WO2009071950A1

WO2009071950A1 - Methods for the detection of a threatened miscarriage

Info

Publication number: WO2009071950A1
Application number: PCT/GB2008/051162
Authority: WO
Inventors: Justin Konje
Original assignee: University Of Leicester
Priority date: 2007-12-07
Filing date: 2008-12-05
Publication date: 2009-06-11
Also published as: GB0723882D0

Abstract

The present invention relates to a method for detecting the outcome of pregnancy in pregnant mammals, particularly humans, presenting with a threatened miscarriage. In particular, the invention relates to a method for diagnosing a threatened miscarriage, comprising detecting the level of an endocannabinoid in a body fluid sample from a subject mammal.

Description

METHODS FOR THE DETECTION OF A THREATENED MISCARRIAGE

FIELD OF THE INVENTION

The present invention relates to a method for detecting the outcome of pregnancy in pregnant mammals, particularly humans, presenting with a threatened miscarriage. Aspects of the invention relate to materials and preparations for use in such methods.

BACKGROUND OF THE INVENTION

Approximately 40-50 percent of all human conceptions are lost before 20 weeks of gestation (Wilcox AJ et al. New EngJMed 1988; 319: 189-194). Factors postulated to be involved include failure of the maternal immune response to pregnancy and an adequate hormonal milieu. Recent animal studies have suggested that the endocannabinoid, anandamide (N-arachidonoyl-ethanolamine, AEA) is critical for the synchronous development of the blastocyst and endometrium in preparation for successful implantation (Wang H, et al. J Clinical Invest 2006; 116:2122-31). Plasma AEA levels are regulated by fatty acid amide hydrolase (FAAH), the enzyme that metabolises AEA into arachidonic acid and ethanolamine (Di Marzo V et al. Nature 1991; 372: 686-91) . Increased FAAH expression and lower AEA levels have been demonstrated at the implantation site and low FAAH expression and high AEA levels at the inter-implantation site prior to successful implantation.

Current markers used to detect an individual at risk of a miscarriage include platelet activating factor (PAF), macrophage inhibitory cytokine 1(MIC 1) and human chorionic gonadotrophin. In particular, human chorionic gonadotrophin is clinically used to predict a risk of miscarriage in the first trimester of pregnancy, with low levels of the hormone being indicative of a high risk of miscarriage (Gaspard U et dXAnn Endocrinol 1984;45(4-5):269-80).

EPl 146128 discloses a method for detecting an individual at risk of miscarriage based on the levels of the enzyme anandamide hydrolase (also called fatty acid amide hydrolase (FAAH)) in lymphocytes. However, there are limitations to this method. The first is that it is dependent of lymphocytes that have to be obtained from whole blood (a process that is cumbersome). The second is the length of time taken to accurately quantify the levels of FAAH from isolated lymphocytes. This document discloses two methods for analysis of FAAH, one of which involves an assessment of enzyme activity through specific radiochromatographic methods and the other involves a measurement of enzyme expression levels by RT-PCR or ELISA. As such, the minimum time required for analysing FAAH from lymphocytes is around 7 hours, excluding the time taken to isolate and purify the lymphocytes.

Furthermore, although the document does disclose a method for detecting an individual at risk of miscarriage, it does not identify the effector that mediates these effects. Thus, although useful as a diagnostic tool, this document does not disclose a potential treatment for individuals at risk of miscarriage. Identification of such an effector would be difficult from this document, since all that is disclosed is an enzyme that has a number of functions and substrates not limited to hydrolysis of the endocannabinoid, anandamide.

Additional and more specific markers of miscarriage, which can be easily, accurately and quickly detected, would therefore be desirable.

SUMMARY OF THE INVENTION

In accordance with the present invention there is provided a method for diagnosing a threatened miscarriage comprising detecting the level of an endocannabinoid in a body fluid sample from a subject mammal, and comparing the detected level with a reference level, an elevated level in the subject mammal being considered indicative of a threatened miscarriage.

For the purposes of the current invention, mammals include, but are not limited to the Order Rodentia, such as mice; Order Lagomorpha, such as rabbits; more particularly the Order Carnivora, including Felines (cats) and Canines (dogs); even more particularly the Order Artiodactyla, Bovines (cows) and Suines (pigs); and the Order Perissodactyla, including Equines (horses); and most particularly the Order Primates, Ceboids and Simoids (monkeys) and Anthropoids (humans and apes). The mammals of one embodiment are humans. When the method is applied to humans the detected level is typically detected at six to twelve weeks gestation. For example, the level may be detected at eight weeks gestation. For other mammals, the relevant stage of gestation may be determined as appropriate.

The endocannabinoid useful in a method described herein can be anandamide (also known as arachidonylethanolamide or AEA). In alternative embodiments of the invention, the endocannabinoid may be 2-arachidonoylglycerol, or palmitoylethanolamide.

The reference level may be based on typical levels of the selected endocannabinoid at six to eight weeks of gestation. The reference level is generally equal to or less than 2.OnM plasma AEA.

The method may further comprise the step of taking a sample from the subject mammal for detection. The sample may be taken from a bodily fluid. The sample may also be taken from blood, plasma, saliva, sputum, urine, cervical or vaginal smears or swabs. Most preferably, the sample is blood or plasma. The detection may be performed in vivo, or preferably in vitro.

Detection of the level of the endocannabinoid may be performed in any suitable manner; for example, by means of HPLC, antibodies, receptors, binding molecules, and the like. In certain embodiments, the detection may involve the use of antibodies, which bind the endocannabinoid; these antibodies may be labelled with fluorescent or radioactive markers to allow detection, or may themselves be subsequently detected with labelled secondary antibodies or enzymes. Alternatively the antibodies may be used to precipitate the endocannabinoid out of solution. The antibodies may be in solution, or affixed to a solid support. Receptors or binding molecules may be used to detect the endocannabinoid; for example, the receptors CBl and CB2 are known to bind to AEA, and may be used in detection of AEA.

In certain embodiments of the invention, there is provided a method for diagnosing a subject mammal at risk of a miscarriage, the method comprising detecting the level of the endocannabinoid in a subject mammal, and comparing the detected level with a reference level, an elevated level in the subject mammal being considered indicative of a risk of miscarriage. The invention also relates to the use of an endocannabinoid in a method of diagnosis.

The invention also provides a kit for use in diagnosis of a subject mammal at risk of a miscarriage, the kit comprising reagents for detection of levels of an endocannabinoid in a sample. The kit may further comprise instructions for use of the reagents. The reagents may comprise antibodies against the endocannabinoid, or may comprise receptors or other binding molecules to the endocannabinoid. The antibodies or binding molecules may be labelled, for example with a fluorescent or radioactive label; alternatively the kit may comprise additional reagents for detection of bound antibodies or binding molecules. The kit may further comprise means for taking a sample from a subject mammal; for example, swabs, syringes, and the like.

According to a further aspect of the present invention, there is provided a method of prevention of a miscarriage, the method comprising reducing the levels of an endocannabinoid in a subject mammal at risk of a miscarriage. The endocannabinoid is preferably AEA. The levels of the endocannabinoid may be reduced by administering antibodies or other binding molecules against AEA; or may be reduced by administering an enzyme that degrades the endocannabinoid, such as FAAH. The levels may also be reduced by administering an activator of an enzyme that degrades the endocannabinoid, such as FAAH. The activator may include pharmacological activators or constitutively active forms of the enzyme. The levels of the endocannabinoid may also be reduced by administering hormones or steroids. More preferably, the levels may be reduced by administering progesterone or human chorionic gonadotrophin, which can up-regulate FAAH activity and subsequently reduce AEA levels. Accordingly, activators of enzymes that degrade endocannabinoids, such as FAAH, include hormones and steroids. Alternatively, the endocannabinoid may be 2-arachidonoylglycerol or palmitoylethanolamide.

The invention also provides for the use of an activator of an enzyme that degrades the endocannabinoid, such as FAAH, in the preparation of a medicament for the prevention of a miscarriage in a subject mammal diagnosed with such a risk. Also provided is a method for inducing an abortion in a subject mammal, the method comprising elevating the levels of an endocannabinoid in a subject mammal. The endocannabinoid is preferably AEA. The method may comprise the step of administering AEA itself, or may comprise administering an analogue thereto. Alternatively the levels of FAAH may be reduced, or a competitor to AEA may be administered to bind to FAAH, so reducing the effective levels thereof. Alternatively, the endocannabinoid may be 2-arachidonoylglycerol or palmitoylethanolamide.

BRIEF DESCRIPTION OF THE FIGURES

These and other aspects of the invention will now be described by way of example only and with reference to the accompanying drawings, which show:

Figure 1 : Characteristics of the study group, to include mean age, gestational age at recruitment, AEA concentration levels, time to miscarriage from recruitment, gestation age at delivery or miscarriage and birth weight of successful pregnancies.

Figure 2: Plasma AEA levels in women with threatened miscarriages that proceed to a live birth in comparison with women that have a miscarriage.

EXAMPLE

This example shows that plasma AEA levels <2nM at presentation with threatened miscarriage, were 100% predictive of a subsequent live birth.

AEA levels were measured in plasma from 45 healthy pregnant non-smokers, body mass index <30kg/M², presenting to the Early Pregnancy Assessment Unit at 6-12 weeks gestation with a threatened miscarriage using a high performance liquid chromatography-mass spectrophotomtery isotope dilution method (see, for example, Habayeb OM, et al. J Clin Endocrinol Metab 2004; 89: 5482-5487). The extraction and quantification was undertaken within 2 hours of blood collection. A power calculation (α= 0.05 and β= 0.8) based on published AEA data (see Habyeb, (2004), ibid, and Maccarrone M, et al. MoI Hum Reprod 2002; 8:188-195) indicated that the minimum number of subjects required in each group that would allow a significant difference in plasma AEA concentration to be observed was six. Ethics Committee approval for the study and signed informed consent from each volunteer were obtained.

The 9 women who subsequently miscarried (miscarriage group) and the 36 who had live births (live birth group) had similar characteristics (Figure 1). However, plasma AEA levels in the miscarriage group were approximately 2.73-fold higher than in the live birth group (3.39 ± 0.29nM v 24 ± 0.12nM {mean ± SEM} PO.0001; Mann- Whitney U-test; Figure 2). All those who miscarried had AEA values <2.0nM. One of the 2 patients with values >2.0nM, developed severe pre-eclampsia and delivered a 0.85Kg growth restricted baby at 33 weeks and the other had an uncomplicated 3.7Kg full term baby. Using an AEA level of 2.OnM as the optimal cut-off point, a single plasma AEA measurement provided a sensitivity of 100% (95%CI 66.37-100) and a specificity of 94% (95% CI 81.34-99.32) with a negative predictive value of 100% and a positive predictive value of 78% for subsequent miscarriage.

Thus, plasma AEA levels <2nM at presentation with threatened miscarriage, were 100% predictive of a subsequent live birth. Nine (75%) of the 12 patients that subsequently miscarried had levels >2.2nM. These results show that systemic AEA levels reflects the uterine distribution of the endocannabinoid system that is so critical to successful implantation and early pregnancy success reported in animals (Wang H, et al J CHn Invest 2006; 116:2122-31).

Claims

CLAIMS:

1. A method for diagnosing a threatened miscarriage, comprising detecting the level of an endocannabinoid in a body fluid sample from a subject mammal, and comparing the detected level with a reference level, an elevated level in the body fluid sample from the subject mammal being considered indicative of a threatened miscarriage.

2. The method of claim 1, wherein the body fluid sample is blood plasma, saliva or urine.

3. The method of claim 1 , wherein the mammal is a human.

4. The method of claim 1- 3, wherein the endocannabinoid is selected from the group comprising 2-arachidonoglycerol, palmitoylethanolamide and anandamide.

5. The method of claim 4, wherein the endocannabinoid is anandamide (AEA).

6. The method of any preceding claim, wherein the reference level is based on typical pregnancy levels for that species of mammal.

7. The method of claim 6, wherein the reference level is based on typical levels at six to twelve weeks' gestation.

8. The method of claim 1-7 wherein the reference level is equal to or less than 2.OnM anandamide (AEA).

9. The method of any preceding claim, further comprising the step of taking a sample from the subject mammal for detection.

10. The method of any preceding claim, wherein the detection is performed in vitro.

11. The method of any preceding claim, wherein the detection is performed by means of antibodies to the endocannabinoid.

12. The use of an endocannabinoid in a method for diagnosing a threatened miscarriage in a mammal, the method comprising detecting the level of the endocannabinoid in a body fluid sample from a subject mammal, and comparing the detected level with a reference level, an elevated level in the subject mammal being considered indicative of a threatened miscarriage.

13. A kit for use in the diagnosis of a threatened miscarriage, the kit comprising a reagent for detection of levels of an endocannabinoid in a sample.

14. The kit of claim 13, further comprising instructions for use of the reagent.

15. The kit of claim 13 or 14, wherein the reagent comprises antibodies against the endocannabinoid.

16. The kit of any of claims 13 to 15, further comprising means for taking a sample from a subject mammal.

17. A method of prevention of a threatened miscarriage in a mammal, the method comprising reducing the levels of an endocannabinoid in a subject mammal being considered at risk of a threatened miscarriage.

18. A method of prevention of a threatened miscarriage in a mammal, the method comprising elevating the levels of the enzyme fatty acid amide hydrolase (FAAH) in a subject mammal being considered at risk of a threatened miscarriage.

19. The use of a fatty acid amide hydrolase (FAAH) activator in the preparation of a medicament for treatment of a threatened miscarriage.

20. The use of claim 19, wherein the activator includes pharmacological activators, constitutively active forms of the enzyme, hormones or steroids.

21. The use of claim 20, wherein the activator is progesterone or human chorionic gonadotrophin.

22. A method of inducing an abortion in a subject mammal, the method comprising elevating the effective levels of an endocannabinoid in a subject mammal.