the concentration of the vasoconstrictor angiotensin II and the Y-axis reflects the contractile response. [Figure 2B]Figure 2B (Example 2) shows concentration-contraction graphs for one patient (patient no. 14) induced by the addition of increasing concentrations of endothelin 1. The X-axis reflects the concentration of the vasoconstrictor endothelin 1 and the Y-axis reflects the contractile response. [Diagram 3] Figure 3 (Example 3) shows the contractile response of tafoxiparin (10 μg / ml) and the control LMWH tinzaparin (10 μg / ml) induced by the addition of increasing concentrations of angiotensin II. The X-axis reflects the concentration of the vasoconstrictor angiotensin II and the Y-axis reflects the contractile response. [Figure 4] Figure 4 (Example 4) shows the contractile response of tafoxiparin (100 μg / ml) and the control LMWH tinzaparin (100 μg / ml) induced by the addition of increasing concentrations of angiotensin II. The X-axis reflects the concentration of the vasoconstrictor angiotensin II and the Y-axis reflects the contractile response. [Diagram 5] 5 (Example 5) shows the contractile responses of tafoxiparin (10 μg / ml) and the control LMWH tinzaparin (10 μg / ml) induced by the addition of increasing concentrations of endothelin-1 (ET-1). The X-axis reflects the concentration of the vasoconstrictor endothelin-1 (ET-1) and the Y-axis reflects the contractile response. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] definition Tafoxiparin is a heparin derivative with the in-house compound code DF01. The INN (International Nonproprietary Name) of DF01 is tafoxiparin sodium. The CAS Registry Number (CAS RN) of tafoxiparin is RN1638190-65-4. DF01 is a depolymerized form of heparin that has essentially no anticoagulant activity.

[0047] More particularly, tafoxiparin is (i) having anti-factor IIa activity of less than 10 IU / mg; (ii) having anti-factor Xa activity of less than 10 IU / mg; (iii) having a weight average molecular weight (Mw) of 4.6 to 6.9 kDa, for example 5.0 to 6.9 kDa; (iv) (Formula I): [ka] (In the formula, [ka] and n is an integer between 2 and 20 such that the polysaccharide chain has 2 to 20 disaccharide units corresponding to a molecular weight between 1.2 and 12 kDa. and having sugars predominantly present in (v) the polysaccharide chain is essentially free of chemically intact non-sulfated iduronic acid and / or glucuronic acid from the pentasaccharide sequence that mediates the anticoagulant effect; (vi) The chemically modified glycosaminoglycan is selected from the group consisting of: [Table 4] and its corresponding molecular weight expressed as cumulative percentage by weight, It is a heparin derivative.

[0048] The predominant polysaccharide chains in tafoxiparin contain 6-12 disaccharide units with molecular weights ranging from 3.6 to 7.2 kDa.

[0049] Tafoxiparin may have a non-reducing terminal unsaturated glucosamine, which is represented in the 1H-NMR spectrum as a signal in the interval 5.0-6.5 ppm, with an intensity (% ratio) of less than 4% relative to the signal at 5.42 ppm from native heparin; and 1 The 1 H-NMR spectrum may give rise to signals at 5.95 ppm and 6.15 ppm.

[0050] The heparin derivative tafoxiparin has the chemical structure: [ka] The glycol splitting residue may also be included.

[0051] The term "treatment or therapy" as used herein takes its usual form in the medical and pharmaceutical arts and includes curative and prophylactic treatment.

[0052] Whenever the expression "therapeutic treatment" or "interventional treatment" is used herein, it refers to the treatment of a pregnant woman who is or has been diagnosed as having (suffering from) pre-eclampsia (PE). This treatment may be a monotherapy with tapoxiparin or a combination therapy including the administration of tapoxiparin and a drug used as a standard treatment for pre-eclampsia (PE).

[0053] Whenever the terms "prophylactic treatment", "preventive treatment" or "prevention treatment" are used herein, it means that tapoxiparin is administered to a pregnant woman who is at risk of developing pre-eclampsia (PE) but has not yet been diagnosed as having (suffering from) pre-eclampsia (PE). This prophylactic treatment may be a monotherapy with tapoxiparin or a combination therapy including tapoxiparin and a drug (drug) used as the standard of care (SOC) for pre-eclampsia (PE).

[0054] The term "monotherapy" as used herein refers to treatment with tafoxiparin alone administered to a woman who is or has been diagnosed with pre-eclampsia (PE) or who is at risk of developing pre-eclampsia (PE). The monotherapy may be a therapeutic therapy (treatment) or a prophylactic therapy (prevention).

[0055] The expression "combination therapy" as used herein refers to a treatment with tafoxiparin in combination with a drug used as a standard treatment for pre-eclampsia (PE). The combination therapy may be used by administering said combination therapy to a woman who is or has been diagnosed with pre-eclampsia (PE) or who is at risk of developing pre-eclampsia (PE). The combination therapy may be a therapeutic therapy (treatment) or a preventative therapy (prevention). Tafoxiparin may be administered in the combination therapy according to the invention before, after (add-on) or simultaneously with the administration of a drug used as a standard treatment for pre-eclampsia (PE).

[0056] As used herein, the expression "tafoxiparin as (used as) add-on therapy" is defined as a combination therapy in which tafoxiparin is administered to a pregnant woman already undergoing standard treatment for pre-eclampsia (PE). Add-on therapy may be therapeutic (i.e., treatment) or preventative (i.e., prophylactic).

[0057] The expression "tafoxiparin administered prior to the administration of a drug used as a standard treatment for pre-eclampsia (PE)" is defined as a combination therapy in which tafoxiparin is administered prior to the administration of a drug used as a standard treatment in pre-eclampsia (PE).

[0058] As used herein, the term "add-on therapy" includes administration of tafoxiparin prior to administration of a drug used as a standard treatment for pre-eclampsia (PE), or administration of tafoxiparin following administration of a drug used as a standard treatment for pre-eclampsia (PE).

[0059] As used herein, the term "co-administration" means that tafoxiparin and a drug (drug) used as a standard treatment for pre-eclampsia (PE) are administered simultaneously to a woman in need of pre-eclampsia treatment.

[0060] When acetylsalicylic acid (ASA) is used as standard therapy, it may be used in one aspect of the invention for women who have been diagnosed with pre-eclampsia (PE) in a previous pregnancy. In an aspect of the invention, acetylsalicylic acid (ASA) may be used as standard therapy for women who are or have been diagnosed with pre-eclampsia (PE) in an ongoing pregnancy. In an aspect of the invention, acetylsalicylic acid (ASA) may be used as standard therapy for women who are at risk of developing pre-eclampsia (PE).

[0061] The term "standard of care" (SOC) as used herein is defined as a procedure or treatment recommended by national and international societies, field guidelines, and medical and regulatory agencies for use in pregnant women at risk of developing pre-eclampsia (PE) or diagnosed with pre-eclampsia (PE). Standard of care may be useful for women who have been diagnosed with pre-eclampsia (PE) in a previous pregnancy, or who are or have been diagnosed with pre-eclampsia (PE) in an ongoing pregnancy, or who are at risk of developing pre-eclampsia (PE).

[0062] The term "pregnant woman" means a pregnant woman who has not been previously pregnant or given birth, or a pregnant woman who has been previously pregnant and given birth.

[0063] The terms "standard of care", "standard of care agent", and "standard of care medication" may be used interchangeably and have the same meaning.

[0064] Examples of standard of care medications commonly used in the treatment of pre-eclampsia are acetylsalicylic acid (ASA), antihypertensive agents, corticosteroids, and anticonvulsant medications.

[0065] In one aspect of the present invention, corticosteroids may be used as a standard treatment drug when a mother suffers from preeclampsia (PE) during pregnancy, putting the baby at risk of being born prematurely with reduced lung function. Examples of corticosteroids that may be useful are dexamethasone or prednisolone. The use of corticosteroids in women with preeclampsia is referred to as prenatal treatment.

[0066] Acetylsalicylic acid (ASA) is an over-the-counter analgesic recognized in countries such as the EU and the United States under the trade name Aspirin® (IUPAC name: 2-acetyloxybenzoic acid).

[0067] The term "pre-eclampsia (PE)" is defined as a condition in a pregnant woman with a blood pressure (BP) of 140 / 90 or more, for example 160 / 100 or more, and a urinary albumin / creatinine value of ≥ 30 mg / mmol. Proteinuria (≥ 2+ by urine dipstick) can be a further indicator of pre-eclampsia (PE). Pre-eclampsia is defined herein by Poon LC et al.: The International Federation of Gynecology and Obstetrics (FIGO) initiative on pre-eclampsia: A pragmatic guide for first-trimester screening and prevention; Int J Gynecol Obstet 2019; 145(Suppl.1) 1-33 DOI: 10.1002 / ijgo.12802. However, the definition of pre-eclampsia made herein should not disqualify the meaning of the term "pre-eclampsia" if this condition is redefined at a later time by medical professionals in the art.

[0068] Preeclampsia (PE) is a pregnancy-specific vascular disease characterized by de novo maternal hypertension and organ damage, more specifically pregnancy-induced hypertension with proteinuria (i.e., increased protein in the urine) that typically develops after 20 weeks of pregnancy. Reduced uterine blood flow leads to placental ischemia, which causes the placenta to release antiangiogenic factors such as sFlt-1, and subsequently leads to PE. Preeclampsia increases the risk of poor outcomes for both the mother and the baby. Severe disease can result in red blood cell destruction, low platelet counts, liver damage, kidney failure, edema, shortness of breath and / or impaired vision due to fluid in the lungs. If left untreated, seizures can occur, at which point this is known as eclampsia. Risk factors for preeclampsia include early diagnosis of preeclampsia, obesity, history of hypertension, advanced age, and diabetes. Preeclampsia is more frequent in women with their first pregnancy and when carrying twins. The underlying mechanisms involve abnormalities in angiogenesis in the placenta, among other factors. Most cases are diagnosed before birth. In rare cases, pre-eclampsia may begin in the post-natal period.

[0069] The definition of pre-eclampsia (PE) is also associated with a number of short-term and long-term perinatal and neonatal complications for the baby, including death. Short-term complications may be fetal growth restriction (FGR), oligohydramnios, intrauterine fetal death (IUFD), preterm birth, low Apgar scores, abnormal FHR at delivery, or the need for NICU (neonatal intensive care unit) admission. Long-term complications may be cerebral palsy, low IQ, hearing loss, visual impairment, insulin resistance, diabetes, coronary artery disease, or hypertension (Poon LC et al.: The International Federation of Gynecology and Obstetrics (FIGO) initiative on pre-eclampsia: A pragmatic guide for first-trimester screening and prevention; Int J Gynecol Obstet 2019;145(Suppl.1):1-33DOI:10.1002 / ijgo.12802).

[0070] Although the acute maternal symptoms resolve after birth, women are often left with permanent vascular damage. Pre-eclampsia is associated with an increased risk of future chronic hypertension, cardiovascular disease (CVD), cerebrovascular disease, and mortality.

[0071] Historically, both hypertension (>140 / 90 in women after 20 weeks' gestation) and protein in the urine were required to make the diagnosis, but some definitions also include those with hypertension and associated organ dysfunction.The exact mechanisms by which preeclampsia and other disorders of pregnancy confer long-term cardiovascular risk are unclear.

[0072] The term "HELLP syndrome" is a life-threatening pregnancy complication that is considered a variant of preeclampsia. It usually develops late in pregnancy or, in some cases, after birth. H stands for hemolysis, which is the destruction of red blood cells, EL stands for elevated liver enzymes, and LP stands for low platelet count. HELLP syndrome can be difficult to diagnose, especially in the absence of high blood pressure and protein in the urine. The symptoms may also be misdiagnosed as gastritis, influenza, acute hepatitis, gallbladder disease, or other conditions.

[0073] The term "obese" is defined as a person (in the present invention, a woman) with a BMI (Body Mass Index) of 30 or more (as defined by the US National Institutes of Health (NIH)).

[0074] The term "prediabetes" refers to a person (in this invention, a woman) who has elements of the metabolic syndrome, characterized by elevated blood glucose levels below the threshold for diagnosing diabetes. Although usually asymptomatic, prediabetes often has obesity (especially abdominal or visceral obesity), dyslipidemia with high triglycerides and / or low HDL cholesterol, and high blood pressure. It is also associated with an increased risk of cardiovascular disease (CVD). Since health complications related to type 2 diabetes often occur before the diagnosis of diabetes, prediabetes is more accurately considered an early stage of diabetes (American Diabetes Association (January 2017). Classification and diagnosis of diabetes". Diabetes Care. 40(Suppl 1):S11-S24. doi:10.2337 / dc17-S005. PMID 27979889).

[0075] The terms "diabetes" or "diabetic" refer to a group of metabolic disorders characterized by high blood glucose levels over a prolonged period of time (World Health Organization. Archived from the original on March 31, 2014. Retrieved April 4, 2014). Symptoms include frequent urination, increased thirst, and increased hunger. If left untreated, diabetes can lead to many complications (WHO. October 2013. Archived from the original on August 26, 2013. Retrieved March 25, 2014). Acute complications may include diabetic ketoacidosis, hyperosmolar hyperglycemic state, or death. Serious long-term complications include cardiovascular disease, stroke, chronic kidney disease, foot ulcers, nerve damage, and eye damage.

[0076] The expression "postpartum treatment" (treatment initiated after birth) is defined herein as treatment (preventive or therapeutic treatment) of a woman who has given birth and is at risk of developing cardiovascular disease or other diseases that may result from pre-eclampsia or eclampsia, or who has already been diagnosed with cardiovascular disease or other diseases that may result from pre-eclampsia or eclampsia. Such treatment may be initiated at any time after birth and may involve a duration of such treatment of up to 1 week, up to 2 weeks, up to 3 weeks, up to 4 weeks, up to 5 weeks, up to 6 weeks, up to 7 weeks, up to 8 weeks, up to 9 weeks, up to 10 weeks, or the duration of such postpartum treatment for pre-eclampsia (PE) as determined by the responsible physician of said woman.

[0077] Treatment of pre-eclampsia (PE) as described herein may be continued after delivery in a further aspect of the invention, and the duration of such treatment may be for up to 1 week, up to 2 weeks, up to 3 weeks, up to 4 weeks, up to 5 weeks, up to 6 weeks, up to 7 weeks, up to 8 weeks, up to 9 weeks, up to 10 weeks, or for any suitable time as determined by the woman's treating physician.

[0078] As used herein, the phrase "increasing the exchange of the placental circulation with the maternal (spiral artery) circulation" means maintaining or improving the ability of the umbilical vein to carry fresh, oxygenated, nutrient-rich blood that circulates back to the fetal systemic circulation.

[0079] The terms preeclampsia, pre-eclampsia or PE are used interchangeably throughout this specification and claims and have the same meaning.

[0080] Pharmaceutical Formulations and Routes of Administration The heparin derivative tafoxiparin used according to the invention can be administered in a pharmaceutical composition suitable for systemic administration, for example parenteral administration, such as subcutaneous administration, intravenous injection, infusion or intramuscular administration.

[0081] The tapoxiparin used according to the invention may also be administered by topical administration. Examples of topical administration that may be useful when administering tapoxiparin according to the invention are oral, vaginal or rectal administration.

[0082] For parenteral administration, tafoxiparin may be incorporated into a solution or suspension, which may also contain one or more adjuvants, such as water for injection, physiological saline, volatile oils, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents, sterile diluents such as antibacterial agents, antioxidants, chelating agents, buffers and agents for adjusting osmolarity. Parenteral pharmaceutical preparations may be filled into ampoules, vials, disposable syringes or may be injectable devices, e.g., for self-administration.

[0083] In one aspect of the invention, the dose of tafoxiparin used according to the invention may be selected from any one of the following daily doses: 300-2000 mg per day; 400-2000 mg / day; 500-2000 mg / day; 600-2000 mg / day; 700-2000 mg / day; 800-2000 mg / day; 900-2000 mg / day; 1000-2000 mg / day; 1100-2000 mg / day; 1200-2000 mg / day; 1300-2000 mg / day; 1400-2000 mg / day; 1500-2000 mg / day; 1600-2000 mg / day; 1700-2000 mg / day; 1800-2000 mg / day; and 1900-2000 mg / day.

[0084] In one aspect of the invention, the dose of tafoxiparin used according to the invention may be selected from any one of the following daily doses: 350-2000 mg per day; 450-2000 mg / day; 550-2000 mg / day; 650-2000 mg / day; 750-2000 mg / day; 850-2000 mg / day; 950-2000 mg / day; 1050-2000 mg / day; 1150-2000 mg / day; 1250-2000 mg / day; 1350-2000 mg / day; 1450-2000 mg / day; 1550-2000 mg / day; 1650-2000 mg / day; 1750-2000 mg / day; 1850-2000 mg / day; and 1950-2000 mg / day.

[0085] In embodiments of the invention, the dose of tafoxiparin used in accordance with the present invention may be selected from any one of the following daily doses: 300-1000 mg / day; 400-1000 mg / day; 500-1000 mg / day; 600-1000 mg / day; 700-1000 mg / day; 800-1000 mg / day; and 900-1000 mg / day.

[0086] In embodiments of the invention, the dose of Tafoxiparin used in accordance with the present invention may be selected from any one of the following daily doses: 350-1000 mg / day; 450-1000 mg / day; 550-1000 mg / day; 650-1000 mg / day; 750-1000 mg / day; 850-1000 mg / day; and 950-1000 mg / day.

[0087] In one embodiment of the invention, the dose of tafoxiparin used according to the invention is 50-300 mg per day, for example 60-300 mg / day, 70-300 mg / day, 80-300 mg / day, 90-300 mg / day, 100-300 mg / day, 110-300 mg / day, 120-300 mg / day, 130-300 mg / day, 140-300 mg / day, 150-300 mg / day, 160-300 mg / day, 170-300 mg / day, 180-300 mg / day, 190-300 mg / day, 200-300 mg / day, 210-300 mg / day, 220-300 mg / day, 230-300 mg / day, 240-300 mg / day, 250-300 mg / day, 260-300 mg / day, 270-300 mg / day, 280-300 mg / day, 290-300 mg / day, 300-300 mg / day, 310-300 mg / day, 320-300 mg / day, 330-300 mg / day, 340-300 mg / day, 350-300 mg / day, 360-300 mg / day, 370-300 mg / day, 380-300 mg / day, 390-300 mg / day, 400-400 mg / day, 410-400 mg / day, 420-400 mg / day, 430-400 mg / day, 440-400 mg / day, 450-400 mg / day, 0-300mg / day, 170-300mg / day, 180-300mg / day, 190-300mg / day, 200-300mg / day, 210-300mg / day, 220-300mg / day, 230-300mg / day, 240-300mg / day, 250-300mg / day, 260-300mg / day, 270-300mg / day, 280-300mg / day, or 290-300mg / day.

[0088] In an embodiment of the invention, the dose of tafoxiparin used according to the invention is 50-100 mg / day, for example 50 mg / day, 60 mg / day, 70 mg / day, 80 mg / day, 90 mg / day, or 100 mg / day. In an embodiment of the invention, the dose of tafoxiparin used according to the invention is 300 mg / day, 200 mg / day, or 150 mg / day.

[0089] In an embodiment of the invention, the dose of Tafoxiparin used according to the invention is up to 150 mg / day, such as up to 100 mg / day, or up to 75 mg / day.

[0090] In one embodiment of the invention, administration may be once a day. In an embodiment of the invention, administration may be twice a day. In an embodiment of the invention, administration may be three times a day. For example, a daily dose of 300 mg may be divided by administering tafoxiparin as three 100 mg doses or two 150 mg doses. A daily dose of 150 mg may be divided as two 75 mg doses or three 50 mg doses. A daily dose of 100 mg may be divided as two 50 mg doses. EXAMPLES

[0091] Manufacturing of Tafoxiparin Tafoxiparin for use according to the present invention may be prepared by following the synthetic procedures described in Examples 1-9 of published patent application WO 2013 / 095279 or Examples 1-3 of WO 2014 / 202982.

[0092] In the myograph experiments performed below, tafoxiparin was provided as a 150 mg / mL solution by Dilafor AB, Sweden. [Table 5]

[0093] Biological testing During the pregnancy-associated syndrome preeclampsia (PE), there is an increased release of placental syncytiotrophoblast extracellular vesicles (STBEVs) and free fetal hemoglobin (HbF) into the maternal circulation. Internalization of STBEVs into primary human coronary artery endothelial cells (HCAECs) and import of placenta-specific miRNAs from STBEVs into the endoplasmic reticulum and mitochondria of these recipient cells has been investigated by Cronqvist T et al.: Scientific Reports 7:4558;DOI:10.1038 / s41598-017-04468-0. miRNA-mediated effects on gene expression may contribute to endothelial reprogramming that may underlie the increased risk of cardiovascular disease later in life reported for women with PE as well as the oxidative and endoplasmic reticulum stress described in PE (Cronqvist T et al.: Scientific Reports 7:4558; DOI:10.1038 / s41598-017-04468-0).

[0094] Cha Han et al. reported in Haematologica 2020, vol. 105(6):1686-1694 that elevated levels of circulating placenta-derived extracellular vesicles (pcEVs) were consistently associated with preeclampsia. The authors reported that pcEVs from injured placentas induced a preeclampsia-like state in mice by inducing endothelial injury, vasoconstriction, and hypercoagulability. This pcEV-induced state was prevented by enhancing EV clearance. Thus, the rates of pcEV production and clearance can be used to assess the risk of preeclampsia and may be a novel therapeutic target for preeclampsia.

[0095] By preventing vascular constriction during pregnancy and thereby reducing blood pressure, improved placental circulation is established, reducing the risk of intrauterine growth restriction (IUGR). Normalization of blood pressure and fetal growth may reduce the number of preterm births. A long-term maternal effect may be the prevention of endothelial cell reprogramming, thereby preventing vascular stiffening after preeclampsia (PE).

[0096] The aim of this study was to determine whether and how vesicles from women with preeclampsia could modify the contractile response to angiotensin II (AngII) and endothelin 1 (ET-1) in subcutaneous arteries removed from women who had just given birth by Caesarean section. We investigated whether the drug tapoxiparin could modify the contractile response.

[0097] Abbreviation The following abbreviations have been used in the examples and figures: "P" refers to arterial segments exposed to vesicles in pregnant women without PE. "PE" refers to an artery or arterial segment exposed to vesicles in a pregnant woman with PE. "PE-tafoxi" refers to arteries or arterial segments treated with tafoxiparin and then with vesicles from pregnant women with PE. "PE+Tinzaparin" means arteries or arterial segments treated with tinzaparin followed by vesicles from pregnant women with PE. "P+tafoxi" refers to arteries or arterial segments treated with tafoxiparin followed by vesicles from pregnant women without PE. "P+Tinzaparin" means arteries or arterial segments treated with tinzaparin followed by vesicles from pregnant women without PE. "Tafoxi" means that the artery or artery segment was treated with tapoxiparin alone (ie, no addition of vesicles). "Blank" means that the artery or arterial segment was not subjected to any treatment. Substance P is a potent vasodilator of 11 amino acid residues well known to those skilled in the art.

[0098] Myograph Experiment Syncytiotrophoblasts (STBs) of the placenta release extracellular vesicles (STB-EVs) that communicate physiological and pathological placental signals to maternal organs. The release of STB-EVs is increased in preeclampsia (PE) (Sammar et al. Placenta 66 (2018) pp. 17-25).

[0099] The myograph model used arteries from the subcutaneous tissue of women undergoing Caesarean section. The contractile effect of arteries containing vesicles derived from the placenta of preeclamptic women was compared to that of arteries containing vesicles derived from the placenta of non-preeclamptic women. We also investigated the possible effect on contractility of pretreatment with tapoxiparin before adding the vesicles. Tinzaparin (Innohep®, Leo Pharma Denmark), a low molecular weight heparin (LMWH), was used as a control drug.

[0100] The multiwire myograph system was designed to examine highly isometric active and passive responses to small vessels (Hogestatt et al., Acta Physiol 1983, 117, 49-61) and is now being applied to abdominal subcutaneous vessels (Edvinsson et al., Acta Physiol 2014;210:811-22; Edvinsson et al., Microcirculation 2016;23:438-46).

[0101] In the myograph experiments performed herein, tapoxiparin (DF01) was provided by Dilafor AB, Sweden.

[0102] Placental perfusion and sample collection Dual ex-vivo perfusion of human placental cotyledons isolated from normal (n=5) and PE pregnancies (n=5) is performed as described in Southcombe, J., Tannetta, D., Redman, C. & Sargent, I. The immunomodulatory role of syncytiotrophoblast microvesicles. PLoS One6, e20245 (2011). Briefly, perfusion experiments consist of one equilibration phase of 30 min, from which maternal perfusate is discarded. After equilibration, the maternal circuit is closed and the placenta is perfused for 3 h. Only perfusions with fetal return rates >80% are included. Perfusate is collected from the maternal side at the end of the 3 h perfusion and used for isolation and analysis of STBEVs. The placental perfusion method is chosen to collect normal and PE STBEVs in this study due to the high vesicle yields obtainable using this method.

[0103] Isolation of STBEVs (small vesicles) Isolation of STBEV is performed using successive centrifugation steps. After perfusion, the maternal perfusate is centrifuged at 600×g or 1500×g to remove red blood cells and the supernatant is frozen and stored at −80° C. until further analysis. The supernatant is centrifuged at 3500×g and +4° C. for 30 min to remove cellular debris. The 3500×g supernatant is ultracentrifuged at 110000×g and +4° C. for an additional 3 h and the pelleted material is used for further studies and is referred to as STBEV.

[0104] Size distribution of STBEVs is analyzed using Nanoparticle Tracking Analysis (NTA). NTA is performed using a NanoSight LM10 (Malvern Instruments, Malvern, UK) equipped with a 488 nm laser. Normal (n=5) and PE (n=5) STBEVs are diluted in 1x PBS and analyzed. Measurements are performed in triplicate for 90 seconds using manual gain, camera level set to 10 or 11, and detection threshold set to 3. Resulting files are analyzed using NTA3.2.16 software (Malvern Instruments). Protein concentration of STBEVs is determined using a NanoDrop Spectrophotometer ND-1000 (NanoDrop Technologies, Wilmington, USA) or using the Pierce™ BCA Protein Assay (Thermo Scientific, Rockford, USA). STBEVs are resuspended in 1x phosphate buffered saline (PBS) and stored at -80°C.

[0105] The vesicles used vary from experiment to experiment and are pooled in different ratios depending on protein concentration, however, no "potency" is measured for each batch of pooled vesicles.

[0106] The mean vesicle size of normal and PE STBEV was 186±22 nm and 185±6 nm, respectively. No significant differences in size distribution were observed between normal and PE STBEV. Comparison between the vesicle concentration of STBEV samples estimated using NTA and the protein concentration measured using NanoDrop analysis showed that the protein concentrations of normal and PE STBEV were 2.97±1.17 mg / mL and 2.68±2.04 mg / mL, respectively.

[0107] According to NTA, the concentration of vesicles was 1.18 × 10 09 ±8.20×10 08 and 9.63 x 10 08 ±8.04×10 08 particles / mL. The number of vesicles / particles was strongly correlated with protein concentration (r=0.91). (Placental syncytiotrophoblast extracellular vesicles enter primary endothelial cells through clathrin-mediated endocytosis. Cronqvist T, Erlandsson L, Tannetta D, Hansson SR. Placenta. 2020 Oct;100:133~141. doi:10.1016 / j.placenta.2020.07.006. Epub 2020 Jul 11. PMID:32980046).

[0108] Contraction experiment Preparation of blood vessels [arterial segments] and contraction experiments Subcutaneous arteries were removed from the abdominal subcutaneous fat during Caesarean section and immediately placed in cold DMEM medium. Arteries were dissected in cold Na-Krebs buffer at pH 7.3-7.4 and cut into ring segments of 1-2 mm length as previously described. The segments were attached to two wires of 40 μm diameter and connected to a device sensitive to vascular tension. The stretching force properties of the arteries were recorded with a Mulvany-Halpern myograph. The mounted arterial segments were immersed in a temperature-controlled (+37 °C) tissue bath containing a bicarbonate-based buffer and gassed with carbogen to pH 7.3-7.4. Each segment was stretched to 90% of its normal inner circumference, the size each segment would have, under a transmural pressure of 100 mmHg. The contractility of each arterial segment was confirmed by exposing it to 60 mM KCl buffer twice or three times and testing for viability. After washing out of each bath, tension returned to baseline. Once stabilized, different molecules or receptor agonists are added to the bath in a concentration-dependent manner and the vasomotor response of the blood vessels is observed.

[0109] In some experiments, endothelial function was tested by precontracting blood vessels with 30 mM potassium buffer to provide a stable tension and then administering substance P (10 -6 or 10 -5 Endothelial function was assessed by exposure to 100 mg / kg of ethanol (M).

[0110] The arteries used in each experiment were from different women with different medical histories, ages, and genetic backgrounds, factors that may affect the vascular endothelium and its responsiveness to drugs such as angiotensin and endothelin, exposure to vesicles, and to the LMWHs tinzaparin and tafoxiparin, respectively.

[0111] Vascular ring segments from a single individual have been reported to have high variability in basic control tests such as contractility measured by KCl treatment, endothelial function measured by 5-HT-induced preconcentration, and dilation in response to carbachol treatment.

[0112] Arterial segments were tested by exposure to cumulative concentrations of the agonists angiotensin II or endothelin 1. Previous studies have shown that this results in contraction that is blocked by the respective antagonists, angiotensin AT1 and endothelin ET A In some experiments, 30 min after adding vesicles to the tissue bath, angiotensin II (angiotensin 1 and 2 receptor agonist, concentration range 10 -12 ~10 -6 Concentration-contraction curves were obtained by cumulative application of 10 μg / mL of endothelin A and B receptor agonists. After washing and tension returning to baseline, agonists were added. After washing and tension returning to baseline, endothelin 1 was added (endothelin A and B receptor agonist, concentration range 10 μg / mL). -11 ~10 -7 M).

[0113] Preprocessing. The vessels were pretreated with the compounds tafoxiparin (Dilafor AB, Sweden) and tinzaparin (Inohep®, Leo Pharma Denmark) for 30 min each, resulting in a final concentration in the bath of 10 ug / ml and 100 ug / ml of tafoxiparin and tinzaparin, respectively. After 30 min, non-preeclamptic vesicles as control (P) or preeclamptic (PE) vesicles were added at a concentration of 200 μg (protein concentration 40 μg / mL). In patient no. 13, UP15 (internal code) was used as control vesicles and UP18 (internal code) as PE vesicles. In patient no. 14, UP8 (internal code) was used as control vesicles and UP17 as PE vesicles.

[0114] In vitro vasoconstriction experiment Thirty minutes after adding the vesicles to the tissue bath, angiotensin II (10 -3 A concentration-contraction curve was obtained by cumulative application of A9525 (Sigma-Aldrich human angiotensin II, powder, CAS number 4474-91-3) dissolved in sodium chloride to a concentration of 10 M. -12 ~10 -6After washing and tension returning to baseline, ET-1 (ALX-155-001, Enzo Life Sciences) was added and recordings were made (endothelin A and B receptor agonist, concentration range 10 -11 ~10 -7 M).

[0115] calculation The threshold for contractile responses to 60 mM potassium-rich bicarbonate buffer (Krebs buffer) was 0.5 mN (normalized value, 100%), using the average of two or three responses to potassium buffer to obtain a constant assessment of contractility. If data did not meet the threshold for contractile responses to potassium-rich buffer, they were excluded from data processing.

[0116] The maximum contraction induced by an agonist is expressed as the maximal contractility (Emax) and logEC50 represents the agonist concentration that produces half the maximum response. The x-axis of the concentration-contraction graphs is logarithmically transformed. Graphs for angiotensin II and endothelin 1 are presented. Endothelial function was calculated by taking the 30 nM contraction as 100%, and relaxation to substance P indicates that the endothelium is functional (in this case about 30% relaxation to the 30 mM induced contraction).

[0117] As shown in the experimental results and figures below, the presence of PE vesicles results in stronger contraction than control vesicles / non-PE vesicles (P).

[0118] Example 1 patient 13 In patient #13, the internal diameter of the arteries was 200-250 μm. Endothelial function was intact in most segments except one.

[0119] Addition of tinzaparin (Inohep®) at 10 μg / ml prior to treatment / incubation of arteries with PE vesicles reduced the maximal contraction induced by application of angiotensin II by 23%, but not the ET-1-induced contraction (E max) is the same, but logEC 50 was decreased by 35% (extrapolated value). Tinzaparin did not appear to have a reducing effect on contraction in the presence of control vesicles.

[0120] "Control" in Tables 1a and 1b below refers to non-preeclamptic vesicles (P). [Table 6] [Table 7]

[0121] The results are also shown in Figures 1A and 1B, which show concentration-contraction graphs for patient #13 in the presence of angiotensin II (Figure 1A) and endothelin 1 (Figure 1B), with the underline indicating the agonist concentration. The left bar is the contraction relative to that obtained with 60 mM potassium buffer. In the graphs, "P" represents non-preeclamptic vesicles, "Tinza" represents the LMWH tinzaparin, "PE" represents preeclamptic vesicles, and "Control" represents the absence of any agent other than the agonist angiotensin II or endothelin 1 in the tissue bath.

[0122] Example 2 patient 14 In patient number 14, arteries ranged from 150 to 350 μm in diameter and endothelial function was intact in all but two vessels, which were excluded.

[0123] Arteries from patient 14 were treated with both tinzaparin (control) and tapoxiparin, respectively. Addition of tinzaparin at a concentration of 10 μg / ml before treatment / incubation of vessels with PE vesicles reduced the maximum contraction induced by angiotensin II by 14% and ET-1 by 15%. The presence of tinzaparin along with control vesicles increased contraction compared to vesicles alone. In this case, there is no control vessel to compare to.

[0124] Addition of tapoxiparin at a concentration of 10 μg / ml reduced the contraction induced by angiotensin II by 79% and by ET-1 by 56% in the presence of PE vesicles. In control P vesicles, pretreatment with tapoxiparin had little or no effect on the contractions mediated by AngII and ET-1.

[0125] "P" in Tables 2a and 2b below means non-preeclamptic vesicles. [Table 8] [Table 9]

[0126] Results are also shown in Figures 2A and 2B, which show concentration-contraction graphs for patient number 14 induced by the addition of increasing concentrations of angiotensin II (Figure 2A) and endothelin 1 (Figure 2B). Contractile responses are given as percent contraction relative to the contraction induced by 60 mM potassium buffer (=100%). In the graphs, "P" stands for non-preeclamptic vesicles, "Tinza" stands for the LMWH tinzaparin, "PE" stands for preeclamptic vesicles, and "Tafoxi" stands for tapoxiparin.

[0127] Example 3 In this study, angiotensin II (AngII) was used as the vasoconstrictor. Data were pooled from four patients.

[0128] After preincubation with tafoxiparin at a concentration of 10 μg / ml or the control drug tinzaparin at a concentration of 10 μg / ml, followed by treatment with vesicles from PE placenta, a contractile response was induced in the blood vessels by angiotensin II.

[0129] The presence of PE vesicles results in stronger contractions at cumulative concentrations of AngII compared to control vesicles (P). Preincubation with tafoxiparin affects the contractile effect of AngII in the presence of PE. Preincubation with tinzaparin (Innohep®) affects the contractile response of AngII in the presence of PE. [Table 10]

[0130] The following statistical principle was used: Wilcoxon t-test. PE vs. PE + tafoxiparin = 0.0039; AUC - 31.5%; PE vs. PE + tinza = 0.0039, AUC - 27.3%.

[0131] The results are also shown in FIG.

[0132] Example 4 In this study, angiotensin II (AngII) was used as the vasoconstrictor. Data were pooled from five patients.

[0133] After preincubation with tafoxiparin at a concentration of 100 μg / ml or the control drug tinzaparin at a concentration of 100 μg / ml, followed by treatment with vesicles from PE placenta, a contractile response was induced in the blood vessels by angiotensin II.

[0134] The presence of PE vesicles resulted in stronger contractions at cumulative concentrations of AngII compared to control vesicles (P). Preincubation with tafoxiparin significantly reduced AngII-induced contractions. Preincubation with the control drug tinzaparin (Inohep®) significantly reduced AngII-induced contractions. [Table 11]

[0135] The following statistical principle was used: Wilcoxon t-test. PE vs. PE + Tafoxiparin = ** p=0.027; AUC-28%. PE vs. PE+Cinza = * ;P=0.0273;AUC-27%.

[0136] The results are also shown in FIG.

[0137] Example 5 In this study, endothelin-1 (ET-1) was used as a vasoconstrictor. Data were pooled from five patients.

[0138] After preincubation with tafoxiparin at a concentration of 10 μg / ml or the control drug tinzaparin at a concentration of 10 μg / ml, followed by treatment with vesicles from PE placenta (PE), a contractile response was induced in the arteries with increasing concentrations of ET-1.

[0139] In the presence of PE vesicles, ET-1 produces a stronger contractile response compared to the ET-1 response in control vesicles (P). Preincubation with tafoxiparin revealed significantly decreased contraction to ET-1. Preincubation with the control drug tinzaparin (Inohep®) revealed significantly decreased contraction to ET-1. [Table 12]

[0140] The following statistical principle was used: Wilcoxon t-test. PE vs. PE + Tafoxiparin = * p=0.0039; AUC-38%; PE vs. PE+Cinza = * ;p=0.0039;AUC-17%.

[0141] The results are also shown in FIG.

[0142] Example 6 Phase IIa Clinical Trial An exploratory, open-label, Phase IIa clinical pilot study will be conducted in which pregnant women aged ≥18 years diagnosed with preeclampsia between 26 and 32 weeks of gestation will be treated with daily subcutaneous tafoxiparin from the time of diagnosis until delivery.

[0143] A total of 23 subjects will participate in the study, with 15 subjects receiving an active treatment that includes standard of care (SOC) plus tafoxiparin, and 8 subjects receiving a control treatment that includes standard of care alone. The duration of the study will be up to 28 days from the time of diagnosis. Treatment can alternatively be extended until delivery (i.e., treatment continues from the time of diagnosis until delivery). After delivery, there will be an 8-week follow-up period.

[0144] The main objective of this study Safety and tolerability will be assessed by the frequency and characteristics of adverse events and serious adverse events, complete and symptom-based physical assessments, vital signs, safety blood samples (hematology and clinical chemistry), and rates of withdrawals from the study and / or study medication.

[0145] Secondary objectives of this study To evaluate the efficacy of tapoxiparin treatment for pre-eclampsia. For the purpose of this study, pre-eclampsia is defined as hypertension 160 / 100 and proteinuria (measured by urine dipstick ≧2+ or albumin / creatinine ≧30 mg / mmol).

[0146] The following secondary (efficacy) endpoints will be monitored and evaluated: Change in diastolic blood pressure from baseline to end of treatment and at 8-week follow-up; change in aortic pulse wave velocity (PWV) as measured by carotid-femoral PWV (cfPWV) at baseline, 2, 7, and 28 days after treatment, and at 8-week follow-up; change in cardiac output; change in soluble Fms-like tyrosine kinase 1 (sFlt-1); change in placental growth factor (PlGF); change in sFlt-1 / PlGF ratio; number of days of pregnancy maintained; extent of antihypertensive treatment (number of drugs and dose); change in fetal growth estimated by ultrasound; change in umbilical cord blood flow; change in uterine artery blood flow; proportion of subjects with placental abruption; proportion of infants with birth weight ≦5th percentile; birth Percentage of infants with birth weight in the 6th to 10th percentile; percentage of subjects with fetal loss after 28 weeks of gestation; percentage of subjects with eclampsia; percentage of subjects with oliguria; percentage of subjects referred to the intensive care unit (ICU) during the study; percentage of subjects experiencing preterm birth; percentage of subjects undergoing caesarean section (CS); maternal and fetal indications for CS; percentage of subjects undergoing instrumental birth (vacuum extraction (VE) / forceps); percentage of subjects with postpartum haemorrhage (PPH) >2000 ml; fetal outcome as assessed by birth weight, Apgar score, acidosis (pH<7.10) and / or arterial or venous cord blood base excess <-12 mmol / L, and indication for referral to the neonatal intensive care unit (NICU).

[0147] Test drug Tafoxiparin is supplied as a 150 mg / mL solution by Dilafor AB, Sweden.

[0148] Study medication will be administered subcutaneously by site personnel every 24 ± 3 hours. Daily study treatment will consist of tafoxiparin 300 mg (2 × 1 mL, tafoxiparin 150 mg / ml) administered subcutaneously (SC) for 4 weeks unless there are clinical indications for discontinuation or continuation of treatment. All subjects will receive standard treatment for preeclampsia, including ASA 150 mg orally (OD) and antihypertensive treatment according to clinical practice.

[0149] Medications (including over-the-counter or prescription medications, and / or herbal supplements) or other specific relevant categories that participants are taking at the time of enrollment or will receive during the study will be recorded.

[0150] The following medications are prohibited before and during the study: Use of medications that interfere with hemostasis, including heparin / LMWH, nonsteroidal anti-inflammatory drugs (NSAIDs), warfarin, and vitamin K antagonists, for 3 weeks prior to enrollment (baseline) and during the study. Use of acetylsalicylic acid (ASA) during early pregnancy or more than 1 week prior to entering the study was prohibited.

[0151] Biomarkers monitored during the study: I. Changes in soluble Fms-like tyrosine kinase 1 (sFlt-1). sFlt1 is elevated in preeclampsia. II. Alterations in Placental Growth Factor (PlGF) Placental growth factor (PlGF) is an increasingly important molecule in the prediction, diagnosis and treatment of pre-eclampsia. III. Changes in the sFlt-1 / PlGF ratio. In pregnant women before the onset of preeclampsia, the ratio of sFlt-1 to PlGF is elevated.

[0152] Selection Criteria (a) Inclusion To participate in this study, subjects had to meet all of the following inclusion criteria: nulliparous women aged 18-45 years who were diagnosed with preeclampsia and were scheduled for hospitalization, the gestational age had to be between 26 and 32 weeks (inclusive) and confirmed by ultrasound examination by the 21st week of gestation, and the pregnancy had to be a singleton pregnancy. (b) Exclusion Subjects suffering from and / or diagnosed with any of the following diseases and / or conditions will be excluded from the study: subjects unable to understand written and verbal instructions in the local language; presence of placenta previa; pathological CTG at enrollment; diabetes mellitus; preeclampsia; HELLP syndrome (hemolysis, elevated liver enzymes, and thrombocytopenia); known coagulopathy (Leiden heterozygote); current use of drugs that interfere with hemostasis (including heparin / LMWH, oral anticoagulant medications, nonsteroidal anti-inflammatory drug (NSAID) compounds, and vitamin K antagonists); ongoing treatment for essential hypertension; ongoing treatment for HIV or acute hepatitis; standard heparin and known allergy to heparin and / or the LMWH heparin; history of heparin-induced thrombocytopenia (platelet count ≦100,000); current drug or alcohol abuse that, in the opinion of the investigator, would prevent participation in the study; unsuitability for participation in the study due to intercurrent illness or severe organ impairment, in the opinion of the investigator; current participation in other interventional pharmaceutical treatment trials; fear of needles that, in the opinion of the investigator, would affect compliance with study medication; severe pulmonary edema; idiopathic thrombocytopenia; chronic kidney disease; chronic liver impairment; systemic lupus erythematosus; and / or antiphospholipid syndrome.

[0153] Methods and procedures for the tapoxiparin treatment group I. Screening Subjects are eligible for the study if they are between 26 and 32 weeks (inclusive) of gestation, have been diagnosed with pre-eclampsia, meet all inclusion criteria, have no exclusion criteria, have a physical examination including vital signs, a medical history, and have signed an "informed consent" form. Subjects will undergo the following activities: informed consent, inclusion and exclusion criteria, vital signs, physical examination including height and weight, medical history, blood pressure (BP) recording as indicated, CTG recording, blood samples for hematology and clinical chemistry.

[0154] II. Baseline Prior to treatment, subjects undergo the following activities: inclusion and exclusion criteria, physical examination, blood pressure measurement as indicated, CTG recording, fetal ultrasound, and blood samples for umbilical cord blood flow and maternal uterine artery blood flow, biomarkers of pre-eclampsia, and PWV recording. Women are then randomized to study treatment and undergo their first study treatment.

[0155] III. During treatment Study medication will be administered subcutaneously by site personnel every 24 ± 3 hours. Daily study treatment will consist of tafoxiparin 300 mg (2 × 1 mL) administered subcutaneously (SC) for 4 weeks unless there are clinical indications for discontinuation or continuation of treatment. All subjects will receive standard treatment for preeclampsia including ASA 150 mg OD and antihypertensive treatment according to clinical practice.

[0156] If pre-eclampsia worsens during or after the 4 weeks of treatment, the subject will be managed according to clinical practice. If pre-eclampsia improves during the 4 weeks of tapoxiparin treatment, treatment may be continued off-main protocol until delivery. If this occurs, it must be registered in the eCRF.

[0157] During the treatment period, the following will be performed and recorded: daily blood pressure measurements; CTG recordings; fetal ultrasound, umbilical cord blood flow and maternal uterine artery blood flow on days 7, 14, 21 and 28; blood samples for hematology and clinical chemistry on days 7, 14, 21 and 28 of treatment; blood samples for biomarkers of pre-eclampsia on days 2, 7, 14, 21 and 28 of treatment; PWV recordings after days 2, 7, 14 and 28 of treatment; cardiac output after days 2, 7, 14, 21 and 28 of treatment; daily checks of treatment compliance; documentation of antihypertensive treatment on days 2, 7, 14, 21 and 28 of treatment; AE / SAE tests; blood samples for hematology including platelet count will be taken and analyzed after administration of the 5th dose on day 5 and must be tested before administration of the 6th dose; concomitant medication records.

[0158] IV. After birth The following activities will be performed in the hospital: blood pressure measurement; blood samples for hematology and clinical chemistry; documentation of antihypertensive treatment; documentation of the infant's condition; AE / SAE testing and concomitant medications.

[0159] V. At the time of discharge Performs the following activities in the hospital: blood pressure measurements as ordered; PWV measurements; cardiac output recording; documentation of infant's condition; antihypertensive treatment recording; AE / SAE testing and concomitant medications.

[0160] VI. Follow-up Follow-up management will be performed in the hospital clinic 8 weeks (± 1 week) after discharge and the following investigations will be performed: blood pressure measurement; blood samples for hematology and clinical chemistry; PWV measurement; cardiac output; documentation of the infant's condition; records of antihypertensive treatment; AE / SAE tests and concomitant medications.

[0161] Subjects in the control group undergo the same procedures listed above as subjects in the treatment group, except for the treatment with tafoxiparin.

Claims

1. A pharmaceutical formulation for the treatment of pre-eclampsia (PE), comprising the heparin derivative tafoxiparin.

2. 2. The pharmaceutical formulation of claim 1, wherein the treatment is a combination therapy with a standard therapy for pre-eclampsia (PE).

3. 3. The pharmaceutical formulation of claim 2, wherein acetylsalicylic acid (ASA) is used as the standard treatment for pre-eclampsia (PE).

4. 4. The pharmaceutical formulation according to claim 2 or 3, wherein antihypertensive agents are used as standard treatment for pre-eclampsia (PE).

5. 5. The pharmaceutical formulation according to claim 4, wherein the antihypertensive agent is selected from any one of diuretics, calcium channel blockers, ACE inhibitors, adrenoceptor antagonists, renin inhibitors and endothelial receptor blockers.

6. The pharmaceutical formulation according to any one of claims 2 to 5, wherein corticosteroids are used as standard treatment for pre-eclampsia (PE).

7. A pharmaceutical formulation according to any one of claims 2 to 6, wherein magnesium compounds such as magnesium sulphate are used as standard treatment for pre-eclampsia (PE).

8. The pharmaceutical formulation according to any one of claims 1 to 7, which is administered prior to or simultaneously with the administration of standard treatment for pre-eclampsia (PE).

9. The pharmaceutical formulation of any one of claims 1 to 7, administered as add-on therapy to a subject already undergoing standard treatment for pre-eclampsia (PE).

10. The pharmaceutical formulation according to any one of claims 1 to 9, which is administered parenterally.

11. The pharmaceutical formulation according to any one of claims 1 to 9, which is administered by topical administration.

12. The pharmaceutical formulation of claim 10, wherein parenteral administration is intravenous, intramuscular or subcutaneous administration.

13. 12. The pharmaceutical formulation of claim 11, wherein the local administration is oral, vaginal or rectal administration.

14. The pharmaceutical preparation according to any one of claims 1 to 13, wherein the treatment is initiated upon diagnosis of PE.

15. The pharmaceutical preparation according to any one of claims 1 to 14, wherein the treatment is terminated at birth.

16. The pharmaceutical preparation according to any one of claims 1 to 14, wherein the treatment is terminated at a time point between 4 and 8 weeks after birth.

17. The pharmaceutical formulation according to any one of claims 1 to 16, wherein the treatment is initiated upon a positive pregnancy test.

18. The pharmaceutical preparation according to any one of claims 1 to 17, wherein the treatment is in women suffering from HELLP syndrome.

19. The pharmaceutical formulation according to any one of claims 1 to 17, wherein the treatment is in women suffering from eclampsia.

20. The pharmaceutical formulation according to any one of claims 1 to 19, wherein the treatment is in obese, pre-diabetic or diabetic women.

21. The pharmaceutical formulation according to any one of claims 1 to 20, wherein the treatment is a therapeutic treatment.

22. The pharmaceutical formulation according to any one of claims 1 to 20, wherein the treatment is a preventative treatment.

23. The pharmaceutical formulation according to any one of claims 1 to 22, wherein the treatment is the postpartum treatment of pre-eclampsia.

24. The pharmaceutical formulation according to any one of claims 1 to 22, wherein the treatment is until birth.

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