Disclosed herein are methods for treating a pregnancy related hypertensive disorder, such as pre-eclampsia and eclampsia, using combinations of compounds that alter soluble endoglin and sFlt-1 expression levels or biological activity. Also disclosed are methods for treating a pregnancy related hypertensive disorder, such as pre-eclampsia and eclampsia, using compounds that increase endothelial nitric oxide synthase levels or biological activity.

Description of the Invention

SUMMARY OF THE INVENTION

We have discovered methods for diagnosing and treating pregnancy related hypertensive disorders, including pre-eclampsia and eclampsia.

Using gene expression analysis, we have discovered that levels of soluble endoglin (sEng) are markedly elevated in placental tissue samples from pregnant women suffering from pregnancy complications associated with hypertension, including pre-eclampsia. Using western blotting, we have also discovered that soluble endoglin protein levels are elevated in blood serum samples taken from women with a pregnancy related hypertensive disorder, such as pre-eclampsia or eclampsia. Soluble endoglin may be formed by cleavage of the extracellular portion of the membrane bounds form by proteolytic enzymes. We have discovered that the soluble endoglin detected in these samples contains a minimum of the first 381 amino acids (excluding the leader peptide, 406 including the leader peptide) of the amino terminal portion of the full-length endoglin. Excess soluble endoglin in pre-eclampsia may be depleting the placenta of necessary amounts of these essential angiogenic and mitogenic factors by preventing binding of TGF-.beta.1 to T.beta.RII on endothelial cells leading to decreased signaling, as described herein. We have also discovered that soluble endoglin interferes with TGF-.beta.1 signaling and endothelial nitric oxide synthase (eNOS) activation in endothelial cells, thereby disrupting key homeostatic mechanisms necessary for maintenance of vascular health. We demonstrate that soluble endoglin prevents binding of TGF-.beta.1 to T.beta.RII on endothelial cells leading to decreased signaling. Since circulating TGF-.beta.1 is complexed with latency associated peptide and latent TGF-.beta.1 binding protein, it cannot bind its receptors, unless activated. It is therefore likely that soluble endoglin only inhibits TGF-.beta.1 effects locally where active TGF-.beta.1 is generated. Taken together, these data suggest a crucial role for endoglin in linking TGF-.beta. receptor activation to nitric oxide (NO) synthesis. In addition, our functional studies suggest that soluble endoglin and sFlt1 act in concert to induce vascular damage and HELLP syndrome by interfering with TGF-.beta.1 and VEGF signaling respectively, likely via inhibition of the downstream activation of NOS.

In the present invention, compounds that bind to or neutralize soluble endoglin are used to reduce the elevated levels of soluble endoglin and to treat pregnancy complications associated with hypertension, including pre-eclampsia or eclampsia. For example, antibodies directed to soluble endoglin as well as RNA interference and antisense nucleobase oligomers directed to lowering the levels of biologically active soluble endoglin are also provided. The invention also features the use of any compound (e.g., polypeptide, small molecule, antibody, nucleic acid, and mimetic) that decreases soluble endolin levels or biological activity or that increases the level or biological activity of TGF-.beta., NOS, and prostacyclin (PGI.sub.2) either alone or in combination with each other or with any compound that decreases the level of sFlt-1 or increases the level or activity of VEGF or PlGF (see for example, U.S. Patent Application Publication Numbers 20040126828, 20050025762, and 20050170444 and PCT Publication Numbers WO 2004/008946 and WO 2005/077007) to treat or prevent pregnancy related hypertensive disorders, such as pre-eclampsia or eclampsia in a subject. The invention also features methods for measuring levels of soluble endoglin, either alone or in combination with sFlt-1, VEGF, PlGF, TGF-.beta., eNOS, or PGI.sub.2, as a detection tool for early diagnosis and management of a pregnancy related hypertensive disorder, including pre-eclampsia and eclampsia.

Accordingly, in a first aspect, the invention features a method of treating or preventing a pregnancy related hypertensive disorder in a subject, that includes administering to the subject (i) a compound capable of decreasing soluble endoglin expression levels or biological activity and (ii) a compound capable of decreasing sFlt-1 expression levels or biological activity, for a time and in an amount sufficient to treat or prevent the pregnancy related hypertensive disorder. Pregnancy related hypertensive disorder include, for example, pre-eclampsia, eclampsia, gestational hypertension, chronic hypertension, HELLP syndrome, and pregnancy with a small for gestational age (SGA) infant. Preferably, the pregnancy related hypertensive disorder is pre-eclampsia or eclampsia.

Assays for soluble endoglin or sFlt-1 expression levels or biological activity are known in the art. Preferred compounds will decrease soluble endoglin or sFlt-1 expression levels or biological activity by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more. Non-limiting examples of compounds capable of decreasing soluble endoglin expression levels or biological activity include any compound that specifically binds soluble endoglin, for example, a purified soluble endoglin antibody, a soluble endoglin antigen-binding fragment, or a growth factor (e.g., TGF-.beta.1, TGF-.beta.3, activin A, Bone Morphogenic Protein (BMP)-2 and BMP-7).

Additional examples of a compound capable of decreasing soluble endoglin expression levels or biological activity include any compound that inhibits a proteolytic enzyme (e.g., a matrix metalloproteinase (MMP), cathepsin, and elastase) or a compound that increases the level of a growth factor capable of binding to soluble endoglin. Growth factors such as TGF-.beta.1, TGF-.beta.3, activin A, BMP-2, BMP-7, or fragments thereof, are examples of compounds that increases the level of a growth factor capable of binding to soluble endoglin as are cyclosporine, alpha tocopherol, methysergide, bromocriptine, and aldomet.

Non-limiting examples of a compound capable of decreasing sFlt-1 expression levels or biological activity include a compound capable of specifically binding to sFlt-1, such as a purified sFlt-1 antibody or an sFlt-1 antigen-binding fragment; compounds that increase the level of a growth factor capable of binding to sFlt-1, such as nicotine, theophylline, adenosine, nifedipine, minoxidil, and magnesium sulfate, VEGF (e.g., VEGF121, VEGF165, or a modified form of VEGF), PlGF, or fragments thereof.

In preferred embodiments of the above method, a compound capable of decreasing soluble endoglin expression levels or biological activity or a compound capable of decreasing sFlt-1 expression levels or biological activity, or both, can also increase nitric oxide synthase (NOS) activity by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more. Assays for NOS activity are known in the art and described herein.

In a second aspect, the invention features a method of treating or preventing a pregnancy related hypertensive disorder in a subject, that includes the step of administering to the subject a compound capable of increasing the expression level or biological activity of NOS, for a time and in an amount sufficient to treat or prevent the pregnancy related hypertensive disorder in the subject. Desirably, the NOS is eNOS. In one embodiment, the compound is a compound that increases the phosphorylation of Ser 1177 of eNOS, such as VEGF (e.g., VEGF121, VEGF165, or a modified form of VEGF), or biologically active fragments thereof, or PlGF or biologically active fragments thereof. In another embodiment, the compound is a compound that increases the dephosphorylation of Thr495 of eNOS, such as TGF-.beta.1 or TGF-.beta.3, activin A, BMP-2, and BMP-7. In another embodiment, the compound is a compound that prevents a reduction in the levels of eNOS or increases the stability of eNOS.

Optionally, the method further includes administering to the subject a compound capable of reducing soluble endoglin expression or biological activity, wherein the administering is sufficient to treat or prevent the pregnancy related hypertensive disorder in the subject. Non-limiting examples of a compound capable of reducing soluble endoglin expression or biological activity include a purified antibody that specifically binds soluble endoglin or a soluble endoglin antigen-binding fragment or a compound that inhibits a proteolytic enzyme selected from the group consisting of a matrix metalloproteinase (MMP), cathepsin, and elastase, or growth factors such as TGF-.beta.1, TGF-.beta.3, activin A, BMP-2, BMP-7, or fragments thereof.

Optionally, the method further includes administering to the subject a compound capable of reducing sFlt-1 expression or biological activity, wherein the administering is sufficient to treat or prevent the pregnancy related hypertensive disorder in the subject. Non-limiting examples of a compound capable of reducing soluble endoglin expression or biological activity include a purified antibody that specifically binds sFlt-1 or a sFlt-1 antigen binding fragment, or a growth factor such as VEGF (e.g., VEGF121, VEGF165, or a modified form of VEGF), PlGF, or fragments thereof.

For any of the above methods, the method can further include the step of administering to a subject an anti-hypertensive compound. In preferred embodiments of any of the above methods, the subject is a pregnant human, a post-partum human, or a non-human (e.g., a cow, a horse, a sheep, a pig, a goat, a dog, and a cat).

As described below, we have discovered that deregulation of both the soluble endoglin/TGF-.beta. and the sFlt-1/VEGF/PlGF signaling pathways can act together to further the pathology of the pregnancy related hypertensive disorder. Therefore, the invention also features combinations of the methods described herein with any of the therapeutic, diagnostic, or monitoring methods described in U.S. Patent Application Publication Numbers 20040126828, 20050025762, 20050170444, and 2006/0067937 and PCT Publication Numbers WO 2004/008946, WO 2005/077007, and WO 06/034507.

DETAILED DESCRIPTION

We have discovered that soluble endoglin levels are elevated in blood serum samples taken from women with a pregnancy related hypertensive disorder, such as pre-eclampsia or eclampsia. Soluble endoglin may be formed by cleavage of the extracellular portion of the membrane bound form by proteolytic enzymes. The lack of detection of alternate splice variants in placenta and the partial peptide sequence of purified soluble endoglin as described herein suggest that it is an N-terminal cleavage product of full-length endoglin. Excess soluble endoglin may be depleting the placenta of necessary amounts of these essential angiogenic and mitogenic factors. We have discovered that excess circulating concentrations of soluble endoglin and sFlt1 in patients with preeclampsia contribute to the pathogenesis of pre-eclampsia and other pregnancy related hypertensive disorder. We have also discovered that soluble endoglin interferes with TGF-.beta.1 and TGF-.beta.3 binding to its receptor leading to decreased signaling such as a reduction in eNOS activation in endothelial cells, thereby disrupting key homeostatic mechanisms necessary for maintenance of vascular health. These data suggest a crucial role for endoglin in linking TGF-.beta. receptor activation to NO synthesis. In addition, we have discovered that soluble endoglin and sFlt1 act in concert to induce vascular damage and pregnancy related hypertensive disorders, such as pre-eclampsia or eclampisa, by interfering with TGF-.beta.1 and VEGF signaling respectively, likely via inhibition of the downstream activation of eNOS.

The present invention features the use of therapeutic agents that interfere with soluble endoglin binding to growth factors, agents that reduce soluble endoglin expression or biological activity, or agents that increase levels of growth factors, can be used to treat or prevent pregnancy related hypertensive disorders, such as pre-eclampsia or eclampsia in a subject. Such agents include, but are not limited to, antibodies to soluble endoglin, oligonucleotides for antisense or RNAi that reduce levels of soluble endoglin, compounds that increase the levels of growth factors, compounds that prevent the proteolytic cleavage of the membrane bound form of endoglin thereby preventing the release of soluble endoglin, and small molecules that bind soluble endoglin and block the growth factor binding site. Additionally or alternatively, the invention features the use of any compound (e.g., polypeptide, small molecule, antibody, nucleic acid, and mimetic) that increases the level or biological activity of TGF-.beta., eNOS, and PGI.sub.2 to treat or prevent pregnancy related hypertensive disorders, such as pre-eclampsia or eclampsia in a subject. Additionally, the invention features the use of any compound that decreases the level of sFlt-1 or increases the level or activity of VEGF or PlGF (see for example, U.S. Patent Application Publication Numbers 20040126828, 20050025762, and 20050170444 and PCT Publication Numbers WO 2004/008946 and WO 2005/077007) in combination with any of the therapeutic compounds described above to treat or prevent pregnancy related hypertensive disorders, such as pre-eclampsia or eclampsia in a subject. In addition, the invention features the use of soluble endoglin, eNOS, TGF-.beta., of PGI.sub.2, either alone or in combination, as a diagnostic marker of pregnancy related hypertensive disorders, including pre-eclampsia and eclampsia.

While the detailed description presented herein refers specifically to soluble endoglin, TGF-.beta.1, eNOS, sFlt-1, VEGF, or PlGF, it will be clear to one skilled in the art that the detailed description can also apply to family members, isoforms, and/or variants of soluble endoglin, TGF-.beta., eNOS, sFlt-1, VEGF, or PlGF.

Diagnostics

We have discovered that soluble endoglin levels are elevated in blood serum samples taken from women with a pregnancy related hypertensive disorder, such as pre-eclampsia or eclampsia. Soluble endoglin starts rising 6-10 weeks before clinical symptoms of preeclampsia. Accordingly, a diagnostic test measuring soluble endoglin and sFlt1, optionally in combination with PlGF, preferably free PlGF in the serum will have enhanced sensitivity and specificity, and provide a powerful tool in the prevention of preeclampsia-induced mortality. The diagnostic test can also include measuring the levels of free VEGF; TGF-.beta. family members, preferably TGF-.beta.1, TGF-.beta.3, free activin-A, BMP2, BMP7; NOS, preferably eNOS; or PGI2, either alone or in any combination thereof.

While the methods described herein refer to pre-eclampsia and eclampsia specifically, it should be understood that the diagnostic and monitoring methods of the invention apply to any pregnancy related hypertensive disorder including, but not limited to, gestational hypertension, pregnancy with a small for gestational age (SGA) infant, HELLP, chronic hypertension, pre-eclampsia (mild, moderate, and severe), and eclampsia.

Levels of soluble endoglin, either free, bound, or total levels, are measured in a subject sample and used as an indicator of pre-eclampsia, eclampsia, or the propensity to develop such conditions.

A subject having pre-eclampsia, eclampsia, or a predisposition to such conditions will show an increase in the expression of a soluble endoglin polypeptide. The soluble endoglin polypeptide can include full-length soluble endoglin, degradation products, alternatively spliced isoforms of soluble endoglin, enzymatic cleavage products of soluble endoglin, and the like. An antibody that specifically binds a soluble endoglin polypeptide may be used for the diagnosis of pre-eclampsia or eclampsia or to identify a subject at risk of developing such conditions. One example of an antibody useful in the methods of the invention is a monoclonal antibody against the N-terminal region of endoglin that is commercially available from Santa Cruz Biotechnology, Inc. (cat #sc-20072). Additional examples include antibodies that specifically bind the extracellular domain of endoglin (e.g., amino acids 1 to 437 of endoglin, amino acids 1 to 587 of endoglin, or any of the amino acid sequences shown in bold and underlined in FIG. 30B (see Original Patent)). A variety of protocols for measuring an alteration in the expression of such polypeptides are known, including immunological methods (such as ELISAs and RIAs), and provide a basis for diagnosing pre-eclampsia or eclampsia or a risk of developing such conditions.

Increased levels of soluble endoglin are a positive indicator of pre-eclampsia or eclampsia. For example, if the level of soluble endoglin is increased relative to a reference (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more), or increases over time in one or more samples from a subject, this is considered a positive indicator of pre-eclampsia or eclampsia. Additionally, any detectable alteration in levels of soluble endoglin, sFlt-1, VEGF, or PlGF relative to normal levels is indicative of eclampsia, pre-eclampsia, or the propensity to develop such conditions. Normally, circulating serum concentrations of soluble endoglin range from 2-7 ng/ml during the non-pregnant state and from 10-20 ng/ml during normal pregnancy. Elevated serum levels, greater than 15 ng/ml, preferably greater than 20 ng/ml, and most preferably greater than 25 ng/ml or more, of soluble endoglin is considered a positive indicator of pre-eclampsia or eclampsia.

In one embodiment, the level of soluble endoglin is measured in combination with the level of sFlt-1, VEGF, or PlGF polypeptide or nucleic acid, or any combination thereof. Methods for the measurement of sFlt-1, VEGF, and PlGF are described in U.S. Patent Application Publication Numbers 20040126828, 20050025762, and 20050170444 and PCT Publication Numbers WO 2004/008946 and WO 2005/077007, hereby incorporated by reference in their entirety. In additional preferred embodiments, the body mass index (BMI) and gestational age of the fetus is also measured and included the diagnostic metric.

In another embodiment, the level of TGF-.beta.1, TGF-.beta.3, or eNOS polypeptide or nucleic acid is measured in combination with the level of soluble endoglin, sFlt-1, VEGF, or PlGF polypeptide or nucleic acid. Antibodies useful for the measurement of TGF-.beta.1 and .beta.3 polypeptide levels are commercially available, for example, from Abcam, Abgent, BD Biosciences Pharmingen, Chemicon, GeneTex, and R&D Systems. The level of PGI.sub.2 can also be used in combination with the level of any of the above polypeptides. PGI.sub.2 levels can be determined, for example, using the PGI.sub.2 receptor as a binding molecule in any of the diagnostic assays described above, or using, for example, the urinary prostacyclin colorimetric ELISA kit (Assay Designs). Antibodies useful for the measurement of eNOS polypeptide levels are commercially available, for example, from Research Diagnostics Inc., Santa Cruz, Cayman Chemicals, and BD Biosciences.

In one embodiment, a metric incorporating soluble endoglin, sFlt-1, VEGF, or PlGF, or any combination therein, is used to determine whether a relationship between levels of at least two of the proteins is indicative of pre-eclampsia or eclampsia. In one example, the metric is a PAAI (sFlt-1/VEGF+PlGF), which is used, in combination with soluble endoglin measurement, as an anti-angiogenic index that is diagnostic of pre-eclampsia, eclampsia, or the propensity to develop such conditions. If the level of soluble endoglin is increased relative to a reference sample (e.g., 1.5-fold, 2-fold, 3-fold, 4-fold, or even by as much as 10-fold or more), and the PAAI is greater than 10, more preferably greater than 20, then the subject is considered to have pre-eclampsia, eclampsia, or to be in imminent risk of developing the same. The PAAI (sFlt-1/VEGF+PlGF) ratio is merely one example of a useful metric that may be used as a diagnostic indicator. It is not intended to limit the invention. Virtually any metric that detects an alteration in the level of soluble endoglin, sFlt-1, PlGF, or VEGF, or any combination thereof, in a subject relative to a normal control may be used as a diagnostic indicator. Another example is the following soluble endoglin anti-angiogenic index: (sFlt-1+0.25(soluble endoglin polypeptide))/PlGF. An increase in the value of the soluble endoglin metric over time or compared to a reference sample or value is a diagnostic indicator of pre-eclampsia or eclampsia. A soluble endoglin index above 100, preferably above 200 is a diagnostic indicator of pre-eclampsia or eclampsia. Another example are the following indexs: (soluble endoglin+sFlt-1)/PlGF or sFlt-1.times. soluble endoglin. In addition, the metric can further include the level of TGF-.beta.1, TGF-.beta.3, PGI.sub.2, or eNOS polypeptide. Any of the metrics can further include the BMI of the mother or the GA of the infant.

Standard methods may be used to measure levels of soluble endoglin, VEGF, PlGF, or sFlt-1 polypeptide in any bodily fluid, including, but not limited to, urine, serum, plasma, saliva, amniotic fluid, or cerebrospinal fluid. Preferably, free VEGF or free PlGF is measured. Such methods include immunoassay, ELISA, western blotting using antibodies directed to soluble endoglin, VEGF, PlGF or sFlt-1, and quantitative enzyme immunoassay techniques such as those described in Ong et al. (Obstet. Gynecol. 98:608-611, 2001) and Su et al. (Obstet. Gynecol., 97:898-904, 2001). ELISA is the preferred method for measuring levels of soluble endoglin, VEGF, PlGF, or sFlt-1. Preferably, soluble endoglin is measured.

Oligonucleotides or longer fragments derived from an endoglin, sFlt-1, PlGF, or VEGF nucleic acid sequence may be used as a probe not only to monitor expression, but also to identify subjects having a genetic variation, mutation, or polymorphism in an endoglin, sFlt-1, PlGF, or VEGF nucleic acid molecule that are indicative of a predisposition to develop the pre-eclampsia or eclampsia. Such methods are described in detail in Abdalla et al., Hum. Mutat. 25:320-321 (2005), U.S. Patent Application Publication No. 2006/0067937 and PCT Publication No. WO 06/034507. Preferred oligonucleotides will hybridize at high stringency to the extracellular domain of endoglin or to any nucleic acid sequence encoding any of the peptides shown in bold and underlined in FIG. 30B (see Original Patent).

The measurement of any of the nucleic acids or polypeptides described herein can occur on at least two different occasions and an alteration in the levels as compared to normal reference levels over time is used as an indicator of pre-eclampsia, eclampsia, or the propensity to develop such conditions.

In one example, the level of a soluble endoglin polypeptide or nucleic acid present in the bodily fluids of a subject having pre-eclampsia, eclampsia, or the propensity to develop such conditions may be increased by as little as 10%, 20%, 30%, or 40%, or by as much as 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more relative to levels in a normal control subject or relative to a previous sampling obtained from the same bodily fluids of the same subject. In another example, the level of a soluble endoglin polypeptide or nucleic acid in the bodily fluids of a subject having pre-eclampsia, eclampsia, or the propensity to develop such conditions may be altered by as little as 10%, 20%, 30%, or 40%, or by as much as 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% over time from one measurement to the next.

The level of sFlt-1, VEGF, or PlGF measured in combination with the level of soluble endoglin in the bodily fluids of a subject having pre-eclampsia, eclampsia, or the propensity to develop such conditions may be altered by as little as 10%, 20%, 30%, or 40%, or by as much as 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more relative to the level of sFlt-1, VEGF, or PlGF in a normal control. The level of sFlt-1, VEGF, or PlGF measured in combination with the level of soluble endoglin in the bodily fluids of a subject having pre-eclampsia, eclampsia, or the propensity to develop such conditions may be altered by as little as 10%, 20%, 30%, or 40%, or by as much as 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% over time from one measurement to the next.

In one embodiment, a subject sample of a bodily fluid (e.g., urine, plasma, serum, amniotic fluid, or cerebrospinal fluid) is collected early in pregnancy prior to the onset of pre-eclampsia symptoms. In another example, the sample can be a tissue or cell collected early in pregnancy prior to the onset of pre-eclampsia symptoms. Non-limiting examples of tissues and cells include placental tissue, placental cells, circulating endothelial cells, and leukocytes such as monocytes. In humans, for example, maternal blood serum samples are collected from the antecubital vein of pregnant women during the first, second, or third trimesters of the pregnancy. Preferably, the assay is carried out during the first trimester, for example, at 4, 6, 8, 10, or 12 weeks, or any interval therein, or during the second trimester, for example at 14, 16, 18, 20, 22, or 24 weeks, or any interval therein. In one example, the assay is carried out between 13 and 16 weeks of pregnancy. Such assays may also be conducted at the end of the second trimester or the third trimester, for example at 26, 28, 30, 32, 34, 36, or 38 weeks, or any interval therein. It is preferable that levels of soluble endoglin be measured twice during this period of time. For the diagnosis of post-partum pre-eclampsia or eclampsia, assays for soluble endoglin may be carried out postpartum. For the diagnosis of a predisposition to pre-eclampsia or eclampsia, the assay is carried out prior to the onset of pregnancy or prior to the development of symptoms of pre-eclampsia or eclampsia. In one example, for the monitoring and management of therapy, the assay is carried out during the pregnancy after the diagnosis of pre-eclampsia, and/or during therapy.

In one particular example, serial blood samples can be collected during pregnancy and the levels of soluble endoglin polypeptide determined by ELISA. In another example, a sample is collected during the second trimester and early in the third trimester and in increase in the level of soluble endoglin from the first sampling to the next is indicative of pre-eclampsia or eclampsia, or the propensity to develop either.

The invention also include the measurement of any ligands of soluble endoglin (e.g., TGF-.beta.1, TGF-.beta.3, activin-A, BMP-2, and BMP-7) ligand in a bodily fluid from a subject, preferably urine, and an alteration (e.g., increase or decrease) in the level of the soluble endoglin ligand is indicative of pre-eclampsia or eclampsia.

In veterinary practice, assays may be carried out at any time during the pregnancy, but are, preferably, carried out early in pregnancy, prior to the onset of pre-eclampsia symptoms. Given that the term of pregnancies varies widely between species, the timing of the assay will be determined by a veterinarian, but will generally correspond to the timing of assays during a human pregnancy.

The diagnostic methods described herein can be used individually or in combination with any other diagnostic method described herein for a more accurate diagnosis of the presence of, severity of, or estimated time of onset of pre-eclampsia or eclampsia. In addition, the diagnostic methods described herein can be used in combination with any other diagnostic methods determined to be useful for the accurate diagnosis of the presence of, severity of, or estimated time of onset of pre-eclampsia or eclampsia.

The diagnostic methods described herein can also be used to monitor and manage pre-eclampsia or eclampsia in a subject. In one example, a therapy is administered until the blood, plasma, or serum soluble endoglin level is less than 25 ng/ml or until the serum endoglin levels return to the baseline level determined before onset of pre-eclampsia or eclampsia. In another example, if a subject is determined to have an increased level of soluble endoglin relative to a normal control then the therapy can be administered until the serum PlGF level rises to approximately 400 pg/mL or a return to baseline level prior to onset of pre-eclampsia or eclampsia. In this embodiment, the levels of soluble endoglin, sFlt-1, PlGF, and VEGF, or any and all of these, are measured repeatedly as a method of not only diagnosing disease but monitoring the treatment and management of the pre-eclampsia and eclampsia.

Diagnostic Kits

The invention also provides for a diagnostic test kit. For example, a diagnostic test kit can include binding agents (e.g., polypeptides or antibodies) that specifically bind to soluble endoglin and means for detecting, and more preferably evaluating, binding between the binding agent and the soluble endoglin polypeptide. For detection, either the binding agent or the soluble endoglin polypeptide is labeled, and either the binding agent or the soluble endoglin polypeptide is substrate-bound, such that soluble endoglin polypeptide-binding agent interaction can be established by determining the amount of label attached to the substrate following binding between the binding agent and the soluble endoglin polypeptide. A conventional ELISA is a common, art-known method for detecting antibody-substrate interaction and can be provided with the kit of the invention. Soluble endoglin polypeptides can be detected in virtually any bodily fluid including, but not limited to urine, serum, plasma, saliva, amniotic fluid, or cerebrospinal fluid. The invention also provides for a diagnostic test kit that includes a soluble endoglin nucleic acid that can be used to detect and determine levels of soluble endoglin nucleic acids. A kit that determines an alteration, for example, an increase, in the level of soluble endoglin polypeptide relative to a reference, such as the level present in a normal control, is useful as a diagnostic kit in the methods of the invention.

The diagnostic kits of the invention can include antibodies or nucleic acids for the detection of sFlt-1, VEGF, or PlGF polypeptides or nucleic acids as described U.S. Patent Application Publication Numbers 20040126828, 20050025762, and 20050170444 and PCT Publication Numbers WO 2004/008946 and WO 2005/077007.

In another embodiment, the kit can also include binding agents for the detection of TGF-.beta.1, TGF-.beta.3, or eNOS polypeptide. Antibodies useful for the measurement of TGF-.beta.1 and .beta.3 polypeptide levels are commercially available, for example, from Abcam, Abgent, BD Biosciences Pharmingen, Chemicon, GeneTex, and R&D Systems. Antibodies useful for the measurement of eNOS polypeptide levels are commercially available, for example, from Research Diagnostics Inc., Santa Cruz, Cayman Chemicals, and BD Biosciences. Binding agents for the detection of PGI.sub.2 levels can also be included and include for example the PGI.sub.2 receptor, or fragments thereof, as a binding molecule in any of the diagnostic assays described above, or using, for example, the urinary prostacyclin colorimetric ELISA kit (Assay Designs). Antibodies useful for the measurement of eNOS polypeptide levels are commercially available, for example, from Research Diagnostics Inc. A kit that determines an alteration, for example, a decrease, in the level of eNOS, TGF-.beta.1 or .beta.3 polypeptide or PGI.sub.2 relative to a reference, such as the level present in a normal control, is useful as a diagnostic kit in the methods of the invention.

Desirably, the kit includes any of the components needed to perform any of the diagnostic methods described above. For example, the kit desirably includes a membrane, where the soluble endoglin binding agent or the agent that binds the soluble endoglin binding agent is immobilized on the membrane. The membrane can be supported on a dipstick structure where the sample is deposited on the membrane by placing the dipstick structure into the sample or the membrane can be supported in a lateral flow cassette where the sample is deposited on the membrane through an opening in the cassette.

The diagnostic kits also generally include a label or instructions for the intended use of the kit components and a reference sample or purified proteins to be used to establish a standard curve. In one example, the kit contains instructions for the use of the kit for the diagnosis of a pregnancy related hypertensive disorder, such as pre-eclampsia, eclampsia, or the propensity to develop pre-eclampsia or eclampsia. In yet another example, the kit contains instructions for the use of the kit to monitor therapeutic treatment or dosage regimens for the treatment of pre-eclampsia or eclampsia. The diagnostic kit may also include a label or instructions for the use of the kit to determine the PAAI or soluble endoglin anti-angiogenesis index of the subject sample and to compare the PAAI or soluble endoglin anti-angiogenesis index to a reference sample value. It will be understood that the reference sample values will depend on the intended use of the kit. For example, the sample can be compared to a normal reference value, wherein an increase in the PAAI or soluble endoglin anti-angiogenesis index or in the soluble endoglin value is indicative of pre-eclampsia or eclampsia, or a predisposition to pre-eclampsia or eclampsia. In another example, a kit used for therapeutic monitoring can have a reference PAAI or soluble endoglin anti-angiogenesis index value or soluble endoglin value that is indicative of pre-eclampsia or eclampsia, wherein a decrease in the PAAI or soluble endoglin anti-angiogenesis index value or a decrease in the soluble endoglin value of the subject sample relative to the reference sample can be used to indicate therapeutic efficacy or effective dosages of therapeutic compounds. A standard curve of levels of purified protein within the normal or positive reference range, depending on the use of the kit, can also be included.

Therapeutics

The present invention features methods and compositions for treating or preventing pre-eclampsia or eclampsia in a subject. Given that levels of soluble endoglin are increased in subjects having pre-eclampsia, eclampsia, or having a predisposition to such conditions, any compound that decreases the expression levels and/or biological activity of a soluble endoglin polypeptide or nucleic acid molecule is useful in the methods of the invention. Such compounds include TGF-.beta.1, TGF-.beta.3, activin-A, BMP2, or BMP7, that can disrupt soluble endoglin binding to ligands; a purified antibody or antigen-binding fragment that specifically binds soluble endoglin; antisense nucleobase oligomers; and dsRNAs used to mediate RNA interference. Additional useful compounds include any compounds that can alter the biological activity of soluble endoglin, for example, as measured by an angiogenesis assay. Exemplary compounds and methods are described in detail below. These methods can also be combined with methods to decrease sFlt-1 levels or to increase VEGF or PlGF levels or decrease sFlt-1 levels as described in PCT Publication Number WO 2004/008946 and U.S. Patent Publication Nos. 20040126828 and 20050170444. In addition, any compound that increases the level or biological activity of TGF-.beta.1 or 3, eNOS, or PGI2. Exemplary compounds and methods are described in detail below. It should be noted the results described herein indicate that the soluble endoglin and sFlt-1 pathways may function in a cooperative manner to further the pathogenesis of pre-eclampsia or eclampsia. Therefore, the invention includes any combination of any of the methods or compositions described herein for the treatment or prevention of a pregnancy related hypertensive disorder. For example, a compound that targets the soluble endoglin pathway (e.g., downregulates soluble endoglin expression or biological activity or upregulates TGF-.beta., eNOS, or PGI.sub.2 expression or biological activity) can be used in combination with a compound that targets the sFlt-1 pathway (e.g., downregulates sFlt-1 expression or biological activity or upregulates VEGF or PlGF expression of biological activity) for the treatment or prevention of a pregnancy related hypertensive disorder.

Therapeutics Targeting the TGF-.beta. Signaling Pathway

TGF-.beta. is the prototype of a family of at least 25 growth factors which regulate growth, differentiation, motility, tissue remodeling, neurogenesis, wound repair, apoptosis, and angiogenesis in many cell types. TGF-.beta. also inhibits cell proliferation in many cell types and can stimulate the synthesis of matrix proteins. Unless evidenced from the context in which it is used, the term TGF-.beta. as used throughout this specification will be understood to generally refer to any and all members of the TGF-.beta. superfamily as appropriate. Soluble endoglin binds several specific members of the TGF-.beta. family including TGF-.beta.1, TGF-.beta.3, activin, BMP-2 and BMP-7, and may serve to deplete the developing fetus or placenta of these necessary mitogenic and angiogenic factor. The present invention features methods of increasing the levels of these ligands to bind to soluble endoglin and to neutralize the effects of soluble endoglin.

Soluble Endoglin Ligands as Therapeutic Compounds

In a preferred embodiment of the present invention, purified forms of any soluble endoglin ligand such as TGF-.beta. family proteins, including but not limited to TGF-.beta.1, TGF-.beta.3, activin-A, BMP2, and BMP7, are administered to the subject in order to treat or prevent pre-eclampsia or eclampsia.

Purified TGF-.beta. family proteins include any protein with an amino acid sequence that is homologous, more desirably, substantially identical to the amino acid sequence of TGF-.beta.1 or TGF-.beta.3, or any known TGF-.beta. family member, that can induce angiogenesis. Non-limiting examples include human TGF-.beta.1 (Cat #240-B-002) and human TGF-.beta.3 (Cat #243-B3-002) from R & D Systems, MN.

Therapeutic Compounds that Inhibit Proteolytic Cleavage of Endoglin

We have identified a potential cleavage site in the extracellular domain of endoglin where a proteolytic enzyme could cleave the membrane bound form of endoglin, releasing the extracellular domain as a soluble form. Our sequence alignments of the cleavage site suggest that a matrix metalloproteinase (MMP) may be responsible for the cleavage and release of soluble endoglin. Alternatively, a cathepsin or an elastase may also be involved in the cleavage event. MMPs are also known as collagenases, gelatinases, and stromelysins and there are currently 26 family members known (for a review see Whittaker and Ayscough, Cell Transmissions 17:1 (2001)). A preferred MMP is MMP9, which is known to be up-regulated in placentas from pre-eclamptic patients (Lim et al., Am. J Pathol. 151:1809-1818, 1997). The activity of MMPs is controlled through activation of pro-enzymes and inhibition by endogenous inhibitors such as the tissue inhibitors of metalloproteinases (TIMPS). Inhibitors of MMPs are zinc binding proteins. There are 4 known endogenous inhibitors (TIMP 1-4), which are reviewed in Whittaker et al., supra. One preferred MMP inhibitor is the inhibitor of membrane type-MMP1 that has been shown to cleave betaglycan, a molecule that shares similarity to enodglin (Velasco-Loyden et al., J. Biol. Chem. 279:7721-7733 (2004)). In addition, a variety of naturally-occurring and synthetic MMP inhibitors have been identified and are also reviewed in Whittaker et al., supra. Examples include antibodies directed to MMPs, and various compounds including marimastat, batimastat, CT1746, BAY 12-9566, Prinomastat, CGS-27023A, D9120, BMS275291 (Bristol Myers Squibb), and trocade, some of which are currently in clinical trials. Given the potential role of MMPs, cathepsins, or elastases in the release and up-regulation of soluble endoglin levels, the present invention also provides for the use of any compound, such as those described above, known to inhibit the activity of any MMP, cathepsin, or elastase involved in the cleavage and release of soluble endoglin, for the treatment or prevention of pre-eclampsia or eclampsia in a subject.

Therapeutic Compounds that Increase Soluble Endoglin Binding Proteins

The present invention provides for the use of any compound known to stimulate or increase blood serum levels of soluble endoglin binding proteins, including but not limited to TGF-.beta.1, TGF-.beta.3, activin-A, BMP2, and BMP7, for the treatment or prevention of pre-eclampsia in a subject. These compounds can be used alone or in combination with the purified proteins described above or any of the other methods used to increase TGF-.beta. family proteins protein levels described herein. In one example, cyclosporine is used at a dosage of 100-200 mg twice a day to stimulate TGF-.beta.1 production.

Therapeutic Compounds that Alter the Anti-Angiogenic Activity of Soluble Endoglin

Additional therapeutic compounds can be identified using angiogenesis assays. For example, pre-eclamptic serum having elevated levels of soluble endoglin are added to a matrigel tube formation assay will induce an anti-angiogenic state. Test compounds can then be added to the assay and a reversion in the anti-angiogenic state by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more indicates that the compound can reduce the biological activity of soluble endoglin and is useful as a therapeutic compound.

Therapeutic Compounds that Increase the Levels or Biological Activity of NOS

NOS is a complex enzyme containing several cofactors, a heme group which is part of the catalytic site, an N-terminal oxygenase domain, which belongs to the class of haem-thiolate proteins, and a C-terminal reductase domain which is homologous to NADPH:P450 reductase. NOS produces NO by catalysing a five-electron oxidation of a guanidino nitrogen of L-arginine (L-Arg).

eNOS activation involves a coordinated increase in Ser1177 phosphorylation and Thr495 dephosphorylation. We have discovered that TGF-.beta.1 dephosphorylates eNOS at Thr495, which is necessary to increase the Ca2+ sensitivity and enzyme activity and may work synergistically with VEGF, which activates eNOS by phosphorylating Ser1177.

Accordingly, any compound (e.g., polypeptide, nucleic acid molecule, small molecule compound, or antibody) that increases the level (e.g., by increasing stability, transcription or translation, or decreasing protein degradation) or biological activity of NOS, particularly eNOS, or any compound that prevents the downregulation of eNOS activity is useful in the methods of the invention. Such compounds include purified NOS, preferably eNOS, or biologically active fragments thereof, nucleic acids encoding NOS, preferably eNOS, or biologically active fragments thereof, statins, vanadate, hepatocyte growth factor, phosphoinositide 3-kinase (PI3K), Akt, VEGF, TGF-.beta.1, or any other compound that increases Ser1177 phosphorylation or Thr495 dephosphorylation or both. Nitric oxide is synthesized from L-arginine by nitric oxide synthase located in endothelial and other cells. Nitric oxide can also be generated by application of various nitric oxide donors such as sodium nitroprusside, nitroglycerin, SIN-1, isosorbid mononitrate, isosorbid dinitrate, and the like. Accordingly, compounds that increase (e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) the level or biological activity of NOS can optionally be administered in combination with L-arginine or a nitric oxide donor (e.g., sodium nitroprusside, nitroglycerin, isosorbidmononitrate, and isosorbo dinitrate). NOS activity can be assayed by standard methods known in the art, including but not limited to the citrulline assay and other assays described in U.S. Patent Application Publication No. 20050256199, the entire disclosure of which is herein incorporated by reference. The Thr495 residue of eNOS is located within the calmodulin (CaM)-binding domain of eNOS. Agonist-induced dephosphorylation of eNOS at Thr495 increases the binding of CaM to the enzyme (Fleming et al., Circ Res. 2001, 88: E68-75), thereby increasing its calcium sensitivity and activation. In addition to TGF-.beta.1 described herein, other agonists that have been shown to cause Thr495 dephosphorylation of eNOS including bradykinin, histamine and VEGF. Thr495 dephosphorylation can be enhanced by the protein kinase C (PKC) inhibitor Ro 31-8220 (Calbiochem) or after PKC downregulation using phorbol 12-myristate 13-acetate (PMA) (Sigma Aldrich). Moreover, agonist-induced dephosphorylation of Thr495 has been shown to be Ca.sup.2+/calmodulin-dependent and inhibitable by calyculin A (Sigma Aldrich), a protein phosphatase 1 (PP 1) inhibitor (Fleming I, et al. Circ Res. 2001, 88: E68-75). Additional compounds that effect eNOS dephosphorylation at Thr495 include histamine and bradykinin (Sigma Aldrich).

Therapeutic Compounds that Increase the Levels or Biological Activity of PGI.sub.2

Prostacyclin is a member of the family of lipid molecules known as eicosanoids. It is produced in endothelial cells from prostaglandin H2 (PGH2) by the action of the enzyme prostacyclin synthase. PGI.sub.2 biological activity includes inhibition of platelet aggregation, relaxation of smooth muscle, reduction of systemic and pulmonary vascular resistance by direct vasodilation, and natriuresis in kidney.

PGI.sub.2 is an anti-thrombotic factor that is stimulated by both VEGF and TGF-.beta.1. PGI.sub.2 biological activity includes inhibition of platelet aggregation and relaxation of vascular smooth muscle and assays for PGI.sub.2 biological activity include any platelet aggregation assay or other PGI2 assay known in the art such as those described in Jakubowski et al., Prostaglandins 47:404 (1994). The invention features the use of any compound that increases (e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) the level or activity of PGI.sub.2, as measured by standard assays known in the art including but not limited to PGI.sub.2 mimetics, iloprost, cicaprost, and aspirin. Additional compounds are known in the art and examples are described in U.S. Pat. No. 5,910,482, the entire disclosure of which is herein incorporated by reference.

Purified Proteins

For any of the purified proteins, or fragment thereof, the proteins are prepared using standard methods known in the art. Analogs or homologs of any of the therapeutic proteins described above are also included and can be constructed, for example, by making various substitutions of residues or sequences, deleting terminal or internal residues or sequences not needed for biological activity, or adding terminal or internal residues which may enhance biological activity. Amino acid substitutions, deletions, additions, or mutations can be made to improve expression, stability, or solubility of the protein in the various expression systems. Generally, substitutions are made conservatively and take into consideration the effect on biological activity. Mutations, deletions, or additions in nucleotide sequences constructed for expression of analog proteins or fragments thereof must, of course, preserve the reading frame of the coding sequences and preferably will not create complementary regions that could hybridize to produce secondary mRNA structures such as loops or hairpins which would adversely affect translation of the mRNA.

Any of the therapeutic compounds of the invention (e.g., polypeptide, antibodies, small molecule compounds) can also include any modified forms. Examples of post-translational modifications include but are not limited to phosphorylation, glycosylation, hydroxylation, sulfation, acetylation, isoprenylation, proline isomerization, subunit dimerization or multimerization, and cross-linking or attachment to any other proteins, or fragments thereof, or membrane components, or fragments thereof (e.g., cleavage of the protein from the membrane with a membrane lipid component attached). Modifications that provide additional advantages such as increased affinity, decreased off-rate, solubility, stability and in vivo or in vitro circulating time of the polypeptide, or decreased immunogenicity and include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, Creighton, "Proteins: Structures and Molecular Properties," 2d Ed., W. H. Freeman and Co., N.Y., 1992; "Postranslational Covalent Modification of Proteins," Johnson, ed., Academic Press, New York, 1983; Seifter et al., Meth. Enzymol., 182:626-646, 1990; Rattan et al., Ann. NY Acad. Sci., 663:48-62, 1992) are also included. The peptidyl therapeutic compound of the invention can also include sequence variants of any of the compounds such as variants that include 1, 2, 3, 4, 5, greater than 5, or greater than 10 amino acid alterations such as substitutions, deletions, or insertions with respect to wild type sequence. Additionally, the therapeutic compound of the invention may contain one or more non-classical amino acids. Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, .alpha.-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, omithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, .beta.-alanine, fluoro-amino acids, designer amino acids such as .beta.-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

Additional post-translational modifications encompassed by the invention include, for example, e.g., N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue.

In addition, chemically modified derivatives of the therapeutic compounds described herein, which may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see U.S. Pat. No. 4,179,337) are also included. The chemical moieties for derivitization may be selected from water soluble polymers such as, for example, polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like. The compound may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.

The polymer may be of any molecular weight, and may be branched or unbranched. For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 kDa (the term "about" indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing. Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog). As noted above, the polyethylene glycol may have a branched structure. Branched polyethylene glycols are described, for example, in U.S. Pat. No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72, (1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750, (1999); and Caliceti et al., Bioconjug. Chem. 10:638-646, (1999), the disclosures of each of which are incorporated by reference.

Any of the therapeutic compounds of the present invention (e.g., polypeptide, antibodies, or small molecule compounds) may also be modified in a way to form a chimeric molecule comprising the therapeutic compound fused to another, heterologous polypeptide or amino acid sequence, such as an Fc sequence, a detectable label, or an additional therapeutic molecule. In one example, an anti-soluble endoglin antibody can be a peptide fused to an Fc fusion protein.

For any of the polypeptides, including antibodies, that are used in the methods of the invention, the nucleic acids encoding the polypeptides or antibodies, or fragments thereof, are also useful in the methods of the invention using standard techniques for gene therapy known in the art and described herein. The invention also includes mimetics, based on modeling the 3-dimensional structure of a polypeptide or peptide fragment and using rational drug design to provide potential inhibitor compounds with particular molecular shape, size and charge characteristics. Following identification of a therapeutic compound, suitable modeling techniques known in the art can be used to study the functional interactions and design mimetic compounds which contain functional groups arranged in such a manner that they could reproduced those interactions. The designing of mimetics to a known pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a lead compound. This might be desirable where the active compound is difficult or expensive to synthesize or where it is unsuitable for a particular method of administration, e.g. peptides are not well suited as active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal. Mimetic design, synthesis and testing may be used to avoid randomly screening large number of molecules for a target property. The mimetic or mimetics can then be screened to see whether they reduce or inhibit soluble endoglin levels or biological activity and further optimization or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.

Therapeutic Nucleic Acids

Recent work has shown that the delivery of nucleic acid (DNA or RNA) capable of expressing an endothelial cell mitogen such as VEGF to the site of a blood vessel injury will induce proliferation and reendothelialization of the injured vessel. While the present invention does not relate to blood vessel injury, these general techniques for the delivery of nucleic acid to endothelial cells can be used in the present invention for the delivery of nucleic acids encoding soluble endoglin binding proteins, such as TGF-.beta.1, TGF-.beta.3, activin-A, BMP2 and BMP7, or eNOS. The techniques can also be used for the delivery of nucleic acids encoding proteins, such as those described above, known to inhibit the activity of any MMP, cathepsin, or elastase involved in the cleavage and release of soluble endoglin, for the treatment or prevention of pre-eclampsia or eclampsia in a subject. These general techniques are described in U.S. Pat. Nos. 5,830,879 and 6,258,787 and are incorporated herein by reference.

In the present invention the nucleic acid may be any nucleic acid (DNA or RNA) including genomic DNA, cDNA, and mRNA, encoding a soluble endoglin binding proteins such as TGF-.beta.1, TGF-.beta.3, activin-A, BMP2 and BMP7, or eNOS. The nucleic acids encoding the desired protein may be obtained using routine procedures in the art, e.g. recombinant DNA, PCR amplification.

Modes for Delivering Nucleic Acids

For any of the nucleic acid applications described herein, standard methods for administering nucleic acids can be used. Examples are described in U.S. Patent Application Publication No. 20060067937 and PCT Publication No. WO 06/034507.

Therapeutic Nucleic Acids that Inhibit Soluble Endoglin Expression

The present invention also features the use of antisense nucleobase oligomers to downregulate expression of soluble endoglin mRNA directly. By binding to the complementary nucleic acid sequence (the sense or coding strand), antisense nucleobase oligomers are able to inhibit protein expression presumably through the enzymatic cleavage of the RNA strand by RNAse H. Preferably the antisense nucleobase oligomer is capable of reducing soluble endoglin protein expression in a cell that expresses increased levels of soluble endoglin. Preferably the decrease in soluble endoglin protein expression is at least 10% relative to cells treated with a control oligonucleotide, preferably 20% or greater, more preferably 40%, 50%, 60%, 70%, 80%, 90% or greater. Methods for selecting and preparing antisense nucleobase oligomers are well known in the art. For an example of the use of antisense nucleobase oligomers to downregulate VEGF expression see U.S. Pat. No. 6,410,322, incorporated herein by reference. Methods for assaying levels of protein expression are also well known in the art and include western blotting, immunoprecipitation, and ELISA.

The present invention also features the use of RNA interference (RNAi) to inhibit expression of soluble endoglin. RNA interference (RNAi) is a recently discovered mechanism of post-transcriptional gene silencing (PTGS) in which double-stranded RNA (dsRNA) corresponding to a gene or mRNA of interest is introduced into an organism resulting in the degradation of the corresponding mRNA. In the RNAi reaction, both the sense and anti-sense strands of a dsRNA molecule are processed into small RNA fragments or segments ranging in length from 21 to 23 nucleotides (nt) and having 2-nucleotide 3' tails. Alternatively, synthetic dsRNAs, which are 21 to 23 nt in length and have 2-nucleotide 3' tails, can be synthesized, purified and used in the reaction. These 21 to 23 nt dsRNAs are known as "guide RNAs" or "short interfering RNAs" (siRNAs).

The siRNA duplexes then bind to a nuclease complex composed of proteins that target and destroy endogenous mRNAs having homology to the siRNA within the complex. Although the identity of the proteins within the complex remains unclear, the function of the complex is to target the homologous mRNA molecule through base pairing interactions between one of the siRNA strands and the endogenous mRNA. The mRNA is then cleaved approximately 12 nt from the 3' terminus of the siRNA and degraded. In this manner, specific genes can be targeted and degraded, thereby resulting in a loss of protein expression from the targeted gene. siRNAs can also be chemically synthesized or obtained from a company that chemically synthesizes siRNAs (e.g., Dharmacon Research Inc., Pharmacia, or ABI).

The specific requirements and modifications of dsRNA are described in PCT Publication No. WO01/75164, and in U.S. Patent Application Publication No. 20060067937 and PCT Publication No. WO 06/034507, incorporated herein by reference.

Soluble Endoglin Based Therapeutic Compounds Useful in Early Pregnancy

Inhibition of full-length endoglin signaling has been shown to enhance trophoblast invasiveness in villous explant cultures (Caniggia I et al, Endocrinology, 1997, 138:4977-88). Soluble endoglin is therefore likely to enhance trophoblast invasiveness during early pregnancy. Accordingly, compositions that increase soluble endoglin levels early in pregnancy in a woman who does not have a pregnancy related hypertensive disorder or a predisposition to a pregnancy related hypertensive disorder may be beneficial for enhancing placentation. Examples of compositions that increase soluble endoglin levels include purified soluble endoglin polypeptides, soluble endoglin encoding nucleic acid molecules, and compounds or growth factors that increase the levels or biological activity of soluble endoglin.

Assays for Gene and Protein Expression

The following methods can be used to evaluate protein or gene expression and determine efficacy for any of the above-mentioned methods for increasing soluble endoglin binding protein levels, or for decreasing soluble endoglin protein levels.

Blood serum from the subject is measured for levels of soluble endoglin, using methods such as ELISA, western blotting, or immunoassays using specific antibodies. Blood serum from the subject can also be measured for levels of TGF-.beta.1, TGF-.beta.3, activin-A, BMP2, BMP7, or any protein ligand known to bind to soluble endoglin. Methods used to measure serum levels of proteins include ELISA, western blotting, or immunoassays using specific antibodies. In addition, in vitro angiogenesis assays can be performed to determine if the subject's blood has converted from an anti-angiogenic state to a pro-angiogenic state. Such assays are described above in Example 4. A result that is diagnostic of pre-eclampsia or eclampsia is considered an increase of at least 10%, 20%, preferably 30%, more preferably at least 40% or 50%, and most preferably at least 60%, 70%, 80%, 90% or more in the levels of soluble endoglin and a result indicating an improvement in the pre-eclampsia or eclampsia is a decrease of at least 10%, 20%, preferably 30%, more preferably at least 40% or 50%, and most preferably at least 60%, 70%, 80%, 90% or more in the levels of soluble endoglin. Alternatively or additionally, a result that is diagnostic of pre-eclampsia or eclampsia is considered a decrease of at least 10%, 20%, preferably 30%, more preferably at least 40% or 50%, and most preferably at least 60%, 70%, 80%, 90% or more in the levels of eNOS, PGI.sub.2, TGF-.beta.1, TGF-.beta.3, activin-A, BMP2, BMP7, or any protein ligand known to bind to soluble endoglin and a result indicating an improvement in the pre-eclampsia or eclampsia is an increase of at least 10%, 20%, preferably 30%, more preferably at least 40% or 50%, and most preferably at least 60%, 70%, 80%, 90% or more in the levels of eNOS, PGI.sub.2, TGF-.beta.1, TGF-.beta.3, activin-A, BMP2, BMP7, or any protein ligand known to bind to soluble endoglin. A result indicating an improvement in the pre-eclampsia or eclampsia can also be considered conversion by at least 10%, preferably 20%, 30%, 40%, 50%, and most preferably at least 60%, 70%, 80%, 90% or more from an anti-angiogenic state to a pro-angiogenic state using the in vitro angiogenesis assay.

Blood serum or urine samples from the subject can also be measured for levels of nucleic acids or polypeptides encoding eNOS, TGF-.beta.1, TGF-.beta.3, activin-A, BMP2, BMP7, or soluble endoglin. There are several art-known methods to assay for gene expression. Some examples include the preparation of RNA from the blood samples of the subject and the use of the RNA for northern blotting, PCR based amplification, or RNAse protection assays. A positive result is considered an increase of at least 10%, 20%, preferably 30%, more preferably at least 40% or 50%, and most preferably at least 60%, 70%, 80%, 90% or more in the levels of soluble endoglin, TGF-.beta.1, TGF-.beta.3, activin-A, BMP2, BMP7 nucleic acids.

Use of Antibodies for Therapeutic Treatment

The elevated levels of soluble endoglin found in the serum samples taken from pregnant women suffering from pre-eclampsia suggests that soluble endoglin is acting as a "physiologic sink" to bind to and deplete the trophoblast cells and maternal endothelial cells of functional growth factors required for the proper development and angiogenesis of the fetus or the placenta. The use of compounds, such as antibodies, to bind to soluble endoglin and neutralize the activity of soluble endoglin (e.g., binding to TGF-.beta.1, TGF-.beta.3, activin-A, BMP2, BMP7), may help prevent or treat pre-eclampsia or eclampsia, by producing an increase in free TGF-.beta.1, TGF-.beta.3, activin-A, BMP2, and BMP7. Such an increase would allow for an increase in trophoblast proliferation, migration and angiogenesis required for placental development and fetal nourishment, and for systemic maternal endothelial cell health.

The present invention provides antibodies that bind specifically to soluble endoglin. Preferably, the antibodies bind to the extracellular domain of endoglin or to the ligand binding domain. The antibodies are used to neutralize the activity of soluble endoglin and the most effective mechanism is believed to be through direct blocking of the binding sites for TGF-.beta.1, TGF-.beta.3, activin-A, BMP2, or BMP7, however, other mechanisms cannot be ruled out. Preferred antibodies can bind to any one or more of the peptide sequences indicated in bold and underlined in FIG. 30B or to any of the preferred fragments of soluble endoglin (e.g., amino acids 1 to 437, 4 to 437, 40 to 406, or 1 to 587 of human endoglin). Methods for the preparation and use of antibodies for therapeutic purposes are described in several patents including U.S. Pat. Nos. 6,054,297; 5,821,337; 6,365,157; and 6,165,464; U.S. Patent Application Publication No. 2006/0067937; and PCT Publication No. WO 06/034507 and are incorporated herein by reference. Antibodies can be polyclonal or monoclonal; monoclonal humanized antibodies are preferred.

Therapeutic Uses of Antibodies

When used in vivo for the treatment or prevention of pre-eclampsia or eclampsia, the antibodies of the subject invention are administered to the subject in therapeutically effective amounts. Preferably, the antibodies are administered parenterally or intravenously by continuous infusion. The dose and dosage regimen depends upon the severity of the disease, and the overall health of the subject. The amount of antibody administered is typically in the range of about 0.001 to about 10 mg/kg of subject weight, preferably 0.01 to about 5 mg/kg of subject weight.

For parenteral administration, the antibodies are formulated in a unit dosage injectable form (solution, suspension, emulsion) in association with a pharmaceutically acceptable parenteral vehicle. Such vehicles are inherently nontoxic, and non-therapeutic. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Nonaqueous vehicles such as fixed oils and ethyl oleate may also be used. Liposomes may be used as carriers. The vehicle may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives. The antibodies typically are formulated in such vehicles at concentrations of about 1 mg/ml to 10 mg/ml.

Combination Therapies

Optionally, a therapeutic may be administered in combination with any other standard pre-eclampsia or eclampsia therapy; such methods are known to the skilled artisan and include the methods described in U.S. Patent Application Publication Numbers 20040126828, 20050025762, 20050170444, and 2006/0067937 and PCT Publication Numbers WO 2004/008946, WO 2005/077007, and WO 06/034507.

Desirably, the invention features the use of a combination of any one or more of the therapeutic agents described herein. Given our discovery that soluble endoglin and sFlt-1 may act in concert to induce vascular damage and pregnancy related hypertensive disorders by interfering with TGF-.beta.1 and VEGF signaling pathway respectively, possibly converging on the NOS signaling pathway, therapeutic methods of the invention include the administration of a compound that decrease sFlt-1 levels or activity or increase VEGF or PlGF levels or activity in combination with a compound that decreases soluble endoglin levels or activity or increase TGF-.beta., NOS, or PGI2 levels or activity. It will be understood by the skilled artisan that any combination of any of the agents can be used for this purpose. For example, an antibody that specifically binds to soluble endoglin can be administered in combination with VEGF. In another example, a compound that increases TGF-.beta.1 levels or activity can be administered in combination with a compound that increases VEGF or PlGF in order to target both the endoglin and the VEGF pathway. Alternatively, a combination of antibodies against both soluble endoglin and sFlt-1 may be used either directly or in an ex vivo approach (e.g., using a column that is lined with anti-soluble endoglin or sFlt-1 and circulating the patient's blood through the column). Any of these combinations can further include the administration of a compound that increases NOS levels or activity, preferably eNOS, in order to regulate the pathway downstream of the respective receptors.

In addition, the invention provides for the use of any chronic hypertension medications used in combination with any of the therapeutic methods described herein. Medications used for the treatment of hypertension during pregnancy include methyldopa, hydralazine hydrochloride, or labetalol. For each of these medications, modes of administration and dosages are determined by the physician and by the manufacturer's instructions.

Dosages and Modes of Administration

Preferably, the therapeutic is administered during pregnancy for the treatment or prevention of pre-eclampsia or eclampsia or after pregnancy to treat post-partum pre-eclampsia or eclampsia. Techniques and dosages for administration vary depending on the type of compound (e.g., chemical compound, purified protein, antibody, antisense, RNAi, or nucleic acid vector) and are well known to those skilled in the art or are readily determined.

Therapeutic compounds of the present invention may be administered with a pharmaceutically acceptable diluent, carrier, or excipient, in unit dosage form. Administration may be parenteral, intravenous, subcutaneous, oral or local by direct injection into the amniotic fluid. Intravenous delivery by continuous infusion is the preferred method for administering the therapeutic compounds of the present invention. The therapeutic compound may be in form of a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation, or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use.

The composition can be in the form of a pill, tablet, capsule, liquid, or sustained release tablet for oral administration; or a liquid for intravenous, subcutaneous or parenteral administration; or a polymer or other sustained release vehicle for local administration.

Methods well known in the art for making formulations are found, for example, in "Remington: The Science and Practice of Pharmacy" (20th ed., ed. A. R. Gennaro A R., 2000, Lippincott Williams & Wilkins, Philadelphia, Pa.). Formulations for parenteral administration may, for example, contain excipients, sterile water, saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Nanoparticulate formulations (e.g., biodegradable nanoparticles, solid lipid nanoparticles, liposomes) may be used to control the biodistribution of the compounds. Other potentially useful parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. The concentration of the compound in the formulation varies depending upon a number of factors, including the dosage of the drug to be administered, and the route of administration.

The compound may be optionally administered as a pharmaceutically acceptable salt, such as non-toxic acid addition salts or metal complexes that are commonly used in the pharmaceutical industry. Examples of acid addition salts include organic acids such as acetic, lactic, pamoic, maleic, citric, malic, ascorbic, succinic, benzoic, palmitic, suberic, salicylic, tartaric, methanesulfonic, toluenesulfonic, or trifluoroacetic acids or the like; polymeric acids such as tannic acid, carboxymethyl cellulose, or the like; and inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid phosphoric acid, or the like. Metal complexes include zinc, iron, and the like.

Formulations for oral use include tablets containing the active ingredient(s) in a mixture with non-toxic pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents or fillers (e.g., sucrose and sorbitol), lubricating agents, glidants, and anti-adhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc).

Formulations for oral use may also be provided as chewable tablets, or as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium.

The dosage and the timing of administering the compound depends on various clinical factors including the overall health of the subject and the severity of the symptoms of pre-eclampsia. In general, once pre-eclampsia or a predisposition to pre-eclampsia is detected, continuous infusion of the purified protein is used to treat or prevent further progression of the condition. Treatment can be continued for a period of time ranging from 1 to 100 days, more preferably 1 to 60 days, and most preferably 1 to 20 days, or until the completion of pregnancy. Dosages vary depending on each compound and the severity of the condition and are titrated to achieve a steady-state blood serum concentration ranging from 10 to 20 ng/ml soluble endoglin; and/or 1 to 500 pg/mL free VEGF or free PlGF, or both, preferably 1 to 100 pg/mL, more preferably 5 to 50 pg/mL and most preferably 5 to 10 pg/mL VEGF or PlGF, or 1-5 ng of sFlt-1.

The diagnostic methods described herein can be used to monitor the pre-eclampsia or eclampsia during therapy or to determine the dosages of therapeutic compounds. In one example, a therapeutic compound is administered and the PAAI is determined during the course of therapy. If the PAAI is less than 20, preferably less than 10, then the therapeutic dosage is considered to be an effective dosage. In another example, a therapeutic compound is administered and the soluble endoglin anti-angiogenic index is determined during the course of therapy. If the soluble endoglin anti-angiogenic index is less than 200, preferably less than 100, then the therapeutic dosage is considered to be an effective dosage.

Subject Monitoring

The disease state or treatment of a subject having pre-eclampsia, eclampsia, or a predisposition to such a condition can be monitored using the diagnostic methods, kits, and compositions of the invention. For example, the expression of a soluble endoglin polypeptide present in a bodily fluid, such as urine, plasma, amniotic fluid, or CSF, can be monitored. The soluble endoglin monitoring can be combined with methods for monitoring the expression of an sFlt-1, VEGF, or PlGF, TGF-.beta., or eNOS polypeptide or nucleic acid, or PGI2. Such monitoring may be useful, for example, in assessing the efficacy of a particular drug in a subject or in assessing disease progression. Therapeutics that decrease the expression or biological activity of a soluble endoglin nucleic acid molecule or polypeptide are taken as particularly useful in the invention.

Screening Assays

As discussed above, the level of a soluble endoglin nucleic acid or polypeptide is increased in a subject having pre-eclampsia, eclampsia, or a predisposition to such conditions. Based on these discoveries, compositions of the invention are useful for the high-throughput low-cost screening of candidate compounds to identify those that modulate the expression of a soluble endoglin polypeptide or nucleic acid molecule whose expression is altered in a subject having a pre-eclampsia or eclampsia.

Any number of methods are available for carrying out screening assays to identify new candidate compounds that alter the expression of a soluble endoglin nucleic acid molecule. Examples are described in detail in U.S. Patent Application Publication No. 20060067937 and PCT Publication No. WO 06/034507.

In one working example, candidate compounds may be screened for those that specifically bind to a soluble endoglin polypeptide. The efficacy of such a candidate compound is dependent upon its ability to interact with such a polypeptide or a functional equivalent thereof. Such an interaction can be readily assayed using any number of standard binding techniques and functional assays such as immunoassays or affinity chromatography based assays (e.g., those described in Ausubel et al., supra). In one embodiment, a soluble endoglin polypeptide is immobilized and compounds are tested for the ability to bind to the immobilized soluble endoglin using standard affinity chromatography based assays. Compounds that bind to the immobilized soluble endoglin can then be eluted and purified and tested further for its ability to bind to soluble endoglin both in vivo and in vitro or its ability to inhibit the biological activity of soluble endoglin.

In another example, a candidate compound is tested for its ability to decrease the biological activity of a soluble endoglin polypeptide by decreasing binding of a soluble endoglin polypeptide and a growth factor, such as TGF-.beta.1, TGF-.beta.3, activin-A, BMP-2 and BMP-7. These assays can be performed in vivo or in vitro and the biological activity of the soluble endoglin polypeptide can be assayed using any of the assays for any of the soluble endoglin activities known in the art or described herein. For example, cells can be incubated with a Smad2/3-dependent reporter construct. If desired, the cells can also be incubated in the presence of TGF-.beta. to enhance the signal on the Smad2/3 dependent reporter construct. The cells can then be incubated in the presence of soluble endoglin which will reduce or inhibit TGF-.beta.-induced activation of the Smad2/3 dependent reporter construct. Candidate compounds can be added to the cell and any compound that results in an increase of TGF-.beta.-induced activation of the Smad2/3 dependent reporter in the soluble endoglin treated cells as compared to cells not treated with the compound, is considered a compound that may be useful for the treatment of pre-eclampsia or eclampsia.

In another example, the TGF-.beta.-induced dephosphorylation of eNOS at Thr495 can also be used as an assay for changes in soluble endoglin biological activity. In this example, cells are incubated in the presence of soluble endoglin, which as shown in the experiments described below, inhibits the TGF-.beta.1 dephosphorylation of Thr495 of eNOS. Candidate compounds are then added to the cells and the phosphorylation state of Thr495 is determined. Any compound that results in an increase of TGF-.beta.-induced activation of Thr495 dephosphorylation in the soluble endoglin treated cells as compared to cells not treated with the compound, is considered a compound that may be useful for the treatment of pre-eclampsia or eclampsia.
 

Claim 1 of 37 Claims

1. A method of treating a pregnancy related hypertensive disorder in a subject on or after the 20.sup.th week of pregnancy, said method comprising the step of administering to said subject (i) a compound capable of decreasing soluble endoglin expression levels or soluble endoglin biological activity, wherein said compound is an antibody that specifically binds soluble endoglin, or a soluble endoglin antigen binding fragment thereof, or a growth factor that binds to soluble endoglin and (ii) a compound capable of decreasing sFlt-1 expression levels or sFlt-1 biological activity, wherein said compound is an antibody that specifically binds sFlt-1, or an sFlt-1 antigen binding fragment thereof, or a growth factor that binds to sFlt-1, wherein said administering is on or after the 20.sup.th week of the pregnancy of said subject and wherein said administering is for a time and in an amount sufficient to treat said pregnancy related hypertensive disorder in said subject.

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