Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
  • A61K31/737—Sulfated polysaccharides, e.g. chondroitin sulfate, dermatan sulfate
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/74—Synthetic polymeric materials
  • A61K31/765—Polymers containing oxygen
  • A61K31/78—Polymers containing oxygen of acrylic acid or derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P13/00—Drugs for disorders of the urinary system
  • A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/10—Antioedematous agents; Diuretics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/12—Antihypertensives

Definitions

• this invention relates to the extracorporeal treatment, and devices for the extracorporeal treatment, of subjects having a pregnancy related hypertensive disorder.
• Pre-eclampsia is a syndrome of hypertension, edema, and proteinuria that affects 5 to 10% of pregnancies and results in substantial maternal and fetal morbidity and mortality.
• Pre-eclampsia accounts for at least 200,000 maternal deaths worldwide per year.
• the symptoms of pre-eclampsia typically appear after the 20 th week of pregnancy and are usually detected by routine measuring of the woman's blood pressure and urine.
• these monitoring methods are ineffective for diagnosis of the syndrome at an early stage, which could reduce the risk to the subject or developing fetus, if an effective treatment were available.
• Pre-eclampsia can vary in severity from mild to life threatening.
• a mild form of pre-eclampsia can be treated with bed rest and frequent monitoring.
• hospitalization is recommended and blood pressure medication or anticonvulsant medications to prevent seizures are prescribed. If the condition becomes life threatening to the mother or the baby the pregnancy is terminated and the baby is delivered pre-term.
• VEGF vascular endothelial growth factor
• sFlt-1 soluble Flt-1 receptor
• P1GF placental growth factor
• VEGF is an endothelial cell-specific mitogen, an angiogenic inducer, and a mediator of vascular permeability.
• VEGF has also been shown to be important for glomerular capillary repair.
• VEGF binds as a homodimer to one of two homologous membrane-spanning tyrosine kinase receptors, the fms-like tyrosine kinase (Flt-1) and the kinase domain receptor (KDR), which are differentially expressed in endothelial cells obtained from many different tissues.
• Flt- 1 but not KDR, is highly expressed by trophoblast cells which contribute to placental formation.
• PIGF is a VEGF family member that is also involved in placental development. PIGF is expressed by cytotrophoblasts and syncytiotrophoblasts and is capable of inducing proliferation, migration, and activation of endothelial cells.
• PIGF binds as a homodimer to the Flt-1 receptor, but not the KDR receptor. Both PIGF and VEGF contribute to the mitogenic activity and angiogenesis that are critical for the developing placenta.
• Soluble Flt-1 (sFlt-1), which lacks the transmembrane and cytoplasmic domains of the receptor, binds to VEGF with a high affinity but does not stimulate mitogenesis of endothelial cells. Careful regulation of angiogenic and mitogenic signaling pathways is critical for maintaining appropriate proliferation, migration, and angiogenesis by trophoblast cells in the developing placenta.
• the present invention is based on the discovery of extracorporeal methods for treating pregnancy related hypertensive disorders, including pre-eclampsia and eclampsia.
• the invention also features extracorporeal devices that are useful for the treatment of pregnancy-related hypertensive disorders.
• sFlt-1 is known to antagonize VEGF and PIGF by acting as a
• sFlt-1 may be depleting the placenta of necessary amounts of these essential angiogenic and mitogenic factors. Excess sFlt-1 may also lead to pre-eclampsia or eclampsia by disrupting the endothelial cells that maintain the blood-brain barrier and/or endothelial cells lining - the choroid plexus of the brain thus leading to cerebral edema and the seizures seen in eclampsia. Methods that counter the effects of excess sFlt-1, allowing for an increase in the essential angiogenic and mitogenic factors, are useful for the treatment of pregnancy related hypertensive disorders.
• hypertension including pre-eclampsia.
• the invention features a method of treating or ameliorating at least one symptom of a pregnancy related hypertensive disorder in a pregnant subject (e.g., a pregnant subject having elevated sFlt-1) which includes the step of extracorporeally removing sFlt-1 from the pregnant subject using an apheresis procedure, wherein the apheresis procedure includes the use of an agent (e.g., negatively charged polymer or polysaccharide) that binds to positively charged proteins (e.g., lipoprotein, apolipoprotein, fibrinogen, globulin, and C reactive protein), and wherein the apheresis procedure is sufficient to treat or ameliorate at least one symptom of the pregnancy related hypertensive disorder.
• an agent e.g., negatively charged polymer or polysaccharide
• positively charged proteins e.g., lipoprotein, apolipoprotein, fibrinogen, globulin, and C reactive protein
• the invention features a method for reducing the level of sFlt-1 in a bodily fluid of a subject that includes the step of extracorporeally removing sFlt-1 from the pregnant subject using an apheresis procedure, wherein the apheresis procedure includes an agent that binds to positively charged proteins (e.g., lipoprotein, apolipoprotein, fibrinogen, globulin, and C reactive protein), and wherein the apheresis procedure is sufficient to reduce the level of sFlt-1 in a subject in need thereof.
• positively charged proteins e.g., lipoprotein, apolipoprotein, fibrinogen, globulin, and C reactive protein
• the invention features an agent that binds positively charged proteins for use in a method for the treatment of a pregnancy related hypertensive disorder in a pregnant subject, wherein the agent is bound to an extracorporeal device and wherein the method includes an apheresis procedure that includes apheresis of a bodily fluid with the help of the extracorporeal device and wherein the apheresis results in the removal of sFlt-1 from the bodily fluid.
• the agent includes dextran sulfate or anionic polyacrylate.
• the agent that binds to positively charged proteins does not include heparin.
• the pregnant subject has an increase in the sFlt-1 level as compared to a normal reference, standard, or level and the agent includes heparin.
• the apheresis procedure includes drawing the pregnant subject's blood and directing the blood through an extracorporeal circuit that includes the agent.
• the invention features an in vitro method for the removal of sFlt-1 from a bodily fluid from a pregnant subject having a pregnancy related hypertensive disorder, wherein the method includes the apheresing of the bodily fluid using an extracorporeal device having the agent bound thereto and wherein the apheresing results in the removal of sFlt-1 from the body fluid.
• the agent includes dextran sulfate or anionic polyacrylate.
• the agent that binds to positively charged proteins does not include heparin.
• the pregnant subject has an increase in the sFlt-1 level as compared to a normal reference, standard, or level and the agent includes heparin.
• the apheresis procedure includes drawing the pregnant subject's blood and directing the blood through an extracorporeal circuit that includes the agent.
• the invention features the use of an autologous bodily fluid from which sFlt-1 has been reduced ex vivo in the manufacture of a medicament for treating a pregnancy related hypertensive disorder in a pregnant subject.
• the sFlt-1 has been reduced by an apheresis procedure, wherein the apheresis procedure includes the use of an agent that binds to positively charged proteins.
• the agent includes dextran sulfate or anionic polyacrylate.
• the agent that binds to positively charged proteins does not include heparin.
• the pregnant subject has an increase in the sFlt-1 level as compared to a normal reference, standard, or level and the agent is heparin.
• the apheresis procedure includes drawing the pregnant subject's blood and directing the blood through an extracorporeal circuit that includes the agent.
• the autologous bodily fluid is a pregnant subject's blood and the sFlt-1 is removed by directing the blood through an extracorporeal circuit that includes the agent that binds positively charged proteins.
• the agent includes dextran sulfate or anionic polyacrylate, or does not include heparin.
• the pregnant subject has an increase in the sFlt-1 level as compared to a normal reference, standard, or level and the agent includes heparin.
• the sFlt-1 level in the bodily fluid is reduced to 5 ng/ml or less. In another embodiment, the sFlt-1 level in the bodily fluid is reduced to 2 ng/ml or less. In another embodiment, the sFlt-1 level in the bodily fluid is reduced to 1 ng/ml or less.
• the apheresis procedure includes the step of drawing the pregnant subject's blood and directing the blood through an extracorporeal circuit that includes an agent that binds to positively charged proteins (e.g., lipoprotein, apolipoprotein, fibrinogen, globulin, and C reactive protein).
• positively charged proteins e.g., lipoprotein, apolipoprotein, fibrinogen, globulin, and C reactive protein.
• the agent that binds to a positively charged protein includes a negatively charged polymer or a negatively charged polysaccharide (e.g., sulfonated polysaccharide, polystyrene sulfonic acid, and polyacrylic acid).
• the negatively charged polymer includes polystyrene sulfonic acid or polyacrylic acid.
• the negatively charged polysaccharide includes sulfonated polysaccharide.
• Non-limiting examples include a polysaccharide that includes dextran sulfate, hyaluronic acid, chondroitin sulfate, keratin sulfate, or heparin sulfate, particularly wherein the pregnant subject has elevated sFlt-1 levels.
• the agent is immobilized with a water-insoluble porous carrier that includes a hydrophilic porous carrier (e.g., polysaccharide, hydrophilic polymer).
• a hydrophilic porous carrier e.g., polysaccharide, hydrophilic polymer
• the polysaccharide include cellulose, pectin, chitin, agarose, carrageenan, and dextran.
• the hydrophilic polymer include polyethylene glycol, polyvinyl alcohol, polyacrylic acid, and silica gel.
• the water insoluble porous carrier includes cellulose or polyacrylic acid.
• the agent is packed in a cartridge includes the apheresis column.
• apheresis columns that can be used in various embodiments of the invention include a LIPOSORBER column (dextran sulfate; Kaneka); a LIPOSORBER LA-15 column (dextran sulfate; Kaneka); a LIPOSORBER D column (dextran sulfate; Kaneka); a H.E.L.P column, a Therasorb column, or a DALI column (anionic polyacrylate; Fresenius SE).
• the apheresis column is a dextran sulfate or anionic polyacrylate based column.
• the apheresis procedure is done one time, two times, three time, four times, five times, six times, seven times, eight times, nine times, ten times, or as many times as needed during the pregnancy.
• the apheresis procedure reduces sFlt-1 levels in the subject by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more.
• the apheresis procedure reduces sFlt-1 levels in the subject by greater than 61%. The reduction may be a total reduction after all apheresis procedures are complete or a reduction after a single apheresis procedure.
• the apheresis procedure reduces the sFlt-1 levels in the subject to the sFlt-1 levels in a normal control or reference that is matched for gestational age. In another embodiment of any of the above aspects, the apheresis procedure reduces the sFlt-1 levels in the subject to within 20%, 10%, 5%, or 2% of the sFlt-1 levels in a normal control or reference that is matched for gestational age.
• the pregnant subject has more than one symptom of a pregnancy related hypertensive disorder.
• symptoms of a pregnancy related hypertensive disorder are known in the art or described herein.
• the pregnant subject may have an increase in the sFlt-1 level or a decrease in the VEGF or P1GF protein levels as compared to a normal reference, standard, or level.
• the pregnant subject has increased sF]t-l levels relative to a normal reference, standard, or level.
• the normal reference may be a prior sample from the subject or a sample from a subject that does not have a pregnancy related hypertensive disorder.
• the pregnant subject has an sFlt- 1 level of at least 2 ng/ml or at least 5 ng/ml prior to the administering of the apheresis procedure.
• the pregnant subject is at least 17,
• the subject may also be a post-partum subject.
• the pregnant subject may be diagnosed with or at risk of developing pregnancy related hypertensive disorder.
• pregnancy related hypertensive disorders include preeclampsia, eclampsia, gestational hypertension, chronic hypertension, HELLP syndrome, and pregnancy with a small for gestational age (SGA) fetus.
• the pregnancy related hypertensive disorder is pre-eclampsia or eclampsia.
• the method further includes monitoring the pregnancy related hypertensive disorder (e.g., pre-eclampsia or eclampsia) in the subject by measuring the level of sFlt-1 , free VEGF, free P1GF, or soluble endoglin polypeptide in a bodily fluid sample from the subject.
• pregnancy related hypertensive disorder e.g., pre-eclampsia or eclampsia
• the level of sFlt- 1 or soluble endoglin is measured and a decrease in the level of sFlt-1 or soluble endoglin after apheresis as compared to an sFlt-1 or soluble endoglin positive reference, standard, or level indicates an improvement in the pregnancy related hypertensive disorder in the subject.
• the positive reference is from a sample from the same subject earlier in pregnancy or before apheresis.
• the level of free VEGF or free P1GF is measured and an increase in the level of free VEGF or free P1GF relative after apheresis as compared to a positive reference, standard, or level indicates an improvement in the pregnancy related hypertensive disorder in the subject.
• the positive reference is from a sample from the same subject earlier in pregnancy or before apheresis.
• the monitoring is used to determine if the apheresis procedure should be repeated.
• a lack of decrease in sFlt-1 or soluble endoglin levels or a lack of increase in free VEGF or free P1GF levels after the apheresis procedure indicates the need for at least one additional apheresis procedure.
• a lack of decrease or increase can mean no detectable decrease or increase, or a decrease or increase that is insufficient to provide therapeutic benefit.
• a level of sFlt-1 polypeptide less than 5 ng/ml, desirably less than 2 ng/ml indicates an improvement in the pre-eclampsia or eclampsia.
• the apheresis procedure is provided until the level of sFlt-1 in the subject is less than 2 ng/ml.
• a decrease in the sFlt-1 level or an increase in the level of VEGF or PIGF polypeptide or nucleic acid measured during or after administering therapy relative to the value before therapy indicates an improvement in the pre-eclampsia or eclampsia.
• the method includes measuring the levels of sFlt-1 prior to the apheresis procedure and measuring the levels of sFlt-1 during or after the apheresis procedure, wherein the apheresis procedure is administered until the levels of sFlt-1 polypeptide are reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more during or after the apheresis procedure as compared to the sFlt-1 levels prior to the apheresis procedure.
• the sFlt-1 levels are reduced by more than 61%.
• a metric or a ratio may be used to monitor the sFlt-1, VEGF, PIGF, or soluble endoglin levels before, during or after treatment.
• a a pre-eclampsia anti-angiogenic index (PAAI): [sFlt-l/VEGF + PIGF] or sFlt-l/PlGF may be used to monitor the subject's progress during therapy.
• PAAI pre-eclampsia anti-angiogenic index
• a decrease in the PAAI value or sFlt-l/PlGF value as compared to a prior value measured in the same subject indicates an improvement in the preeclampsia or eclampsia.
• a PAAI value less than 20, more preferably less than 10 indicates an improvement in the pre-eclampsia or eclampsia.
• a decrease in the PAAI or sFlt-l/PlGF levels measured during or after therapy can also indicate an effective amount or duration of apheresis treatment.
• the apheresis procedure is carried out such that the PAAI is less than 20.
• the apheresis procedure is carried out such that the PAAI is less than 10.
• the apheresis procedure is carried out such that the sFlt-l/PlGF levels as measured, for example, on the Elecsys platform (Roche), are less than or equal to 80.
• the measuring of the levels of sFlt-1, PIGF, or VEGF is done on two or more occasions and a change in the levels between measurements is used to monitor therapy or to determine appropriate duration or amount of apheresis treatment.
• the PAAI or sFlt-l/PlGF level after apheresis as compared to a positive reference, standard, or level indicates an improvement in the pregnancy related hypertensive disorder in the subject.
• the positive reference is from a sample from the same subject earlier in pregnancy or before apheresis.
• the diagnostic and monitoring methods described herein are used to monitor the subject during therapy or to determine effective therapeutic dosages or to determine the number of treatments needed.
• a decrease in the level of sFlt-1 polypeptide or nucleic acid measured during or after administering therapy relative to the value before therapy indicates an improvement in the pre-eclampsia or eclampsia.
• the clinician may decide to stop apheresis treatment.
• a lack of decrease or an increase in the level of sFlt-1 polypeptide or nucleic acid measured during or after administering therapy relative to the value before therapy may indicate the need for continued apheresis treatment.
• any of the diagnostic methods known in the art or described herein can be used to monitor the pre-eclampsia or eclampsia in the subject before, during, or after treatment.
• the method further includes administering an anticoagulation drug compound or an anti-hypertensive compound to the pregnant subject.
• an anticoagulation drug compound or an anti-hypertensive compound include nicotine, theophylline, adenosine, nifedipine, minoxidil, and magnesium sulfate.
• the bodily fluid is blood, serum, or plasma.
• 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, or a cat).
• the subject is a pregnant human having or at risk for developing a pregnancy related hypertensive disorder.
• the apheresis procedure includes the step of regenerating the apheresis column wherein the step of regenerating the apheresis column includes usage of eluent (e.g., an eluent that includes an inorganic salt solution).
• eluent e.g., an eluent that includes an inorganic salt solution
• the apheresis cartridge that has been reduced the capacity during the treatment is regenerated with eluent that includes 0.5-25 w/v% inorganic salt solution, or saline solution or 3-10 w/v% saline solution.
• 4-6 w/v% saline solution is used as eluent for the regeneration of the apheresis cartridge. The regeneration may occur during or after a treatment with the apheresis procedure.
• the invention also features combinations of the methods described herein with any of the therapeutic, diagnostic, or monitoring methods described in U.S. Patent Numbers 7,335,362; 7,407,659; and 7,435,419; U.S. Patent Application Publication Numbers 2006/0067937 and 2007/0104707; and PCT Publication Numbers WO 2004/008946; WO 2005/077007; WO 2006/034507; and WO
• alteration is meant a change (increase or decrease).
• An alteration can include a change in the expression levels of a gene or polypeptide as detected by standard art known methods such as those described below.
• an alteration includes a 10% change in protein levels, preferably a 25% change, more preferably a 40%, 50%, 60%, 70%, 80%, 90% or greater change in expression levels.
• apheresis By “apheresis,” “hemapheresis,” or “pheresis” is meant the process of removing a specific component from the blood, plasma, serum, or a fraction thereof, of a subject. Apheresis can be used to remove, separate, or collect one or more specific components of the blood, plasma, serum, or a fraction thereof. In general, apheresis includes the withdrawal of blood from the subject's body, removal of one or more components from the blood, and transfusion of the remaining blood back into the subject's body.
• binding is meant a non-covalcnt or a covalent interaction, preferably non-covalent, that holds two molecules together.
• Non-covalent interactions include, but are not limited to, hydrogen bonding, ionic interactions among charged groups, van der Waals interactions, and hydrophobic interactions among non-polar groups. One or more of these interactions can mediate the binding of two molecules to each other. Binding may exhibit discriminatory properties such as specificity or selectivity.
• body mass index is meant a number, derived by using height and weight measurements, that gives a general indication of whether or not weight falls within a healthy range.
• the formula generally used to determine the body mass index is a person's weight in kilograms divided by a person's height in meters squared or weight (kg)/ (height (m)) 2 .
• compound is meant any small molecule chemical compound (peptidyl or non-peptidyl), antibody, nucleic acid molecule, polypeptide, or fragments thereof,
• Endoglin or “Eng,” also known as CD 105, is meant a mammalian growth factor that has endoglin biological activity (see, for example, U.S. Patent Application Publication Nos. 20060067931 and 20070104707; WO2006034507; WO
• soluble endoglin polypeptide or “sEng” is meant any circulating, non- membrane bound form of endoglin which includes at least a part of the extracellular portion of the endoglin protein and is substantially identical (e.g., 60%, 70%, 80%, 90%, 995%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence encoding the extracellular portion of the endoglin protein (see, for example, U.S. Patent Application Publication Numbers 2006/0067937 and 2007/0104707; and PCT Publication Numbers WO 2006/034507 and WO 2008/030283).
• fragment is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or
• a fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, 1800 or more nucleotides or 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 640 amino acids or more.
• gestational age is meant a reference to the age of the fetus, counting from the first day of the mother's last menstrual period usually referred to in weeks.
• gestational hypertension is meant the development of high blood pressure without proteinuria after 20 weeks of pregnancy.
• a "history of pre-eclampsia or eclampsia” is meant a previous diagnosis of pre-eclampsia or eclampsia or pregnancy induced hypertension in the subject themselves or in a related family member.
• IUG intrauterine growth retardation
• a syndrome resulting in a birth weight which is less than the 10th percentile of the predicted fetal weight for the gestational age of the fetus.
• the current World Health Organization criterion for low birth weight is a weight less than 2,500 gm (5 lbs. 8 oz.) or below the 10 th percentile for gestational age according to U.S. tables of birth weight for gestational age by race, parity, and infant sex (Zhang and Bowes, Obstet. Gynecol. 86:200-208, 1995).
• SGA small for gestational age
• Pre-eclampsia is a condition known to be associated with IUGR or SGA.
• pharmaceutically acceptable carrier is meant a carrier that is
• physiologically acceptable to the treated mammal while retaining the therapeutic properties of the compound with which it is administered.
• One exemplary pharmaceutically acceptable carrier substance is physiological saline.
• physiologically acceptable carriers and their formulations are known to one skilled in the art and described, for example, in Remington's Pharmaceutical Sciences, (20 th edition), ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, PA.
• placental growth factor P1GF
• P1GF placental growth factor
• P1GF is expressed by cyto- and syncytiotrophoblasts in the placenta and P1GF biological activities include induction of proliferation, migration, and activation of endothelial cells, particularly trophoblast cells.
• pregnancy related hypertensive disorder any condition or disease or pregnancy that is associated with or characterized by an increase in blood pressure. Included among these conditions are pre-eclampsia (including premature pre-eclampsia, severe pre-eclampsia), eclampsia, gestational hypertension, HELLP syndrome, (hemolysis, elevated liver enzymes, low platelets), abruption placenta, chronic hypertension, pregnancy with intra uterine growth restriction, and pregnancy with a small for gestational age (SGA) fetus. It should be noted that although pregnancy with a SGA fetus is not often associated with hypertension, it is included in this definition.
• pre-eclampsia is meant the multi-system disorder that is characterized by hypertension with proteinuria or edema, or both, glomerular dysfunction, brain edema, liver edema, or coagulation abnomialities due to pregnancy or the influence of a recent pregnancy. All forms of pre-eclampsia, such as premature, mild, moderate, and severe pre-eclampsia are included in this definition. Pre-eclampsia generally
• Pre-eclampsia is generally defined as some combination of the following symptoms: (1) a systolic blood pressure (BP) >140 mmHg and a diastolic BP >90 mmHg after 20 weeks gestation (generally measured on two occasions, 4-168 hours apart), (2) new onset proteinuria (1 + by dipstik on urinalysis, > 300mg of protein in a 24-hour urine collection, or a single random urine sample having a protein/creatinine ratio >0.3), and (3) resolution of hypertension and proteinuria by 12 weeks postpartum.
• BP systolic blood pressure
• diastolic BP >90 mmHg after 20 weeks gestation (generally measured on two occasions, 4-168 hours apart)
• new onset proteinuria (1 + by dipstik on urinalysis, > 300mg of protein in a 24-hour urine collection, or a single random urine sample having a protein/creatinine ratio >0.3
• Severe pre-eclampsia is generally defined as (1) a diastolic BP > 110 mmHg (generally measured on two occasions, 4-168 hours apart) or (2) proteinuria characterized by a measurement of 3.5 grams or more protein in a 24-hour urine collection or two random urine specimens with at least 3+ protein by dipstick.
• pre-eclampsia hypertension and proteinuria generally occur within seven days of each other.
• severe pre-eclampsia severe hypertension, severe proteinuria and HELLP syndrome (hemolysis, elevated liver enzymes, low platelets) or eclampsia can occur simultaneously or only one symptom at a time.
• HELLP syndrome is characterized by evidence of thrombocytopenia ( ⁇ 100000 cells/ ⁇ ), increased LDH (>600 IU/L) and increased AST (>70 IU/L). Occasionally, severe pre-eclampsia can lead to the development of seizures. This severe form of the syndrome is referred to as "eclampsia.” Eclampsia can also include dysfunction or damage to several organs or tissues such as the liver (e.g., hepatocellular damage, periportal necrosis) and the central nervous system (e.g., cerebral edema and cerebral hemorrhage). The etiology of the seizures is thought to be secondary to the development of cerebral edema and focal spasm of small blood vessels in the kidney.
• pre-eclampsia pre-eclampsia with onset of symptoms ⁇ 37 weeks.
• protein or “polypeptide” or “polypeptide fragment” is meant any chain of more than two amino acids, regardless of post-translational modification (e.g., glycosylation or phosphorylation), constituting all or part of a naturally occurring polypeptide or peptide, or constituting a non-naturally occurring
• control or “reference” is meant any sample, standard, or level that is used for comparison purposes.
• a control sample may be a prior sample taken from the same subject (e.g., a sample from a prior time point or prior to the onset of symptoms).
• Non-limiting examples of control samples include: a sample from a pregnant subject or group of subjects not having a pregnancy related hypertensive disorder, such as pre-eclampsia or eclampsia; a subject or group of subjects that is pregnant but the sample was taken early in pregnancy (e.g., in the first or second trimester or before the detection of a pregnancy related hypertensive disorder, such as pre-eclampsia or eclampsia); a subject or group of subjects that is pregnant and has no history of a pregnancy-related hypertensive disorder, such as pre-eclampsia or eclampsia; a subject or group of subjects that is not pregnant; a sample of a purified reference polypeptide at a known normal
• control samples can be prepared from a subject or group of subjects prior to developing or diagnosis with a pregnancy-related hypertensive disorder.
• control standard or level or "normal reference standard or level” is meant a value or number derived from a control sample.
• a control standard or level can be a value or number derived from a normal subject or group of subjects that is matched to the sample subject, for example, by at least one of the following criteria: age, sex, weight, gestational age of the fetus, maternal age, maternal blood pressure prior to pregnancy, maternal blood pressure during pregnancy, BMI of the mother, weight of the fetus, prior diagnosis of pre-eclampsia or eclampsia, and a family history of pre-eclampsia or eclampsia.
• a "positive control" or "positive reference” sample, standard or value is a sample or value or number derived from a subject or group of subjects that is known to have a pregnancy-related hypertensive disorder.
• a positive control sample may be from a subject or group of subjects having a pregnancy-related hypertensive disorder (e.g., pre-eclampsia or eclampsia), that is matched to the sample subject by at least one of the following criteria: gestational age of the fetus, maternal age, maternal blood pressure prior to pregnancy, maternal blood pressure during pregnancy, BMI of the mother, weight of the fetus, prior diagnosis of a pregnancy-related hypertensive disorder, and a family history of a pregnancy related hypertensive disorder.
• An "elevated sFlt-1 level” is an sFlt-1 level that is higher than a normal reference standard or level.
• reduce or inhibit is meant the ability to cause an overall decrease preferably of 10% or greater, more preferably of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater.
• reduce is meant a decrease, preferably of 10% or greater, more preferably of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater, in the level of protein or nucleic acid as compared to an untreated sample.
• the apheresis procedure will be carried out such that the level of sFlt-1 in the blood of a pregnant subject is reduced by at least 10%, 20%, 30%, 40%, 50% or more.
• the sFlt-1 levels can be measured before apheresis is administered and again during or after apheresis for comparison.
• sample is meant a bodily fluid (e.g., blood, serum, plasma, urine, saliva, amniotic fluid, or cerebrospinal fluid), tissue sample, cell or other specimen obtained from a subject.
• sample used for apheresis is a blood sample.
• soluble Flt-1 (sFlt-1 )
• sFlt-1 also known as sVEGF-Rl
• soluble Flt-1 the soluble form of the Flt-1 receptor, that is homologous to the protein defined by GenBank accession number U01 134, NM_001153392, NM_001159920, or EU368830 and that has sFlt-1 biological activity or isoforms or fragments thereof.
• the biological activity of an sFlt-1 polypeptide may be assayed using any standard method, for example, by assaying sFlt-1 binding to VEGF.
• sFlt-1 lacks the transmembrane domain and the cytoplasmic tyrosine kinase domain of the Flt-1 receptor.
• sFlt-1 can bind to VEGF and P1GF with high affinity, but it cannot induce proliferation or angiogenesis and is therefore functionally different from the Flt-1 and KDR receptors.
• sFlt-1 was initially purified from human umbilical endothelial cells and later shown to be produced by trophoblast cells in vivo.
• sFlt-1 can include any sFlt-1 family member or isoform (e.g., alternatively spliced isoforms).
• sFlt-1 can also mean degradation products or fragments that result from enzymatic cleavage of the Flt-1 receptor and that maintain sFlt-1 biological activity.
• telomere binding By “specifically binds” is meant a compound which recognizes and binds a polypeptide of the invention but that does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polypeptide of the invention.
• subject is meant a mammal, including, but not limited to, a human or non-human mammal, such as a cow, a horse, a sheep, a pig, a goat, a dog, or a cat. Included in this definition are pregnant, post-partum, and non-pregnant mammals.
• symptoms of pre-eclampsia is meant any of the following: (1) a systolic blood pressure (BP) >140 mmHg and a diastolic BP >90 mmHg after 20 weeks gestation, (2) new onset proteinuria (1+ by dipstik on urinanaysis, >300mg of protein in a 24 hour urine collection, or random urine protein/creatinine ratio >0.3), and (3) resolution of hypertension and proteinuria by 12 weeks postpartum.
• the symptoms of pre-eclampsia can also include renal dysfunction and glomerular endotheliosis or hypertrophy.
• symptoms of eclampsia is meant the development of any of the following symptoms due to pregnancy or the influence of a recent pregnancy:
• seizures, coma, thrombocytopenia, liver edema, pulmonary edema, and cerebral edema seizures, coma, thrombocytopenia, liver edema, pulmonary edema, and cerebral edema.
• therapeutic amount is meant an amount that when administered to a patient suffering from pre-eclampsia or eclampsia is sufficient to cause a qualitative or quantitative reduction in the symptoms of pre-eclampsia or eclampsia as described herein.
• a “therapeutic amount” can also mean an amount that when administered to a patient suffering from pre-eclampsia or eclampsia is sufficient to cause a reduction in the expression levels of soluble endoglin or sFlt-1 or an increase in the expression levels of VEGF or P1GF as measured by the assays described herein.
• treating or “ameliorating” is meant administering a compound or a pharmaceutical composition or administering an ex vivo therapy (e.g., apheresis) to treat or ameliorate a condition or symptom(s) of the condition (e.g., the symptoms of pregnancy related hypertensive disorders described herein).
• an ex vivo therapy e.g., apheresis
• a condition or symptom(s) of the condition e.g., the symptoms of pregnancy related hypertensive disorders described herein.
• therapeutic treatment refers to administering the treatment to a subject already suffering from a disease to improve the subject's condition.
• the subject is diagnosed with or identified as having a predisposition for developing a pregnancy related hypertensive disorder.
• amelioration or degree of treatment is an improvement in the condition or symptoms of the treated subject of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%, as measured by any standard technique.
• vascular endothelial growth factor is meant a mammalian growth factor that is homologous to the growth factor defined in U.S. Patent Nos. 5,332,671 ; 5,240,848; 5,194,596; and Charnock-Jones et al. (Biol. Reproduction, 48: 1120-1128, 1993), and has VEGF biological activity.
• VEGF exists as a glycosylated homodimer and includes at least four different alternatively spliced isoforms.
• the biological activity of native VEGF includes the promotion of selective growth of vascular endothelial cells or umbilical vein endothelial cells and induction of angiogenesis.
• VEGF includes any VEGF family member or isoform (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF 189, VEGF 165, or VEGF 121) or any modified form of VEGF (Tischer et al., J Biol. Chem, 266, 11947- 1 1954, 1991 ; Neufed et al. Cancer Metastasis 15: 153-158, 1996), which is described in U.S. Patent Nos. 6,447,768; 5,219,739; and 5,194,596, hereby incorporated by reference.
• human VEGF is preferred, the invention is not limited to human forms and can include other animal forms of VEGF (e.g. mouse, rat, dog, or chicken).
• apheresis methods and devices including those previously known and used to remove positively charged proteins, such as low density lipoproteins or apolipoproteins, from blood can be used to remove sFlt-1 from the blood.
• Such apheresis methods and devices can be used to treat or ameliorate symptoms of pregnancy related hypertensive disorders.
• apheresis methods that were used to remove lipoproteins are effective for the specific removal of sFlt-1 because, while most plasma proteins have isoelectric points between 4 and 6, sFltl has an isoelectric point greater than 9.5. Although the apheresis technique may still bind to and remove some LDLs and other positively charged proteins such as fibrinogen, globulin, and C reactive protein, the method will still be selective for sFlt-1 , because of its isoelectric point. Apheresis Methods and Devices
• apheresis includes the removal or withdrawal of blood from the subject's body, removal of one or more components from the blood, and transfusion of the remaining blood back into the subject's body.
• apheresis is used for the removal, desirably selective removal, of sFlt-1 from the blood of a pregnant woman diagnosed with or suffering from symptoms of a pregnancy related hypertensive disorder.
• the present invention includes the use of apheresis methods and reagents (e.g., negatively charged polymers or polysaccharides) previously used for the removal of positively charged proteins, such as low density lipoprotein, fibrinogen, globulin, and C reactive protein.
• apheresis methods and reagents e.g., negatively charged polymers or polysaccharides
• positively charged proteins such as low density lipoprotein, fibrinogen, globulin, and C reactive protein.
• the apheresis methods of the invention generally utilize a cartridge, column, or filter that include agents, e.g., sulfonated polysaccharides, polystyrene sulfonic acids, or polyacrylic acids that are immobilized with a water-insoluble porous carrier, and that selectively bind to and remove sFlt-1 while leaving other blood components intact.
• agents e.g., sulfonated polysaccharide (e.g., hyaluronic acid, chondroitin sulfate, keratin sulfate, heparin sulfate, dextran sulfate), polystyrene sulfonic acid, and polyacrylic acid.
• Water-insoluble porous carriers include hydrophilic porous carriers which can include porous carriers such as but not limited to polysaccharide (e.g., cellulose, pectin, chitin, agarose, carrageenan, dextran) and hydrophilic polymer (e.g., polyethylene glycol, polyvinyl alcohol, polyacrylic acid, silica gel). All of these molecules have been used in apheresis devices and procedures known in the art for the removal of specific blood proteins, e.g., Low Density Lipoprotein, Lipoprotein(a) (Lp (a)) or the plasma proteins including but not limited to globulin, fibrinogen, and C-reactive protein (CRP).
• porous carriers such as but not limited to polysaccharide (e.g., cellulose, pectin, chitin, agarose, carrageenan, dextran) and hydrophilic polymer (e.g., polyethylene glycol, polyvinyl alcohol, polyacryl
• dextran sulfate-mediated apheresis is preferred.
• the agent may be immobilized with a water-insoluble porous carrier.
• the average particle diameter when plasma as a cell-free body fluid is used may be 5 to 1 ,000 ⁇ , preferably 25 tol,000 ⁇ , most preferably 50 to 300 um, and in case of the contact of these carriers with blood is 5 to 1,000 ⁇ , and preferably 250 to 1 ,000 ⁇ , and most preferably 250 to 600 ⁇ .
• the average particle diameter for the water-insoluble porous carrier is 5 to 1 ,000 ⁇ , and preferably 100 to 600 ⁇ , and most preferably 250 to 300 ⁇ .
• the average particle diameter for the water insoluble porous carrier is 5 to 1 ,000 ⁇ , and preferably 250 to 1 ,000 ⁇ , and most preferably 350 to 600 ⁇ .
• Non-limiting examples of such water-insoluble porous carriers include a molecular weight exclusion (i.e., the molecular weight of a molecule having the smallest molecular weight among molecules that cannot enter pores (are excluded) in gel permeation chromatography (Experimental High Performance Liquid
• Non-limiting examples of commercially available apheresis columns or devices known in the art for the removal of positively charged lipoproteins include Liposorber® (dextran sulfate-mediated apheresis; Kaneka Pharma); DL75 (Octanova, Kaneka Pharma); H.E.L.P.TM (heparin mediated apheresis; Braun), TheraSorbTM (Miltenyi); and DALI (anionic polyacrylate; Fresenius SE).
• the plasma is separated from the rest of the blood using a membrane plasma filter.
• the plasma meets the heparin buffer solution or the dextran sulfate buffer solution, at which time a reaction occurs, allowing the sFlt-1 to be removed.
• the sFlt- 1 -reduced plasma can then optionally pass through additional filters to remove additional proteins or contaminating materials.
• the sFlt-1 reduced plasma is then combined with the patient's blood and returned to the patient.
• blood is drawn from the patient's vein and directed to a device that separates plasma from cellular components of the blood.
• the plasma is then directed into an adsorber.
• the various adsorbers contain a specific matrix (e.g., heparin, anionic polyacrylate, or dextran sulfate based) that selectively retain the sFlt- 1 protein. After passing through the adsorber's matrix, the plasma is recombined with the remaining blood components and returned to the patient.
• blood from the patient is circulated extracorporeally using standard apheresis equipment.
• the blood is separated into the cellular elements (red blood cells, white blood cells and platelets) and fluid (plasma) elements using differential centrifugation or a membrane filter.
• the plasma is then pumped through the targeted apheresis device where the sFlt-1 will bind to the immobilized heparin or dextran sulfate.
• the sFlt-1 depleted plasma is then mixed with the cellular blood elements and returned to the patient.
• the apheresis device will be constructed as a cylinder with an inlet to allow plasma to enter at one end, and an outlet at the opposite end to allow the cleaned plasma to exit and be returned to the patient.
• Other device configurations may also be designed and are within the scope of this invention.
• the apheresis cartridge may be employed as a single use device or it may be regenerated and used multiple times.
• the apheresis cartridge that has been reduced the capacity during the treatment is regenerated with eluent include 0.5-25 w/v% inorganic salt solution and as much as 3-10 w/v% saline solution. In desired embodiments, 4-6 w/v% saline solution is used for the regeneration of the apheresis cartridge.
• the heparin-mediated, anionic polyacrylate-mediated, or dextran sulfate-mediated apheresis will remove at least 10%, 15%, 20%, 25%, 30%, 35%, or 40% of the sFlt-1 in the patient's blood and as much as 45%, 50%, 55%, 60%, 65%, 70%), or 75% of the sFlt-1 in the patient's blood. In desired embodiments, 25% or more, 50% or more, 60% or more, or 75% or more of the sFlt-1 is removed from the patient's blood.
• the sFlt-1 is removed such that the subject's sFlt-1 levels return to within 20%, 10%, 5%, 2% or equal to a normal control or reference that is matched for gestational age.
• Standard assays for sFlt-1 levels are known in the art and can be used to measure the level of the sFlt-1 in the patient's blood before, during, or after apheresis.
• the apheresis method results in a clearance of at least 50% of the sFlt-1 protein from the patient's blood as compared to the level before apheresis.
• the pH of the blood is restored to normal biological levels prior to returning to the subject.
• the sFlt-1 protein removed in the methods of the invention may be full-length, or an sFlt-1 fragment thereof (e.g., from enzymatic cleavage or degradation), an isoform of sFlt-1 (e.g., alternatively spliced), free, total, or bound sFlt-1.
• the apheresis procedures of the invention may be carried out after diagnosis of a pregnancy related hypertensive disorder, e.g., pre-eclampsia or eclampsia, and before or after the onset of clinical symptoms of a pregnancy related hypertensive disorder, e.g., pre-eclampsia or eclampsia.
• a patient is identified as being at risk for developing pre-eclampsia and the sFlt-1 levels are measured and monitored. When sFlt-1 levels are increased as compared to normal reference controls, levels, or standard values or curves, the clinician may begin apheresis treatment.
• Treatment may be given once or repeated until symptoms are diminished; sFlt-1 levels are reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more; sFlt-1 levels return to the normal or baseline levels determined early in pregnancy or within 20%, 10%, 5%, or 2% of the normal or baseline levels determined early in pregnancy (e.g., during the first or second trimester); the baby is delivered; the pregnancy is terminated; the subject is deemed to be stable or no longer in need of treatment; or the pregnancy-related hypertensive disorder is fully treated and no longer requires treatment.
• Other parameters that suggest improvement after apheresis therapy include increasing growth of the fetus, prolonging the pregnancy past 34 weeks of gestation, improvement in hypertension and/or improvement in proteinuria.
• treatment begins in the second trimester. In another example, treatment begins in the third trimester. In another example, treatment begins at 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 weeks and is continued until no longer necessary.
• the methods of the present invention may be combined with any other methods for the treatment of a pregnancy related hypertensive disorder (e.g., preeclampsia or eclampsia) known in the art or described herein.
• a pregnancy related hypertensive disorder e.g., preeclampsia or eclampsia
• the apheresis methods of the present invention are combined with therapeutic methods, in vivo or ex vivo, designed to specifically decrease the sFlt-1 levels (e.g., through the use of an sFlt-1 binding peptide such as VEGF or P1GF or an anti-sFlt-1 antibody) or increase the VEGF or P1GF levels in the subject (see for example, U.S.
• Patent Numbers 7,335,362; 7,407,659; and 7,435,419; and PCT Publication Numbers WO 2004/008946 and WO 2005/077007) are combined with therapeutic methods, in vivo or ex vivo, designed to decrease the level of soluble endoglin (e.g., through the use of an anti- soluble endoglin antibody) or combined with the use of any compound (e.g., polypeptide, small molecule, antibody, nucleic acid, and mimetic) that increases the level or biological activity of TGF- ⁇ , eNOS, and PGI 2 (see, for example, U.S. Patent Application Publication Numbers 2006/0067937 and 2007/0104707; and PCT Publication Numbers WO 2006/034507; and WO 2008/030283.)
• the invention features the use of a combination of any one or more of the therapeutic agents described herein.
• therapeutic methods of the invention include apheresis methods to decrease sFlt-1 levels or activity or increase VEGF or P1GF levels or activity in combination with a compound that decreases soluble endoglin levels or activity or increase TGF- ⁇ , 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.
• an antibody that specifically binds to soluble endoglin can be administered in combination with the apheresis methods of the present invention.
• a compound that increases TGF- ⁇ levels or activity can be administered in combination with the apheresis methods of the present invention in order to target both the endoglin and the sFlt-1 pathway.
• a combination of the dextran-based (e.g., dextran sulfate) or heparin-based apheresis methods with a targeted apheresis method using antibodies against sFlt-1 or soluble endoglin or both may also be used.
• apheresis methods of the present may be combined with any methods known to be beneficial during apheresis.
• an anti-coagulant therapy is administered to the subject before, during, or after the apheresis.
• the subject is given fluid (e.g., saline) for fluid replacement during therapy.
• 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.
• modes of administration and dosages are determined by the physician and by the manufacturer's instructions.
• the apheresis methods of the present invention may be carried out with methods for monitoring the pregnancy related hypertensive condition (e.g., preeclampsia or eclampsia) using diagnostic methods known in the art or described herein.
• diagnostic methods include the measurement and detection of sFlt-1 , VEGF, or P1GF as described in U.S. Patent Numbers 7,335,362; 7,407,659; and 7,435,419; and PCT Publication Numbers WO 2004/008946 and WO 2005/077007.
• diagnostic methods include the use of metrics incorporating sFlt- 1, P1GF, VEGF, soluble endoglin, or combinations thereof (e.g., sFflt-l/PlGF) as described in U.S. Patent Numbers 7,335,362; 7,407,659; and 7,435,419; U.S. Patent Application Publication Numbers 2006/0067937 and 2007/0104707; and PCT Publication Numbers WO 2004/008946; WO 2005/077007; WO 2006/034507; and WO 2008/030283.
• metrics incorporating sFlt- 1, P1GF, VEGF, soluble endoglin, or combinations thereof e.g., sFflt-l/PlGF
• apheresis therapy is administered until the blood, plasma, or serum sFlt-1 level is less than 5 ng/ml, desirably less than 2 ng/ml, or until the sFlt-1 levels return to the baseline level determined before onset of pre-eclampsia or eclampsia.
• the apheresis therapy can be administered until the serum P1GF level rises to approximately 400 pg/mL or the serum P1GF levels return to baseline level prior to onset of pre-eclampsia or eclampsia.
• the levels of soluble endoglin, sFlt-1, P1GF, 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.
• porous cellulose beads (a molecular weight exclusion limit for globular proteins of 5,000,000, a particle diameter of 45 to 105 ⁇ ), 100 ml of water, 50 ml of 2N sodium hydroxide, and 17 ml of epichlorohydrin were mixed and reacted at 40°C for 2 hours. After the reaction, the beads were thoroughly washed with water to give epoxidized cellulose beads. To the obtained epoxidized cellulose beads, 107 ml of 58% aqueous solution of dextran sulfate sodium (a sulfur content of 18%) was added to adjust to pH 9 and the whole was shaken at 45°C for 22 hours.
• Insect cell recombinant human sFlt-1 (manufactured by RDI) was adjusted to a predetermined concentration with phosphate buffered saline (PBS)/0.1% BSA.
• the dextran sulfate sodium-immobilized cellulose beads were equilibrated with physiological saline. Into a test tube, 0.5 ml of the beads was placed, and excess physiological saline was removed. Into the test tube, 3 ml of human serum containing human sFlt-1 at about 4500 pg/ml was added, the whole was shaken at 37°C for 2 hours, and then 2.5 ml of supernatant was removed (supernatant A).
• Adsorption rate (%) (C c i - C a i)/C c i x 100
• Dextran sulfate-immobilized cellulose beads were obtained in the same manner as that described in Example 1 except that porous cellulose beads (; molecular weight exclusion limit for globular proteins of 20,000,000, a particle diameter of 45 to 105 ⁇ ) were used. Evaluation of the obtained adsorbent B was also carried out in the same manner as that in Example 1.
• adsorbent C Dextran sulfate-immobilized cellulose beads (adsorbent C) were obtained in the same manner as that described in Example 1 except that porous cellulose beads (an average particle diameter of 195 ⁇ ) were used. Evaluation of the obtained , adsorbent C was also carried out in the same manner as that in Example 1.
• Example 4 Dextran sulfate-immobilized cellulose beads (adsorbent C) were obtained in the same manner as that described in Example 1 except that porous cellulose beads (an average particle diameter of 195 ⁇ ) were used. Evaluation of the obtained , adsorbent C was also carried out in the same manner as that in Example 1.
• Example 4 Dextran sulfate-immobilized cellulose beads (adsorbent C) were obtained in the same manner as that described in Example 1 except that porous cellulose beads (an average particle diameter of 195 ⁇ ) were used. Evaluation of the obtained , adsorbent C was also carried out in the same manner as that in Example 1.
• porous cellulose beads a molecular weight exclusion limit for globular proteins of 50,000,000, an average particle diameter of about 450 ⁇
• 22 ml of water 22 ml of water
• 31 ml of 4N aqueous sodium hydroxide solution 32 ml of
• a solution of 0.77 g of L-tryptophan dissolved in 50 ml of a diluted aqueous NaOH solution was added and the whole was reacted at 50°C for 8 hours.
• the beads were thoroughly washed with water and an aqueous sodium chloride solution to give dextran sulfate and tryptophan-immobilized cellulose beads (adsorbent D). Evaluation of the obtained adsorbent D was carried out in the same manner as that in Example 1.
• the calculated isoelectric point for sFltl is 9.78.
• sFltl protein would be predominantly positively charged.
• Most plasma proteins have isoelectric points between 4-6 suggesting that at physiological pH they are negatively charged.
• an extracorporeal column containing negatively charged material would bind sFltl and therefore would be useful to remove sFltl in patients with pre-eclampsia.
• Dextran sulfate columns may be of therapeutic use in patients with preeclampsia, particularly those characterized by high circulating sFltl .

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