Detection and Treatment of Pregnancy Complications Comprising Determining Sialyl Lewis Antigens and Administering hCG

Abstract

Disclosed herein is a method of identifying and/or addressing incipient preeclampsia in a patient-subject by the steps of (a) performing a bioassay to determine the level of at least one sialyl Lewis antigen in a said patient-subject at about 25 weeks of pregnancy or earlier; (b) performing a bioassay to determine the level of at least one sialyl Lewis antigen in a pregnant non-preeclampsia one or more subjects at about 30 weeks of pregnancy or later, wherein said at least one sialyl Lewis antigen assay is for a sialyl Lewis antigen assayed in step (a) is and if more than one subject is assayed, averaging said results; and (c) managing said patient-subject for preeclampsia, if said level of at least one sialyl Lewis antigen of step (a) is at or greater than about 20% above the level of such silalyl Lewis antigen assayed in step (b).

Claims

1. A method of identifying and/or addressing incipient preeclampsia in a patient-subject by the steps of by the steps of (a) performing a bioassay to determine the level of at least one sialyl Lewis antigen in a said patient-subject at about 25 weeks of pregnancy or earlier; (b) performing a bioassay to determine the level of at least one sialyl Lewis antigen in a pregnant non-preeclampsia one or more subjects at about 30 weeks of pregnancy or later, wherein said at least one sialyl Lewis antigen assay is for a sialyl Lewis antigen assayed in step (a) is and if more than one subject is assayed, averaging said results; and (c) managing said patient-subject for preeclampsia, if said level of at least one sialyl Lewis antigen of step (a) is at or greater than about 20% above the level of such silalyl Lewis antigen assayed in step (b).

2. The method of claim 1, wherein step (a) is performed at about 20 weeks of pregnancy or earlier.

3. The method of claim 2, wherein step (a) is performed at about 10 weeks of pregnancy or earlier.

4. A method of identifying and/or addressing incipient preeclampsia in a patient-subject by the steps of by the steps of (a) performing a bioassay to determine the level of least two or more sialyl Lewis antigens in a said patient-subject at about 25 weeks of pregnancy or earlier; (b) performing a bioassay to determine the level of at least two or more sialyl Lewis antigens of step (a) in a pregnant non-preeclampsia one or more subjects at about 30 weeks of pregnancy or later, wherein said at least two or more sialyl Lewis antigens is for a sialyl Lewis antigen assayed in step (a) is and if more than one subject is assayed, averaging said results as to each antigen separately; (c) managing said patient-subject for preeclampsia, if the sum level of at two or more sialyl Lewis antigens of step (a) is at or greater than about 30% above the level of the sum level of silalyl Lewis antigens assayed in step (b).

5. The method of claim 4, wherein three sialyl Lewis antigens are the subject of the bioassay of step (a) and step (b) wherein step (c) comprises managing said patient-subject for preeclampsia, if the sum level of said three sialyl Lewis antigens of step (a) is at or greater than about 50% above the level of the sum level of the sum of the silalyl Lewis antigens levels assayed in step (b).

6. A method of determining incipient preeclampsia at 20 weeks of pregnancy or earlier by the steps of (a) performing a bioassay to determine the level of at least one sialyl Lewis antigen in a female subject with a diagnosis of preeclampsia about 30 weeks of pregnancy or later, said result being a first comparator; (b) performing a bioassay to determine the level of said at least one sialyl Lewis antigen of stem (a) in a pregnant non-preeclampsia subject at about 30 weeks of pregnancy or later said result being a second comparator; (c) performing a bioassay to determine the level of sialyl Lewis antigen in a subject being tested for incipient preeclampsia; and, (d) managing said patient-subject for preeclampsia, if, upon comparing the level of sialyl Lewis antigens of (c) with said first and second comparators, wherein a sialyl Lewis antigen of level closer to the level of (a) than (b) is predictive of incipient preeclampsia

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0072] FIG. 1 is a graph of serum and amniotic fluid hCG exhibiting expression of sLex and sLea as compared to urine hCG and hCG secreted in supernatants from choriocarcinoma cells BeWo or Jeg 3.

[0073] FIG. 2. is a graph of sialyl Lewis antigen (sLe.sup.Y, sLe.sup.a and sLe.sup.x) expression on preeclampsia serum (PES)-hCG as compared to normal pregnancy serum (NPS)-hCG.

[0074] FIG. 3 is a graphic quantification of sialyl Lewis antigens in serum.

[0075] FIG. 4A are displays fetal size (upper panel) and fetal weights (lower panel).

[0076] FIG. 4B presents blood pressure data

[0077] FIG. 4C presents mouse subject proteinuria levels.

[0078] FIG. 5A shows renal pathology by H&E staining of the glomerulus

[0079] FIG. 5B is a graph of production of sFlt-1.

[0080] FIG. 5C is a graph of sEng in pregnant IL-10.sup.7″ mice.

DETAILED DESCRIPTION OF THE INVENTION

[0081] In the practice of this invention it is to be understood that the detection of PE (or related GPs) in instances where the pathology is effectively treated is the detection of precursor indicia before 20 weeks of gestation. For convenience the notation “sLe<20 w” is used to denote sialyl Lewis antigen elevation before 20 weeks of pregnancy. In this context. “elevation” will be understood to mean up-regulation or increase of at least about 20% of each of sialyl Lewis antigen alone as compared to the average control of the respective sialyl Lewis antigen antigen, or more than 30% if two sialyl Lewis antigen levels are combined, and 50% increase if three are combined. There are a number of bioassays suitable to determine such levels and other assays are being developed.

[0082] The practice of the invention, in one embodiment, is characterized by finding altered carbohydrate patterns, with specific reference to by the excess or elevated presence of sialyl Lewis antigens on preeclampsia hCG as compared to normal pregnancy hCG. Another aspect is the therapeutic use of sialyl Lewis antigen-free hCG in mitigating the symptoms associated with pregnancy complications such as preeclampsia. (Expert Opin Drug Deliv. 2012 August:9(8):893-900. Epub 2012 Jun. 18.)

hCG Can Rescue Pregnancy.

[0083] Without being bound by any particular theory, in IL-10″ mice, it is believed that the mode of action is by subverting production of anti-angiogenic factors and by replenishing uterine immune cells. Deglycosylated hCG is not reported as able to bind to mannose receptors on uNK cells, again emphasizing the importance of carbohydrate patterns in the function of hCG. Given the functional associations co-regulated by hCG, IL-10 and Treg migration, dysregulated hCG effects uterine Tregs and contributes to preeclampsia. Particularly noted is therapeutic administration of CG. CH with a less antigenic presentation is useful. This includes recombinant hCG. Intravenous administration is noted. Dosing with recombinant hCG, i.v. from between about 50 I.U. to about 500 I.U with particular reference to dosages between about 100 I.U and 200 I.U. is noted. Prefilled pens for administration of recombinant human chorionic gonadotropin (r-hCG) are available and useful in the practice of this invention.

EXEMPLIFICATION

Example 1

Quantification of Sialyl Lewis Antigens on hCG in Different Biological Fluids

[0084] Ninety-six-well microtitre plates (Maxisorp, Nunc) were coated with 50 μI rabbit anti-human hCG antibody (5 μg/ml in PBS, Dako A0231) at 4° C. overnight. The wells were washed three times with PBS, pH 7.2 containing 0.05% Tween 20, blocked for 1 hour with washing buffer containing 1% BSA and washed again three times.

[0085] 50 μI of pregnancy serum/amniotic fluid/urine/cell culture supernatant samples or hCG (5 μg/ml) were added and incubated for 1.5 hours at room temperature and washed three times. 50 μI of sLe.sup.x (Calbiochem, KM93) or sLe.sup.a (Calbiochem, KM 231) recognizing antibodies were added at concentration of 1 μg/ml. The wells were incubated for 1.5 hours at room temperature and washed three times. 50 μI of HRP-conjugated rabbit anti-mouse antibody (Dako, PO260) was added to each well, incubated for 1.5 hours, washed three times, developed with DMB and color development was followed by measuring the absorbance at 492 nm/630 nm. Wells without hCG served as controls.

Example 2

Quantification of Sialyl Lewis Antigens from Preeclampsia Serum hCG and Normal Pregnancy Serum hCG

[0086] Ninety-six-well microtitre plates (Maxisorp. Nunc) were coated with 50 μI rabbit anti-human hCG antibody (5 μ9/.Math.τ.Math.I in PBS, Dako A0231) at 4° C. overnight. The wells were washed three times with PBS, pH 7.2 containing 0.05% Tween 20, blocked for 1 hour with washing buffer containing 1% BSA and washed again three times.

[0087] 50 μI of human normal (n=15) or preeclampsia diagnosed pregnancy serum (n=14) obtained from blood collected at 32-36 weeks of pregnancy were added and incubated for 1.5 hours at room temperature and washed three times.

[0088] 50 μI of sLe.sup.x (Calbiochem, KM93) or sLe.sup.a (Calbiochem, KM 231) or Le.sup.y or Thomsen-Friedenreich antigen (Glycotope) recognizing antibodies were added at concentration of 5 μg/ml in PBS. The wells were incubated for 1.5 hours at room temperature and washed three times.

[0089] 50 μI of HRP-conjugated rabbit anti-mouse antibody (Dako, PO260) was added to each well, incubated for 1.5 hours, washed three times, developed with DMB and color development was followed by measuring the absorbance at 492 nm/630 nm. The mean absorbance obtained with multiple normal pregnancy hCG were considered as 100%.

[0090] As seen in the FIG. 2, sialyl Lewis antigen (LeY, sLeA and sLeX) expression is significantly higher on preeclampsia serum (PES)-hCG as compared to normal pregnancy serum (NPS)-hCG. As seen in FIG. 2, there was 21.6% up-regulation of sLe.sup.a (P=0.002), 32% up-regulation of sLe.sup.x (P=0.019) and 21.6% up-regulation of Le.sup.Y in the 32nd-36th week of gestation (P=0.021) in preeclamptic hCG compared to normal pregnancy hCG. The increase in sialyl Lewis antigen expression in PES-hCG was independent of the serum levels of β-hCG.

Example 3

Quantification of Sialyl Lewis Antigens in Serum hCG Collected Before the Onset of Preeclampsia

[0091] Ninety-six-well microtitre plates (Maxisorp, Nunc) were coated with 50 μI rabbit anti-human hCG antibody (5 μ9/.Math.ηI in PBS, Dako A0231) at 4° C. overnight. The wells were washed three times with PBS, pH 7.2 containing 0.05% Tween 20, blocked for 1 hour with washing buffer containing 1% BSA and washed again three times.

[0092] 50 μI of human serum obtained from blood collected at 12-14 weeks of pregnancy who later either went on have normal pregnancy (n=8) or were diagnosed with preeclampsia (n=8) were added and incubated for 1.5 hours at room temperature and washed three times. 50 μI of sLe.sup.x (Calbiochem, KM93) or sLe.sup.a (Calbiochem, KM 231) or Le.sup.y or Thomsen-Friedenreich (TF) antigen (Glycotope) recognizing antibodies were added at concentration of 5 μg/ml in PBS.

[0093] The wells were incubated for 1.5 hours at room temperature and washed three times. 50 μI of HRP-conjugated rabbit anti-mouse antibody (Dako, PO260) was added to each well, incubated for 1.5 hours, washed three times, developed with DMB and color development was followed by measuring the absorbance at 492 nm/630 nm. The mean absorbance obtained with multiple normal pregnancy hCG were considered as 100%.

[0094] As seen in the FIG. 3, sialyl Lewis antigen (sLe.sup.a and sLe.sup.x) expression is significantly elevated on preeclampsia serum (PES)-hCG collected at 12-14 weeks of pregnancy before the clinical diagnosis of disease as compared to normal pregnancy serum (NPS)-hCG. The increase in sialyl Lewis antigen expression in PES-hCG was independent of the serum levels of β-hCG. 11.2% upregulation in SLe.sup.a and 224% upregulation in SLe.sup.x expression in the 10th-12th week of gestation in preeclamptic hCG compared to normal pregnancy hCG. The expression of the TF antigen is not significantly changed in preeclamptic hCG compared to normal control hCG.

Example 4

Rescue of Preeclampsia-Like Features (IUGR, Hypertension and Proteinuria) by Sialyl Lewis Antigen-Free hCG (Functional hCG) in Mouse Model

[0095] Pregnant IL-10.sup.7″ mice were injected (gestational day 10, i.p) with either normal pregnancy serum (NPS) or PE serum (PES) with or without sialyl Lewis antigen-free hCG (urine or recombinant).

[0096] On gestational day 17, blood pressure and fetal weight were recorded. Urinary albumin and creatinine was measured in 24-hour urine samples using commercial ELISA kits. Proteinuria is expressed as a ratio of albumin and creatinine.

[0097] As seen in FIG. 4, functional hCG treatment reverses PES-induced intrauterine growth restriction (IUGR) (A) as reflected by fetal size (upper panel) and fetal weights (lower panel), hypertension (B), and proteinuria (C) in pregnant IL-10.sup.7″ mice. * and .sup.aP<0.05 significance as compared to NPS and PES groups respectively by student's T test

Example 5

Rescue of Preeclampsia-Like Features (Kidney Pathology, Elevated Soluble Fit-1 and Soluble Endoqiin) by Sialyl Lewis Antigen-Free hCG (Functional hCG) in Mouse Model

[0098] Pregnant IL-10.sup.7″ mice were injected (gestational day 10, i.p) with either NPS or PES with or without sialyl Lewis antigen-free hCG (urine or recombinant).

[0099] On gestational day 17, blood was collected by cardiac puncture and serum separated. Serum levels of mouse sFlt-1 & sEng were measured using commercial ELISA kits (R&D Systems).

[0100] As seen in FIG. 5, functional hCG treatment reverses PES-induced renal pathology as shown by H&E staining of glomerulus (A), and excess production of sFlt-1 (B) and sEng (C) in pregnant IL-10.sup.7″ mice. * and .sup.a P<0.05 significance as compared to NPS and PES groups respectively by student's T test

Example 6

Treatment of Pregnant Human with Sialyl Lewis Antigen Elevation Before 20 Weeks of Pregnancy

[0101] A 26 year old female presents at 10 weeks of pregnancy. Her serum is tested by the method of Example 2. The test detects sialyl Lewis antigen sLe levels above 25% average control normal.

[0102] These results are consistent with and predictive of consistent with incipient PE. She is then dosed with recombinant 100 IU hCG, i.v. The pregnancy comes to term without either insufficient trophoblast invasion or marked maternal spiral artery remodeling and inflammation.

Example 7

Treatment of Pregnant Human with Sialyl Lewis Antigen Elevation Before 20 Weeks of Pregnancy

[0103] A 26 year old female presents at 10 weeks of pregnancy. Her serum is tested by the method of Example 2. The test detects sialyl Lewis antigen sLe.sup.Y level of 18% above average control normal, and sLe.sup.x level of 18% above average control normal, and sLe.sup.a level of 15% above average control normal, with a combined percentage of over 50% above average control. These results are consistent with and predictive of incipient PE. She is then managed for preeclampsia. The pregnancy comes to term without either insufficient trophoblast invasion or marked maternal spiral artery remodeling and inflammation.