Patent Application
20110236394
The present invention relates to a method for modulating and monitoring PGF2α levels and activity in a subject in need thereof by modulating AKR1B1 (aldose reductase) levels or its PGFS activity in the subject. AKR1B1 as a therapeutic target, and method for modulating its expression and PGFS activity are provided.
Prostanoids, such as prostaglandins (PGs), thromboxanes (TXs) and prostacyclin (PGI2), are lipid compounds enzymatically derived from free fatty acids (FFAs). All prostanoids contain 20 carbon with a 5-carbon ring. Based on the initial fatty acid from which they are derived, either gamma-dihomolinolenic acid (DGLA), arachidonic acid (AA) or 5,8,11,14,17-eicosapentaenoic acid (EPA), there will be 1, 2 or 3 double bounds in the members of series 1 (ex.: PGE1), series 2 (ex.: PGE2) or series 3 (ex.: PGE3) PGs respectively. The most important FFA in the western diet of both human and farm animals is AA, thus yielding the pro-inflammatory series 2 PGs. PGs are involved in a wide variety of physiological actions and processes, but are especially notorious for their involvement in pain, inflammation, thrombosis and cancer for which they have been a therapeutic target for more than a century. Altering the production of series 2 PGs or their relative proportion with omega-3 FFAs reduces insulin resistance and risks of heart disease.
The first rate-limiting step for the production of series 2 PGs is the release of AA from the cell membrane phospholipids via the phospholipase A2 (PLA2) or successive action of phospholipase C (PLC) and diglycerol lipase enzymes. AA is then converted into prostaglandin H2 (PGH2), the common precursor for all PGs, through the cyclooxygenase and peroxidase activities of prostaglandin H synthase (PGHS) also known as cyclooxygenase (COX). There are three COX isoforms, namely COX-1 (PGHS-1), COX-2 (PGHS-2) and COX-3 (PGHS-3).
COX-1 is a constitutively expressed enzyme localized mainly in the endoplasmic reticulum and involved in normal physiological functions, although it is however suspected of being upregulated in various carcinoma. COX-2 is predominantly localized on the nuclear envelope of the cell and its expression is induced by various growth factors, oncogenes, carcinogens and tumor-promoting phorbol esters. COX-2 has been previously associated with rheumatoid diseases, inflammation and tumorigenesis. COX-3 is a splice variant of COX-1, but its contribution in human physiological function remains to be established.
The various PGs isotypes have different and often opposed physiological effects, but share PGH2 as their common precursor. Therefore, the various COX isoforms, which represent the current therapeutic target of pharmacological control of PG action, do not a priori exhibit a selectivity on the production of a specific PG isotype over another. The production of specific PG isotypes is rather controlled by the various terminal prostaglandin synthases, all of them utilizing PGH2 as a substrate. Some active PGs can also be converted into another active isoform, PGD2 and PGE2 can be converted enzymatically into PGF2α and PGD2 spontaneously into PGJ2.
Prostaglandin F synthase (PGFS) is a terminal prostaglandin synthase that converts PGH2 into prostaglandin F2α (PGF2α). PGF2α has been found to be involved in several physiological processes including, for example, contraction of smooth muscle including uterus and vascular walls, luteolysis, renal filtration and regulation of ocular pressure. It has also been associated with the initiation of menstruation and with the uterine ischemia leading to menstrual pain, while its vasoconstrictive effect is suspected of playing a role in controlling menstruation bleeding. PGF2α has further been associated with premature labor, and recent observations in blood vessels, heart and nerve terminals suggested that it may contribute to complications associated with various diseases and disorders, such as diabetes, osteoporosis and menstrual disorders.
Another major PG is prostaglandin E2 (PGE2), produced from PGH2 by the terminal prostaglandin synthase prostaglandin E synthase (PGES). PGE2 often presents effects opposed to those of PGF2α on many physiological functions and processes, such as luteal function and smooth muscle contraction including that of blood vessels. However, prior art studies on PGs have mostly focused on the contribution of PGE2 to the notorious effects of PGs on pain, inflammation, and cancer, thus emphasizing the development of systemic and non-selective blockade of PG biosynthesis with Non Steroidal Anti-Inflammatory Drugs (NSAIDs) and COX inhibitors.
Positive contribution of PGs to normal physiological function has been described mainly in the female reproductive system, in which they are generally recognized as primary regulators of ovulation, uterine receptivity, implantation and parturition. In this respect, we have previously shown the importance of the balance between the relative effects of PGE2 and PGF2α. To date, most efforts have been concentrated on the identification of the PGES pathway leading to the production of PGE2. Accordingly, the main enzyme responsible for stimulated formation of PGE2 is microsomal PGES-1 (mPGES-1), which is considered as a major therapeutic target in different pathological conditions such as inflammation, pain, fever, anorexia, atherosclerosis, stroke and cancer.
PGF2α can be synthesized from three distinct pathways (
Until now, six enzymes having a PGFS activity have been identified. Of them, three were isolated from the cattle: lung-type PGFS (PGFS1), lung-type PGFS found in liver (PGFS2), and liver-type PGFS, also known as dihydrodiol dehydrogenase 3 (DDBX). The other three PGFS were respectively isolated from human, sheep and Trypanosoma brucei. As a group, these six enzymes belong to the AKR family, with the T. brucei enzyme belonging to the AKR5A subfamily, and the other five to the AKR1C subfamily. With the exception of the T. brucei enzyme, those enzymes also possess a 11-ketoreductase activity, thus giving them the ability to convert PGD2 into 9α,11β-PGF2α, a bioactive enantiomer of PGF2α. Bovine PGFS1 and PGFS2 have a Km value of 120 μM for PGD2 and of 10 μM for PGH2. DDBX possess Km values of 10 μM for PGD2 and of 25 μM for PGH2. The three bovine PGFS are closely related, with PGFS1 and PGFS2 sharing 99% identity, although produced from 2 different genes. DDBX is 86% identical to both PGFS1 and PGFS2.
Previous studies on the regulation of PGFS activity in bovine endometrium led to the conclusion that none of the PGFS of the AKR1c family were responsible for PGF2α production. This led to the identification of the bovine 20α-hydroxysteroid dehydrogenase (HSD) (bovine AKR1B5) as the functional PGFS responsible for PGF2α production in the bovine endometrium (Madore et al., J Biol Chem 278(13); 11205-12, 2003).
Aldo-keto reductases (AKRs) are generally soluble 37 kDa monomeric NAD(P)(H)-dependent oxidoreductases capable of reducing aldehydes and ketones to yield primary and secondary alcohols. The AKR family comprises approximately 140 members sharing minimal sequence identity (less than 40% overall), divided in 15 subfamilies. AKRs having protein sequences sharing more than 60% identity are grouped into subfamilies, with mammalian AKR1 representing the largest of the 15 subfamilies.
The human PGFS AKR1C3 (EC 1.1.1.213, 1.3.1.20 and 1.1.1.62), is an aldo-keto reductase of the 1C family generally associated with a HSD activity. It has been primarily studied for its type V 17β-HSD activity, and in this respect, was found to be expressed in the human endometrium.
Aldehyde reductase (AKR1A1; EC 1.1.1.2) and aldose reductase (AKR1B1; EC 1.1.1.21) are monomeric NADPH-dependent oxidoreductases sharing 51% identity and having wide substrate specificities for carbonyl compounds.
The best known and most widely studied human AKR is the human aldose reductase AKR1B1 (E.C. 1.1.1.21), previously demonstrated to be broadly expressed throughout the body and primarily associated with the polyol pathway (reduction of glucose into sorbitol). AKR1B1 is believed to be involved in metabolic disorders such as diabetes complications and comorbidities.
More recently AKR1B1 has been presented as a detoxification enzyme protecting against toxic aldehydes derived from lipid peroxidation. (Jin Y and Penning T M, Ann Rev Pharmacol Toxicol, 2007). According to this hypothesis, a series of metabolic reactions would deplete the NAD-NADPH pool and could thus explain complications associated with metabolic disorders such as diabetes.
AKR1B1 catalyzes the reduction of various aromatic and aliphatic aldehydes, including the aldehyde form of glucose, which is reduced by AKR1B1 to its corresponding sugar alcohol, sorbitol. Sorbitol can subsequently be metabolized to fructose by sorbitol dehydrogenase. Under normal glycemic conditions, this pathway only plays a minor role in glucose metabolism in most tissues. However, in diabetic hyperglycemia, the cells undergoing insulin-independent uptake of glucose are producing significant quantities of sorbitol. This leads to an accumulation of sorbitol in the cells because of the poor penetration of sorbitol across cellular membrane and its slow metabolism by sorbitol dehydrogenase. The resulting cellular hyperosmotic stress can induce diabetic complications such as neuropathy, retinopathy, and cataracts. Further, recent studies showed that AKR1B1 also possesses a high catalytic activity towards the reduction of lipid peroxides derived from aldehydes and their glutathione conjugates, suggesting that under normal glucose conditions, AKR1B1 could therefore protects the organism against oxidative stress (Obrosova I. G. et al., Curr Vasc Pharmacol 3(3); 267-83, 2005) and electrophilic stress (Barisani D. et al., FEBS Lett 469(2-3); 208-12, 2000).
The reduction of glucose into sorbitol by AKR1B1 has thus been linked to mechanisms involved in diabetes-related disorders, such as cataracts, renal disorders, neuropathies, cardiac ischemia and cerebral ischemia. Accordingly, various AKR1B1 inhibitors have been tested in the prevention of diabetes-related disorders, in order to try to regulate AKR1B1-induced sorbitol formation. However, AKR1B1 inhibitors developed by pharmaceutical companies, such as Tolrestat™, Statil™ and Zopolrestat™, and which are administered for blocking the reduction of glucose into sorbitol in diabetic subjects having neuropathies, have often been found to be associated with undesirable hepatic side-effects (
Numerous and highly complex metabolic reactions therefore appears to be involved in diabetic complications, but the mechanisms underlying AKR1B1 involvement, either from its polyol or lipid peroxidation activities, remains to be established.
NSAIDs and COX-2-specific inhibitors are widely used to treat pain, fever and inflammation. While current therapies with NSAIDs and COX inhibitors can alleviate some of the symptoms by blocking non-selectively the production of all PGs at various degrees, there is a need for a more subtle and targeted approach to treat such conditions. It is known that the production of different PG isoforms or expression of their receptors must be coordinated through crosstalk mechanisms in order to maintain homeostasis. However, under special conditions such as insulin resistance, diabetes, oxidative stress or COX inhibitor therapy, these intrinsic feedback mechanisms can become impaired or inoperative. Overproduction of PGF2α relative to PGE2 could therefore occur in such conditions, which could lead to ischemia for example. Underproduction of PGF2α relative to PGE2 could also occur, for example in the eye, which could ultimately result in an increased ocular pressure. Thus, in conditions where such intrinsic feedback mechanisms are partly or totally inoperative, new drug targets directed toward specific terminal synthases involved in the production of PGs and regulating their activity are highly desirable.
Considering the opposed effects of the various PGs on many physiological processes and functions along with the existence of regulatory crosstalk and feedback mechanisms allowing for a balanced ratio of PGF2α/PGE2, there is therefore a need for a new and more specific therapeutic target allowing fine control of specific PG isotypes production.
PGs are important regulators of female reproductive function and contribute to gynecological disorders. Menstruation depends on an equilibrium between vasoconstrictors such as PGF2α and vasodilators such as PGE2 and nitric oxide (NO). Certain disorders are known to involve a dysregulation of the balance between PGE2 and PGF2α levels. When such an unbalance implies higher levels of vasoconstrictor PGF2α compared to vasodilator PGE2, it has been observed that PGF2α induces sustained muscle contractions that can lead to muscle ischemia (Lundstrom, V., Acta Obstet Gynecol Scand, 1977, 56(3); 167-72). In cases where the balance is dysregulated towards higher PGE2 levels, abundant bleeding have been reported. Therefore, the balance of these two PGs with opposite effects is of primordial importance.
PGF2α shares with TXA2 the ability to contract smooth muscle including that of vascular walls. PGF2α receptors (FP) were recently discovered in the left heart ventricle and coronaries, thus suggesting a possible implication of PGF2α in cardiac ischemia. A similar pattern of expression was described in the human uterus and associated with uterine ischemia leading to menstrual pain. Moreover, following the approval of inhaled insulin for the treatment of diabetes, it was found that absorption was limited by the constriction of bronchi, an effect that could be potentiated by PGF2α. Ocular pressure and renal filtration are additional mechanisms in which PGF2α could play a role.
Because of its notorious role on inflammation and pain, the biosynthetic pathway leading to PGE2 synthesis has been well studied, while the one leading to PGF2α synthesis is poorly documented. The data presented herein describes for the first time the expression of AKR1B1 gene and protein and its functional association with PGF2α production in the human endometrium. Also presented are methods and tools for evaluating the risk of a subject towards AKR1B1-related disorders, and for developing modulators of the PGFS activity of AKR1B1.
AKR1B1 (EC 1.1.1.21) is an aldose reductase that has mainly been associated with the polyol pathway, and more recently with lipid deperoxidation. We have identified that the primary activity of this enzyme is rather a PGFS activity, catalyzing the transformation of PGH2 into PGF2α (
It is an aspect of the present invention to provide a method for decreasing the PGFS activity in a subject, said method comprising the step of administering an AKR1B1 inhibitor to said subject. In accordance with the present invention, the AKR1B1 inhibitor is preferably selected from the group consisting of inhibitor of AKR1B1 PGFS activity, inhibitor of AKR1B1 synthesis, inhibitor of AKR1B1 translation, inhibitor of AKR1B1 post-translational modification, regulator of AKR1B1 transit within the cytoplasm, and activator of AKR1B1 degradation; and is preferably one of a AKR1B1 siRNA and a AKR1B1 antibody. In further accordance with the present invention, the AKR1B1 inhibitor can be co-administered to the subject with at least one of a COX inhibitor, COX-2-specific inhibitor, FP receptor blocker, EP1 receptor blocker, EP3 receptor blocker, and a PGF2α antagonist. In yet further accordance with the present invention, the subject is a human subject.
It is another aspect of the present invention to provide a method for decreasing the levels of PGF2α in a subject, said method comprising the step of administering an AKR1B1 inhibitor to said subject. In accordance with the present invention, the AKR1B1 inhibitor is preferably selected from the group consisting of inhibitor of AKR1B1 PGFS activity, inhibitor of AKR1B1 synthesis, inhibitor of AKR1B1 translation, inhibitor of AKR1B1 post-translational modification, regulator of AKR1B1 transit within the cytoplasm, and activator of AKR1B1 degradation; and is preferably one of a AKR1B1 siRNA and a AKR1B1 antibody. In further accordance with the present invention, the AKR1B1 inhibitor can be co-administered to the subject with at least one of a COX inhibitor, COX-2-specific inhibitor, FP receptor blocker, EP1 receptor blocker, EP3 receptor blocker, and a PGF2α antagonist. In yet further accordance with the present invention, the subject is a human subject.
It is another aspect of the present invention to provide a method for treating or preventing a condition associated to an increase of PGF2α levels or activity in a subject, said method comprising the step of administering an AKR1B1 inhibitor to said subject. In accordance with the present invention, the condition is preferably selected from the group consisting of metabolic disorders, metabolic disorder complications, cardiac ischemia, cerebral ischemia, bronchial constriction, menstrual pain, renal dysfunction and premature labor. In accordance with the present invention, the AKR1B1 inhibitor is preferably selected from the group consisting of inhibitor of AKR1B1 PGFS activity, inhibitor of AKR1B1 synthesis, inhibitor of AKR1B1 translation, inhibitor of AKR1B1 post-translational modification, regulator of AKR1B1 transit within the cytoplasm, and activator of AKR1B1 degradation; and is preferably one of a AKR1B1 siRNA and a AKR1B1 antibody. In further accordance with the present invention, the AKR1B1 inhibitor can be co-administered to the subject with at least one of a COX inhibitor, COX-2-specific inhibitor, FP receptor blocker, EP1 receptor blocker, EP3 receptor blocker, and a PGF2α antagonist. In yet further accordance with the present invention, the subject is a human subject.
It is another aspect of the present invention to provide a method for increasing the PGFS activity in a subject, said method comprising the step of administering an AKR1B1 activator to said subject. In accordance with the present invention, the AKR1B1 activator is preferably selected from the group consisting of activator of AKR1B1 synthesis, activator of AKR1B1 translation, activator of AKR1B1 binding, and inhibitor of AKR1B1 degradation, AKR1B1 gene and AKR1B1 protein; and is preferably one of a nucleic acid encoding for at least the PGFS activity portion of AKR1B1 and a polypeptide having at least the PGFS activity of AKR1B1. In further accordance with the present invention, the AKR1B1 activator can be co-administered to the subject with at least one of a COX activator, COX-2-specific activator, FP receptor activator, EP1 receptor activator, EP3 receptor activator, and a PGF2α agonist. In yet further accordance with the present invention, the subject is a human subject.
It is another aspect of the present invention to provide a method for increasing the levels of PGF2α in a subject, said method comprising the step of administering an AKR1B1 activator to said subject. In accordance with the present invention, the AKR1B1 activator is preferably selected from the group consisting of activator of AKR1B1 synthesis, activator of AKR1B1 translation, activator of AKR1B1 binding, and inhibitor of AKR1B1 degradation, AKR1B1 gene and AKR1B1 protein; and is preferably one of a nucleic acid encoding for at least the PGFS activity portion of AKR1B1 and a polypeptide having at least the PGFS activity of AKR1B1. In further accordance with the present invention, the AKR1B1 activator can be co-administered to the subject with at least one of a COX activator, COX-2-specific activator, FP receptor activator, EP1 receptor activator, EP3 receptor activator, and a PGF2α agonist. In yet further accordance with the present invention, the subject is a human subject.
It is another aspect of the present invention to provide a method for treating or preventing a condition associated to a decrease of PGF2α levels or activity in a subject, said method comprising the step of administering an AKR1B1 activator to said subject. In accordance with the present invention, the condition is preferably selected from the group consisting of hyperglycemia, inflammation and impaired renal function. In accordance with the present invention, the AKR1B1 activator is preferably selected from the group consisting of activator of AKR1B1 synthesis, activator of AKR1B1 translation, activator of AKR1B1 binding, and inhibitor of AKR1B1 degradation, AKR1B1 gene and AKR1B1 protein; and is preferably one of a nucleic acid encoding for at least the PGFS activity portion of AKR1B1 and a polypeptide having at least the PGFS activity of AKR1B1. In further accordance with the present invention, the AKR1B1 activator can be co-administered to the subject with at least one of a COX activator, COX-2-specific activator, FP receptor activator, EP1 receptor activator, EP3 receptor activator, and a PGF2α agonist. In yet further accordance with the present invention, the subject is a human subject.
It is yet another aspect of the present invention to provide a method for diagnosing or predicting the occurrence of a side-effect associated with the use of a COX inhibitor in a subject, said method comprising the steps of
It is yet another aspect of the present invention to provide a method for predicting or diagnosing a side-effect associated with the use of a COX inhibitor in a subject, said method comprising a) establishing a normal activity level of ARK1B1 by measuring the activity level of ARK1B1 in a normal sample, said normal sample being selected from the group consisting of a sample of the subject prior to the use of a COX inhibitor and a plurality of samples from different subjects not using said COX inhibitor; b) taking a sample from said subject following the use of said COX inhibitor; c) measuring the activity level of ARK1B1 in said sample of step b); and d) comparing the measure of the activity level of AKR1B1 of step c) with the normal activity level established at step a); wherein a higher activity level of AKR1B1 in the sample of said subject compared to the normal activity level of AKR1B1 is indicative of a risk of developing or the presence of a side-effect associated with the use of a COX inhibitor by said subject.
It is yet another aspect of the present invention to provide a method for identifying a compound for alleviating a side-effect associated with the use of a COX inhibitor, said method comprising the steps of
It is yet another aspect of the present invention to provide a method for identifying a compound for alleviating a side-effect associated with the use of a COX inhibitor, said method comprising the step of a) providing the COX inhibitor to a cell system; b) providing the compound to the cell system, thereby producing a treated cell system; c) measuring at least one of the expression level and activity level of at least one of AKR1B1 and PGF2α in the treated cell system of step b); and d) comparing the at least one of the expression level and activity level of at least one of AKR1B1 and PGF2α in the treated cell system of step c) with at least one of the expression level and activity level of at least one of AKR1B1 and PGF2α in a non-treated cell system; wherein a lowering of at least one of the expression level or the activity level of at least one of AKR1B1 and PGF2α in the treated cell system with the at least one of the expression level and activity level of at least one of AKR1B1 and PGF2α in the non-treated cell system is indicative of the compound being a compound for alleviating a side-effect associated with the use of the COX inhibitor.
It is yet another aspect of the present invention to provide the use of a PGF/PGE ratio for the identification of a compound alleviating a side-effect associated with the use of a COX inhibitor, wherein said compound induces a decrease in the value of PGF/PGE ratio in a cell treated with said COX inhibitor. In accordance with the present invention, the side-effect associated with the use of the COX inhibitor is selected from the group consisting of cardiovascular side-effect, cardiac ischemia, heart failure, respiratory side-effect, cerebral ischemia, polyneuropathy, vision trouble, kidney dysfunction, menstrual disorders, heartburn, nausea, vomiting, stomach pain, swelling of feet, swelling of ankle, joint pain, muscle pain, weakness, bleeding, persisting sore throat, fever, diarrhea and headache. In yet further accordance with the present invention, the COX inhibitor is a COX-2-specific inhibitor. In yet further accordance with the present invention, the subject is a human subject.
It is yet another aspect of the present invention to provide a human endometrial stromal cell line deposited at the International Depository Authority of Canada under Accession number IDAC 301008-04. It is another aspect of the present invention to provide a human endometrial epithelial cell line deposited at the International Depository Authority of Canada under Accession number IDAC 301008-05. It is a further aspect of the present invention to provide the use of the human endometrial stromal cell line having the Accession number IDAC 301008-04, and/or the human endometrial epithelial cell line having the accession number IDAC 301008-05 for the identification of a compound for alleviating a side-effect associated with the use of a COX inhibitor.
It is yet another aspect of the present invention to provide a method for alleviating a side-effect associated with the use of a COX inhibitor in a subject, said method comprising the step of administrating a PGF2α inhibitor to said subject. In accordance with the present invention, the PGF2α inhibitor is selected from the group consisting of inhibitor of PGF2α synthesis, inhibitor of AKR1B1 PGFS activity, inhibitor of PGF2α binding, FP receptor blocker, EP1 receptor blocker, EP3 receptor blocker, and PGF2α antagonist. In further accordance with the present invention, the side-effect associated with the use of the COX inhibitor is selected from the group consisting of cardiovascular side-effect, cardiac ischemia, heart failure, respiratory side-effect, cerebral ischemia, polyneuropathy, vision trouble, kidney dysfunction, menstrual disorders, heartburn, nausea, vomiting, stomach pain, swelling of feet, swelling of ankle, joint pain, muscle pain, weakness, bleeding, persisting sore throat, fever, diarrhea and headache. In yet further accordance with the present invention, the subject is a human subject, and in yet further accordance the subject has diabetes or insulin resistance.
It is yet another aspect of the present invention to provide a method for diagnosing or predicting at least one of a metabolic disorder, metabolic disorder complication, and a cardiac problem in a subject, said method comprising the steps of
It is yet another aspect of the present invention to provide a method for predicting at least one of a metabolic disorder and a cardiac problem, said method comprising the steps of a) taking a sample from a subject; b) measuring the concentration of PGFM and PGEM in the sample of step a); c) determining the PGFM/PGEM ratio; and d) comparing the PGFM/PGEM ratio of step c) with a normal PGFM/PGEM ratio value reflective of the absence of a metabolic disorder and a cardiac risk; wherein a higher PGFM/PGEM ratio determined from the sample of said subject compared to the normal PGFM/PGEM ratio is indicative of a risk of developing at least one of a metabolic disorder and a cardiac problem by said subject.
It is yet another aspect of the present invention to provide an immunoassay kit for determining a PGFM/PGEM ratio, said immunoassay kit comprising a container with anti-PGFM antibodies and a container with anti-PGEM antibodies.
It is yet another aspect of the present invention to provide a use of a PGF/PGE ratio for diagnosing or predicting at least one of a metabolic disorder and a cardiac problem in a subject, wherein said subject has a higher value of PGF/PGE ratio than a standard PGF/PGE ratio determined from a plurality of subjects not affected by said metabolic disorder or cardiac problem.
Having thus generally described the aspects of the invention, reference will now be made to the accompanying drawings, showing by way of illustration, preferred embodiments thereof, and in which:
The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.
All series 2 PGs originate from the same precursor, PGH2, which is synthesized from AA by the COXs enzymes. Specific terminal prostaglandin synthases can use this common substrate to produce specific PGs, with notably PGES catalyzing the synthesis of PGE2, and PGFS catalyzing the formation of PGF2α (
We evaluated a potential link between the two main COX isoforms, COX-1 and COX-2, and the various PG synthases with stimulators, inhibitors and knock-down experiments using siRNA. We confirmed the association between COX-2, mPGES-1 and PGE2. We also found an association between AKR1B1, both COX-1 and COX-2 and PGF2α, while a knock-down of AKR1B1 led to reduced levels of PGF2α but also of PGE2.
AKR1B1 was isolated and characterized with respect to transformation of glucose into sorbitol. However, this action occurs only in hyperglycemia, such as in diabetic subjects, since the glucose levels associated with normal glycemia conditions are not high enough to constitute a substrate for AKR1B1. In addition of catalyzing the formation of sorbitol from glucose, AKR1B1 also has a detoxificating action on peroxidized lipids, as reported by Srivastava (Srivastava et al., Endocr Rev 26(3); 380-92, 2005). At this point, it is worth noting that PGH2 is a peroxidized lipid. While Srivastava mentions that AKR1B1 exerts its detoxificating action by destroying peroxidized lipids, we rather propose that AKR1B1 has a constitutive physiological role within the organism, converting PGH2 into PGF2α, a biological molecule acting through specific receptors (
The newly identified PGFS AKR1B1, along with the known PGFS AKR1C3, are both present in the endometrium throughout the menstrual cycle (
AKR1B1 has been traditionally associated with reduction of glucose and diabetes-induced oxidative stress. Accordingly, AKR1B3 (the mouse aldose reductase now referred to as mouse AKR1B1) knockout mice have been used to study the pathogenesis of various diseases associated with diabetes mellitus, such as cataract, retinopathy, neuropathy and nephropathy. Reduced pathological responses were observed in these animals despite reduced intrinsic expression of aldose reductase in mice compared with humans. Interestingly, transgenic mice overexpressing human AKR1B1 have been found to be more prone to myocardial ischemic injury whereas knockout mice appeared to be protected against cerebral ischemic injury (Lo A C et al, J Cereb Blood Flow Metab. 2007 August; 27(8):1496-509), but the relationship between AKR1B1 and PGF2α was never established or even suggested.
With regards to the ratio between PGF2α and PGE2, one of the principal mechanism for preserving this ratio is the existence of a retro-feedback mechanism inducing an increase of PGE2 production by mPGES-1 via the early growth response factor 1 (EGR-1) transcription factor. This allows PGF2α produced by AKR1B1 to bind to its own membrane receptor (FP) and stimulate the expression of mPGES-1 (
These observations showed that AKR1B1 could be involved in the regulation of vascular tone under conditions where glucose metabolism is not involved. However, in presence of high glucose levels associated with diabetes, glucose becomes available as a substrate for AKR1B1 and competition among substrates may explain the development of vascular and neurological complications.
In the human endometrium, it has been previously reported that production of PGF2α is higher in late secretory and menstrual phases of the menstrual cycle. We have shown that AKR1B1 gene and protein levels increased significantly during the corresponding periods of menstrual cycle, whereas AKR1C3 does not vary (
We have established that human AKR1B1 is capable of metabolizing PGH2 and synthesizing PGF2α with a high efficiency (
Because of their association with inflammation and other pathological conditions, PGs as a whole are often considered as disorder-related molecules. Moreover, since successful clinical management of PGs is possible with NSAIDs, PGs are generally perceived as a single, unique factor. Accordingly, only two limiting steps are currently acknowledged in the synthesis of PGs: the release of AA from membrane phospholipids by phospholipases, and the generation of the intermediate PG metabolite PGH2 by COXs. However, these steps lead to the synthesis of a common precursor for several bioactive mediators, and not a priori directly to a specific PG isotype. PGs induce a wide variety of responses mediated by receptors, which are distinct for each PG isotype and are using various second messenger systems. For example, TXA2 and PGI2 exert opposing effects on coagulation and vascular tone to regulate hemostasis, while in the reproductive system, opposite actions are observed for PGF2α and PGE2.
In subjects affected with insulin resistance, insulin secretion is increased to maintain normal glucose levels. However, in these subjects, only one component of the insulin receptor is desensitized, corresponding to the PI3K pathway, whereas the other MAPK pathway remains intact. Therefore, higher insulin levels in insulin-resistant subjects are required to maintain normal glucose levels (silent condition), but the expression of insulin-responsive genes, including AKR1B1, are aberrantly expressed in response to these higher insulin levels.
The PGFS activity of AKR1B1 therefore predominates over the reduction of glucose or peroxylipids (
Similarly, the recently developed COX inhibitors Celebrex™ and Vioxx™ are COX-2 selective inhibitors that have proven extremely efficient to reduce pain and inflammation induced by PGE2. Unfortunately, the use of several COX inhibitors has been found to be associated with an increased risk of heart failure, whereas other common NSAIDs acting indistinctly on both COXs, such as naproxen, do not induce such cardiovascular side-effects, although they often induce gastrointestinal side-effects.
In the present study, we have clearly established an association between AKR1B1 expression, PGF2α production and the stimulation of PGE2 production in human endometrial stromal cells stimulated by IL-1β (
COX-2-specific inhibitors such as Vioxx™ are very efficient anti-inflammatory and anti-pain molecules. These drugs act preferentially by blocking the COX-2 pathway, which lowers the PGH2 available as a substrate for mPGES-1, thus decreasing the production of PGE2. In subjects having aberrantly high AKR1B1 levels, for example in subjects affected with a metabolic disorder such as type 2 diabetes, insulin resistance or obesity, and consequently high PGF2α levels, a compensatory mechanism induces PGE2 production in order to maintain an equilibrated PGF2α/PGE2 balance. However, if the same subjects have chronic pain in addition to their metabolic disorder, and these subjects are prescribed a COX-2-specific inhibitor for their chronic pain, PGF2α levels will rise to uncompensated pathogenic levels because the feedback mechanism involving mPGES-1 and subsequent production of PGE2 is rendered inoperative from the action of the COX-2-specific inhibitor. The resulting unbalanced PGF2α/PGE2 ratio can in turn induce a risk of cardiovascular ischemia caused by higher levels of PGF2α in the heart.
Since the feedback mechanisms to regulate the balance between PGF2α and PGE2 are impaired, it is thus imperative that a reduction in PGF2α must be performed in such a subject to prevent the cardiovascular side-effects. Such a regulation can be performed via PGF2α inhibitors, such as, but not limited to, inhibitors of PGF2α synthesis and inhibitors of PGF2α binding to receptors (FP, EP1 and EP3 receptors). Readily available inhibitors of AKR1B1 could be evaluated for their potential utility, despite the fact that these inhibitors have originally been designed for blocking the polyol activity of AKR1B1, and not the PGFS activity. Since AKR1B1 activity is regulated at two different molecular locations, the polyol and PGFS activities may be affected according to totally distinct dynamics. Therefore, the use of an already available AKR1B1 (polyol) inhibitor for blocking the PGFS activity of AKR1B1 is not recommended prior to testing for their capacity to block the novel PGFS activity of AKR1B1, because there are no guarantee that this novel activity will be blocked.
As used herein, the expression “PGFS activity” is intended to encompass a prostaglandin F synthase enzymatic activity as traditionally involving the transformation of PGH2 into PGF2α. A molecule having a PGFS activity, such as ARK1B1, is therefore intended to reflect on the ability of this molecule to catalyze the enzymatic transformation of PGH2 into PGF2α. The result of a molecule having a PGFS activity, provided it is contacted with the adequate substrate in the adequate conditions to exert its activity, is the production of PGF variants, such as PGF2α. This is reflected, in the case of PGF2α, by an augmentation of the PGF2α levels in the immediate environment of the molecule, or by an augmentation of its stable metabolites, such as PGFM, in blood circulation or urine.
The activity level of AKR1B1 is reflective of its PGFS activity in a biological environment, such as in a subject, having access to its PGH2 substrate, and transforming this substrate into PGF2α. The activity level of PGF2α is reflective of its action in a biological environment, such as in a subject, exerting its action directly or via a receptor. The expression level of AKR1B1, or of PGF2α, is reflective of the expression of the gene of AKR1B1, or of PGF2α, that is reflected on the level of AKR1B1 mRNA or protein, or of PGF2α, or of its stable metabolite PGFM. Measurements of expression levels and activity levels are performed according to techniques known in the art.
The expression “PGF/PGE ratio” as used herein is intended to encompass the ratio of prostaglandin F and its variants relative to prostaglandin E and its variants. While this ratio can be expressed as an activity ratio or an expression ratio, it is mainly intended to represent a concentration ratio. Examples of prostaglandin F variants include PGF1α, PGF2α, PGF3α, and PGFM, while examples of prostaglandin E variants include PGE1, PGE2, PGE3, and PGEM. It will be understood that if a ratio is to be used with the concentrations of specific PGF variants, such as both PGF2α and PGFM for example, the ratio must be expressed in relation with concentrations the corresponding PGE variants, such as PGE2 and PGEM in this example, for the ratio to be consistent within the present invention. Further, the PGF/PGE ratio can include almost exclusively PGFM and PGEM, with virtually no PGF2α or PGE2, such as in the case of a PGF/PGE ratio measured in a urine sample for example, wherein most if not all of the native prostaglandins (PGF2α and PGE2) have been degraded before reaching the bladder.
The terms “treatment”, “treating” and the like as used herein are intended to encompass any kind of action performed to a subject having the effect of reducing or removing a cause or a symptom of a condition as defined in the text, including, but not limited to, the administration of a molecule (such as a AKR1B1 inhibitor, a PGF2α agonist, a PGF2α receptor blocker, etc) to the subject.
The terms “administering”, “administration” and the likes as used herein are intended to encompass the administration of the substance of interest into a site of interest in the subject by any means known in the art and suitably adaptable to the substance to be administered. For example, a pharmaceutical composition containing the substance of interest, such as an AKR1B1 inhibitor or a PGF2α receptor blocker for example, can be administered by parenteral, topical, oral, nasal, intrathecal, or local (e.g. as a cream or topical ointment) routes. Preferably, the administration is performed parentally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly. In addition, it will be understood that for the administration of a pharmaceutical composition comprising the substance of interest, the substance of interest has to be dissolved or suspended in an acceptable carrier, preferably an aqueous carrier. To that effect, a variety of aqueous carriers may be used, such as for example water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well-known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration. In addition, the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
The terms “prevention”, “preventing” and the like as used herein are intended to encompass any kind of action performed to a subject having the effect of preventing, stopping or slowing the progression of a condition as described in the text, including, but not limited to, the administration of a molecule (such as a AKR1B1 inhibitor, a PGF2α antagonist, a PGF2α receptor blocker, etc) to the subject. The prevention can be performed in a subject in which the condition has never developed, has started to develop, or is expected to develop.
The terms “prediction”, “predicting” and the like as used herein are intended to reflect on the determination of the risk of a subject to develop a condition, a disorder or a symptom. The prediction can be performed on a normal subject not affected by the condition, disorder or symptom, or on a subject affected by the condition, disorder or symptom, a prediction in the latter case being reflective on the evolution of the condition, disorder or symptom in response to the absence or presence of a treatment.
The terms “diagnosis”, “diagnosing” and the like as used herein are intended to reflect on the identification of a condition, a disorder or a symptom in a subject based on the determination of a physiological parameter, such as but not limited to the expression level of AKR1B1 or the activity level of AKR1B1, and the comparison of that same physiological parameter obtained from a subject known not to be affected by that condition, disorder or symptoms, or with a standard value for that particular physiological parameter.
The terms “alleviation”, “alleviating” and the like as used herein are intended to represent the removal, partial or total, of a side-effect normally occurring as a result of a COX treatment. The alleviation can be partial or total.
The conditions associated to an increase of PGF2α levels as mentioned herein are intended to encompass any kind of condition, disorder or symptom that can be clearly correlated with a general or local increase in PGF variants levels, such as PGF2α levels. Non-limitative examples of such conditions include metabolic disorders, obesity, type 2 diabetes, insulin resistance. Additional non-limitative examples of such conditions include cardiac ischemia, cerebral ischemia, bronchial constriction, kidney dysfunction. Further non-limitative examples of such conditions include menstrual pain or premature labor (
The side-effects associated with the use of a COX inhibitor, or COX inhibitor-associated side-effects, as mentioned herein are intended to encompass any kind of condition, disorder or symptom associated with the use of a COX inhibitor that appeared as a direct or indirect consequence of the COX inhibitor use. These side-effects can be associated with either the dosage or the duration of the COX inhibitor treatment, while the severity of the side-effect is not necessarily directly associated to the dosage or the duration of the COX inhibitor treatment. Non-limitative examples of side-effects associated with the use of a COX inhibitor include cardiovascular side-effects, respiratory side-effects, cardiac ischemia, cardiac failure, cerebral ischemia, polyneuropathy, vision disorder, visual perception trouble, kidney dysfunction, menstrual disorders, heartburn, nausea, vomiting, stomach pain, swelling of foot, swelling of ankle, joint pain, muscle pain, weakness, bleeding, persisting sore throat, diarrhea, headache and fever.
The expression “ARK1B1 inhibitor” as used herein is intended to encompass any molecule that can inhibit or lower AKR1B1 ability to exert its PGFS activity. Molecules exclusively inhibiting or lowering the transformation of glucose into sorbitol by AKR1B1, without affecting its ability to convert PGH2 into PGF2α, or any other PGH variants into its respective PGF variant, are not meant to be included within this term as used herein. Molecules inhibiting or lowering the transformation of glucose into sorbitol by AKR1B1, and also inhibiting or lowering, partially or totally, the ability of AKR1B1 to convert PGH2 into PGF2α, are meant to be included within this term as used herein. Known AKR1B1 inhibitors, such as Sorbinil™, Tolrestat™ and Zopolrestat™, or other AKR1B1 inhibitors mentioned in
The expression “ARK1B1 activator” as used herein is intended to encompass any molecule that can increase or stimulate AKR1B1 ability to exert its PGFS activity. Molecules exclusively increasing or stimulating the transformation of glucose into sorbitol by AKR1B1, without affecting its ability to convert PGH2 into PGF2α, are not meant to be included within this term as used herein. Activators of AKR1B1 transcription, translation or post-translational modifications are encompassed within this expression since they increase AKR1B1 levels available to exert a PGFS activity by stimulating its synthesis. Regulators of AKR1B1 transit within the cytoplasm are included as long as they increase the formation of PGF2α from PGH2 by AKR1B1. Inhibitors of AKR1B1 degradation are also included since they prevent the lowering of the AKR1B1 levels available to form PGF2α. It will be understood that the increase or stimulation of these inhibitors can directly affect the ability of an AKR1B1 molecule to produce PGF2α or generally increase the whole production of PGF2α by AKR1B1 in a biological system. Non-limitative examples of AKR1B1 activators include AKR1B1 gene, vector containing an AKR1B1 gene for at least the portion of the gene encoding for the PGFS activity, AKR1B1 protein and AKR1B1 peptide having the PGFS activity of the AKR1B1 protein.
The term “COX inhibitor” as used herein is intended to encompass any molecule inhibiting the expression of one or more COX enzyme gene, or the action of one or more COX enzyme protein. The COX gene and protein can be any COX, including COX-1, COX-2 or COX-3.
The expression “receptor blocker” as used herein is intended to encompass any molecule blocking the normal action or signaling pathway of a receptor prior or following the binding of its ligand, either by preventing the ligand to bind to the receptor, by preventing the receptor to bind to its ligand, by preventing the ligand-receptor complex from activating its second messenger system, or by preventing the ligand-receptor complex from being internalized in the cell. The receptors aimed to be blocked in the present application can be, for example, receptors having a sufficient affinity with PGF2α for binding with PGF2α and exerting the biological effect of PGF2α. Non-limitative examples of such receptors include FP receptor, EP1 receptor and EP3 receptor. Non-limitative examples of receptor blockers include FP receptor blocker, EP1 receptor blocker and EP3 receptor blocker.
The term “PGF2α antagonist” as used herein is intended to include any molecule that can bind to a PGF2α receptor in place of PGF2α, or that is capable of blocking a biological effect of PGF2α. A PGF2α antagonist can compete for a binding site with an endogenous PGF2α, thus preventing the endogenous PCF2α from exerting its effect.
The expression “subject” as used herein is intended to encompass any mammalian subject, such as for example a human or a dog.
The expression “biological fluid” as used herein is intended to encompass any fluid originating from a mammalian organism, such as for example blood, plasma, urine, saliva, sweat, and menses.
In the bovine endometrium, we previously demonstrated a strong PGFS activity of AKR1B5 recently renamed as bos taurus AKR1B1 (Gene ID: 317748), a new function for this enzyme previously known for its 20a-HSD and glucose metabolism activities (Madore et al., J Biol Chem 278(13); 11205-12, 2003). The human and bovine AKR1B1 both belong to the aldoketoreductase 1B family and share 86% identity or homology. The human AKR1B1 (Gene ID: 231) also known as the aldose reductase is highly expressed in the placenta for glucose metabolism and in the eye and kidney for osmotic regulation.
After identifying the bovine AKR1B1 as a functional PGFS, we have found that AKR1B1 expression was associated with PGF2α production in human endometrial cell lines and in decidualized stromal cells (Chapdelaine et al., Mol Hum Reprod, 12(5); 309-19, 2006). However, expression of AKR1B1 within the human endometrium and its ability to act as a PGF synthases to produce PGF2α remain to be investigated. Therefore, in the present study, we have studied the expression of both AKR1B1 and AKR1C3 at the mRNA and protein levels in non pregnant human endometrium across the menstrual cycle. We have also investigated their ability to produce PGF2α using human endometrial cell lines.
Endometrial biopsies were taken from women aged between 25 to 50 years with regular cycles (21-35 days) without hormonal treatment in the 3 months prior to biopsy collection and undergoing gynecological investigation for infertility or menorrhagia. Informed consent for donation of anonymous endometrial samples was obtained before tissue collection. Biopsies representing functionalis layer were collected with an endometrial curette (Pipelle) and dated according to the stated last menstrual period. The stage of the cycle (proliferative or secretory) was then confirmed by histological examination using the criterion of Noyes (Noyes et al., Fertil Steril 1; 3-25, 1950) and samples with conflicting dating were discarded. Shortly after collection, the tissue was put in cold Hank's solution, placed on ice and brought to the laboratory. The samples were washed, divided and portions processed differentially for RNA and protein analysis.
Analysis of AKR1B1 and AKR1C3 mRNAs was performed by competitive PCR. Briefly, biopsies (N=48) were processed immediately upon reception. RNAs was prepared in TRIzol™ Reagent according to the manufacturer's instructions and samples stored at −80° C. until used for competitive PCR analysis. To generate RNA template competitors, a 100 bp deletion was created in AKR1B1 cDNA contained in pEF6/V5 by cutting with Hpa1 (containing 2 restrictions sites) and self ligation while for AKR1C3 cDNA, a 150 bp deletion was done with Ppum1 and Bsg1 blunted with Klenow followed by self ligation. The resulting recombinants were linearized with Pme1, transcribed into RNA with T7 RNA polymerase, purified on a polyacrylamide gel and RNA quantified at 260 nm.
For competitive PCR analysis of endometrial RNA (20 μg), cDNA first strands were synthesized in presence of AKR1B1 or AKR1C3 RNA competitors with Superscript II™ reverse transcriptase using the following primers: AKR1B1 (344 by amplicon): forward 5′-gatgagtcgggcaatgtggttcc-3′ (SEQ ID NO:1) and reverse 5′-cttggctgcgatcgccttgatcc-3′ (SEQ ID NO:2); AKR1C3 (565 by amplicon): forward 5′-ctaaagccaggtgaggaactttc-3′ (SEQ ID NO:3) and reverse 5′-ctatcactgttaaaatagtggag-3′ (SEQ ID NO:4). PCR amplification was achieved as follows: 94° C. for 20 seconds, 55° C. for 30 seconds and 72° C. for 30 seconds during 35 cycles. Five RTs with different competitor concentrations were performed for both enzymes and PCR products were loaded on 1.5-1.7% agarose gel stained with ethidium bromide and bands quantified by image analysis using the AlphaImager 2000™ software (Alpha Innotech Corporation, San Leandro, Calif.).
For immunohistochemistry, 3 μm tissue sections of human endometrium were taken at different periods of the menstrual cycle, fixed in 4% paraformaldehyde and prepared as paraffin-embedded sections. Slides were deparaffinized in xylene and rehydrated using decreasing grades of ethanol. Endogenous peroxidase activity was blocked with 3% H202 in methanol. Antigen retrieval was done by heating the sections in 1M urea solution for 15 minutes in a microwave oven at medium power. Tissue sections were then blocked with 10% goat serum for 1 hour in a humidified chamber at room temperature followed by an overnight incubation at 4° C. with primary antibodies at optimal dilutions (AKR1B1 1:250 in-house rabbit anti-human), AKR1C3 1:200 (goat polyclonal, Abcam inc., Cambridge, Mass., USA), COX-1 1:500 (rabbit, kindly provided by Dr. S. Kargman, Merck, QC, Canada) and COX-2 1:750(rabbit, kindly provided by Dr. S. Kargman, Merck, QC, Canada)). Non-immune rabbit serum was used as the negative control. The next day, sections were washed in PBS and incubated 30 minutes at room temperature with biotinylated goat anti-rabbit IgG 1:200 (AKR1B1, COX-1 and -2) or rabbit anti-goat IgG 1:200 (AKR1C3) as secondary antibodies (Dako Diagnostic of Canada inc., Mississauga, ON, Canada). After washing with PBS, sections were treated with avidin-biotin-peroxidase complex (Vectastain™ Elite ABC kit, Vector Laboratories Inc., Burlingame, Calif., USA) followed by staining with 3-amino-9-ethyl carbazole. Finally, sections were washed with water and counterstained with Harris hematoxylin reagent (Sigma, Mississauga, Canada). The staining was evaluated subjectively by three blinded observers not involved with the present study, using a scoring system of immunostaining intensity interpreted as absent (O), weak (I), moderate (2), or intense (3). Individual scores for each slide were averaged and expressed as relative expression level.
Specific short interfering RNAs (siRNAs) for AKR1B1 were designed using the TROD (T7 RNAi Oligo Designer) software v. 1.1.2 (Dudek and Picard, Nucleic Acids Res, 32(Web Server issue); W121-3, 2004) designed for facilitating the identification of optimal oligonucleotides for the production of siRNA, with T7 RNA polymerase forward: 5′-aaattgttgagcaggagacggctatagtgagtcgtattacc-3′ (SEQ ID NO:5) and reverse: 5′-aagccgtctcctgctcaacaactatagtgagtcgtattacc-3′ (SEQ ID NO:6), according to the procedure of Donze (Donze et al., nucleic acid research, 2002, 30: e46) with RiboMax™ polymerase kit (Promega, Madison, Wis., USA). The resulting siRNA products were purified by ethanol precipitation and 100 ng/ml were used for transfection of cells grown in 6 or 24-well plates using Lipofectamine™ 2000 (Invitrogen).
Western blot analysis was performed with approximately 20 μg total proteins from cultured cells on a 10% SDS-PAGE gel, followed by electro-transfer onto nitrocellulose membrane. The primary antibodies used for the present study were rabbit AKR1B1 (dilution 1:1000) and COX-2 (dilution 1:10 000) anti-sera and goat AKR1C3 (dilution 1:500) anti-serum. A 8-actin monoclonal antibody (1:5000, Sigma, Mississauga, Ontario, Canada) was used as an internal control. Goat anti-rabbit IgG conjugated with horseradish peroxidase (HRP) (Jackson Immunoresearch Laboratories, West Grove, Pa., USA), rabbit anti-goat IgG HRP and goat anti-mouse IgG HRP were used as secondary antibodies. Chemiluminescence was analyzed with autoradiography films at optimal times of exposure following treatment of the membranes with Renaissance™ reagent (NEN Life Science Products, Boston, Mass., USA).
Northern blot analysis was performed with 20 μg total RNA from endometrial cells in culture on a 1.2% formaldehyde-agarose gel. Following electrophoresis, RNA were transferred overnight onto a nylon membrane in 10× saline-sodium citrate (SSC). The AKR1B1 cDNA probe was generated by labeling the −500 by cDNA fragment with [α-32P]dCTP (3000 Ci/mmol) (Perkin-Elmer Life Sciences, Markham, ON, Canada) using the Ready-To-Go™ DNA labeling Kit (Amersham/Pharmacia). Prehybridization (2-4 hours) and hybridization (overnight) were performed at 45° C. using UltraHyb™ solution (Ambion Inc., Austin, Tex., USA). Blots were then washed twice at 65° C. for 15 minutes in 0.5×SSC and exposed on BioMAX™ films for quantification. 18S ribosomal RNA was used to confirm uniform loading of RNA samples.
For cell culture transfection, immortalized human endometrial stromal cells (HIESC-2) and epithelial cells (HIEEC-22) were cultured in RPMI 1640 without phenol red, containing 50 IU penicillin-streptomycin supplemented with 10% whole fetal bovine serum (FBS). Ten percent dextran-coated charcoal-extracted FBS was used once cells have reached confluency. Knock-down and knock-in transfections of cells with AKR1B1 specific siRNA, AKR1B1 or AKRIC3 cDNAs in pCR3.1 expression vectors were performed with Lipofectamine™ 2000 for 4 hours in culture medium without antibiotic. Thirty-six hours after transfection (24 hours for siRNA transfection), cells were treated for 24 hours with recombinant human interleukin 1β (IL-1β) (1 ng/ml) (R&D Systems, Minneapolis, Minn., USA) or arachidonic acid (AA) 10 μM in RPMI 1640 medium without serum. At the end of the treatment period, the culture medium was recovered and stored at −20° C. until evaluation for PGF2α production.
For evaluation of AKR1B1 enzymatic activity by thin layer chromatography (TLC), recombinant AKR1B1 protein was overexpressed in Escherichia coli, purified, and the enzymatic activity was determined by inserting AKR1B1 in the NdeI restriction site of pET17B. HIS-TAG proteins were produced and purified on Nickel-sepharose column (Novagen). Enzymatic activity was measured by monitoring NADPH degradation at 340 nm. The assays were performed in 1 ml of 50 mM Tris-HCl pH 7.5, 100 μM NADPH with 10 to 100 μg of enzyme and various concentrations of PGH2. Migration was performed in ethyl acetate [110:50:20] water saturated solvent and detection of PGF2α production was achieved by spraying the TLC silica plates with phosphomolybdic acid 10% (v/v) in methanol and cooking the plate at 120° C. for 10 minutes.
Enzymatic immunoassay (EIA) was performed with an acetylcholinesterase-linked PGF2α tracer (Cayman) as described previously (Asselin et al., Biol Reprod 54(2); 371-9, 1996). Sheep anti-PGF2α (Bio-Quant, Ann Arbor, Mich., USA) was used as the selective antibody. Inter-assay and intra-assay coefficients of variations (n=12) were of 16% and 10% respectively. Statistical analysis was performed using ANOVA with Statview™ software (Abacus Concept, California). Values were considered statistically significant for p<0.05.
Analysis of mRNA expression for COX-1, COX-2, AKR1B1 and AKR1C3 was performed by competitive RT-PCR in endometrial biopsies collected at different period of the menstrual cycle (
Immunohistochemical staining for AKR1B1 and AKR1C3 was performed in endometrial samples collected at different phases of the menstrual cycle (
The effect of IL-1β, a known regulator of PG production, on AKR1B1 and AKR1C3 protein and their relative contribution to produce PGF2α was studied in cultured endometrial cells. Western blot analysis shows that when the endometrial cell lines HIESC-2 and HIEEC-22 are treated with IL-1β (1 ng/ml) an increase of COX-2 protein level is associated with an increase of AKR1B1 protein (
For analysis of the PGFS activity of AKR1B1, AKR1B1 recombinant protein was produced in Escherichia coli and purified on a nickel-nitrilotriacetic column. The recombinant protein was found to functionally reduce phenanthrenequinone and NADPH at a rate of 10 nmole/min/mg in presence of 40 μM PGH2 as monitored by absorbance at 340 nm (
Prostaglandins are important regulators of female reproductive function and contribute to gynecological disorders. Normal menstruation depend on an equilibrium between vasoconstrictors such as PGF2α and vasodilators such as PGE2 or nitric oxide (NO). Excessive production of contracting prostaglandins create an ischemia-reperfusion response causing painful menstruation or dysmenorrhea, whereas increased vasodilatation leads to abundant menstrual bleeding. NSAIDs represent the most important and widely used drugs on the market and they are all efficient to treat menstrual disorders at some level. However these drugs act at an early step of biosynthesis common for all PGs and not only the isotype responsible for the pathological response. Because of its notorious role on inflammation and pain, the biosynthetic pathway leading to PGE2 has been studied extensively, but that of PGF2α is poorly documented. The data presented describes for the first time the expression of two gene candidates, AKR1B1 and AKR1C3, and the corresponding proteins, and their functional association with PGF2α production.
In the human endometrium, it has been reported that production of PGF2α is higher in late secretory and menstrual periods of the menstrual cycle (Downie et al., J Physiol 236(2); 465-72, 1974). Accordingly, both AKR1B1 and AKR1C3 enzymes are present in the endometrium throughout the menstrual cycle. By contrast with AKR1B1 expressed in both stromal and glandular epithelial cells and modulated in accordance with endometrial PGF2α production, AKR1C3 expression is constant and completely absent in stromal cells. The absence of the only currently accepted human PGFS, i.e. AKR1C3, in stromal cells was surprising because we and others have shown that human endometrial stromal cells produce high levels of PGF2α that is further stimulated by cytokines such as IL-1β (
We have first demonstrated the ability of the purified recombinant human AKR1B1 to release PGF2α and metabolize PGH2 in vitro in presence of NADPH (
Together, these results show that AKR1B1 is the primary candidate to be considered as the functional PGFS responsible for PGF2α production in the human endometrium (
AKR1B1 has been previously studied, but its contribution to prostaglandin production had never been suspected. AKR1B1 has been traditionally associated with reduction of glucose and diabetes-induced oxidative stress. Accordingly, AKR1B1 knockout mice have been used to study the pathogenesis of various diseases associated with diabetes mellitus such as cataract, retinopathy, neuropathy and nephropathy (Ho et al., Mol Cell Biol 20(16); 5840-6, 2000). Interestingly, transgenic mice overexpressing human AKR1B1 were more prone to myocardial ischemic injury (Hwang et al. Faseb J 18(11); 1192-9, 2004), whereas knockout mice appeared protected against cerebral ischemic injury (Lo et al. J Cereb Blood Flow Metab 27(8); 1496-509, 2007). In hindsight, these observations are compatible with the involvement of AKR1B1 in the regulation of vascular tone by mechanisms distinct from glucose metabolism. Interestingly, this is a documented function of PGF2α and its FP receptor (Norel, Scientific World Journal 7; 1359-74, 2007). If PGFS activity or FP receptors are altered in presence of high glucose levels or aberrant insulin response, it could explain the development of vascular and neurological complications in diabetes.
Because of their association with inflammation and other pathological conditions, prostaglandins as a whole are considered as foes. Moreover, because NSAIDs, a single class of medication, are highly efficient to treat pain, inflammation and menstrual disorders, PGs are treated globally as if they were a single factor. There are two limiting steps in the synthesis of PGs; the liberation of AA from membrane phospholipids by phospholipases and the generation of the intermediate PG metabolite PGH2 by PGH synthases or COXs. These steps are common for all bioactive PGs and not limited a priori to the specific one that drives aberrant responses. PGs induce a wide variety of responses mediated by receptors distinct for each isoform and using several second messenger systems. In the vascular system, TXA2 and PGI2 exert opposing action on coagulation and vascular tone to regulate hemostasis. In the reproductive system the same is often observed for PGF2α and PGE2.
There have been reports showing that some terminal synthases are preferentially associated with a specific COX such as mPGES-1 with COX-2 or mPGES-2 with COX-1 (Ueno et al., Biochem Biophys Res Commun 338(1); 70-6, 2005). Intriguingly, in spite of significant and stimulus sensitive production of PGF2α, no co-localization or association was found between COXs and PGF synthases (Nakashima et al., Biochem Biophys Acta 1633(2); 96-105, 2003). Such associations would imply that inhibition of a specific COX could exert some selectivity on the release of a specific PG. In this respect ASA, the first marketed NSAID (ASPIRIN™) exhibits a slight preference for COX-1 and platelets thus yielding preferential inhibition of TXA2 over PGI2 in the vascular system. Similarly, the recently developed COX inhibitors such as Bextra™ and Vioxx™ are COX-2 selective and have proven extremely efficient to reduce pain and inflammation induced by PGE2 (Zeilhofer, Trends Pharmacol Sci 27(9); 467-74, 2006). Unfortunately, the use of these drugs has been found to be associated with an increased risk of heart failure whereas other common NSAIDs such as ibuprofen and naproxen, act on both COX with no distinction between COX-1 and COX-2 (Rainsford, Inflammopharmacology 13(4); 331-41, 2005). Therefore, acting at the level of terminal synthases responsible for the release of specific PG isotypes appears as a promising avenue to control the release of “bad” PGs while allowing the action of the “good” PGs.
AKR1B1 was first identified as a key enzyme of the polyol pathway and more recently as a detoxification enzyme involved in the reduction of a wide range of carbonyl compound including benzaldehyde derivatives, quinones, sugars and many lipid peroxidation end products such as 4-hydroxy trans-2-nonenal (HNE) and acrolein (Srivastava et al., Endocr Rev 26(3); 380-92, 2005). The present finding that AKR1B1 is a functional PGFS liberating PGF2α, a bioactive metabolite acting on a specific receptor, was unexpected and is highly challenging.
The retroviral vector SSR69 containing SV40 large TAG and a gene resistant to hygromycin was transfected with Effectene™ (Qiagen, Mississauga, ON, Canada) in the mouse amphotropic packaging cell line PA 317. The resulting colonies resistant to hygromycin (800 μg/ml, Roche, Mississauga, ON, Canada) were cultured, and the supernatants containing amphotropic viruses were collected and used to infect, separately, purified stromal and epithelial cells in primary culture. Endometrial cells grown in six-well plates were infected in the presence of polybrene (8 μg/ml, Sigma) for 6 h, and the procedure was repeated 24 h later. The day following the last infection, the cells were trypsinized and seeded in 10 mm dishes in the presence of hygromycin (400 μg/ml). The cultures were grown for 7-8 days until the TAG-infected cells formed colonies while control non-infected cells died in the presence of the antibiotic.
A total of 17 clones (17 colonies) for stromal cells and 50 for epithelial cells were picked by clonal selection (cloning o-ring) and grown in 24-well plates until confluency and then seeded in T-25 flasks. The PD for TAG clones was calculated as follows: n(PD)=log(final cells count)−log(inoculation cell count)/0.301. Because colonies are produced from a single cell, we calculated that at confluency, the initial PD in T-25 flask was 19.2. The TAG clones were maintained in complete culture medium unless specified differently. The clones were then selected according to their growth rate, production of PGs and response to IL-1β.
Two cell lines, one of stromal origin (HIESC-2) (IDAC deposit account number 301008-04) and one of epithelial origin (HIEEC-22) (IDAC deposit account number 301008-05), were selected and characterized thoroughly. Both cell lines produce significant levels of PGE2 and PGF2α that can be stimulated by IL1β. The epithelial cell line HIEEC-22 expresses the two PGFS AKR1C3 and AKR1B1 whereas HIESC-2 expresses only AKR1B1. In both cases, increased PGF2α production is associated with increased expression of COX-2 and AKR1B1. Both cell lines are ideal models for testing the effect of COX inhibitors and NSAIDs on different PG isoforms in an integrated manner (
Following the discovery of AKR1B1 as a major PGFS involved in the synthesis of PGF2α, and since AKR1B1 has been involved in diabetes-associated pathologies, and that its impact on cardiac and cerebral ischemia has been demonstrated in transgenic mice (Hwang Y. C. et al., 2004; Iwata K. et al., 2006; Vikramadithyan R K), we investigated if the PGFS function of AKR1B1 could allow for the identification of PGF2α as a molecule responsible for ischemia and pain.
The PGFS activity of AKR1B1 therefore represents a crucial step in the synthesis of PGF2α from PGH2 released by COX-1 and COX-2. The COX-inhibiting NSAIDs are commonly used in the treatment of headaches and muscle aches. Prior art studies of AKR1B1 mainly focused on its role in polyols synthesis or in lipid detoxification. While AKR1B1 inhibitors have been developed to treat pathological conditions such as diabetic complications, ischemic damage of non-cardiac tissues, and Huntington's disease (U.S. Pat. No. 6,696,407, U.S. Pat. No. 6,127,367, U.S. Pat. No. 6,380,200), the identification of AKR1B1 as a PGFS has never been considered or used as an end issue. Our observation led us to consider AKR1B1 as a new alternate target to regulate PGF2α output associated with pathologic conditions more selectively than NSAIDs used as COX-1 and COX-2-specific inhibitors.
NSAIDs inhibiting COX-1 induce ulcers and other gastric problems, while COX-2-specific inhibitors possess analgesic properties used in the treatment of pain associated with arthritis, rheumatism and inflammation. However, pharmaceutical companies have developed powerful analgesic agents specifically targeting COX-2 for treating arthritis and related disorders without presenting the gastric side effects induced by COX-1 inhibitors. Most of those products, such as Vioxx™ and Bextra™, have been withdrawn from the market for having an increased risk in causing infarctus as a side effect. However, Aspirin™ and ibuprofen (both NSAIDs) are still used for their good analgesic activity.
Considering that ASA (Aspirin™) is the only NSAID clinically proven to exert cardio-protective effects, we hypothesized that this drug could be interacting directly with AKR1B1. The results of a dose-dependent inhibition of AKR1B1 protein with Aspirin™ (1-5 mM) in our cellular model confirmed this hypothesis (
We also demonstrated that the PGFS activity of AKR1B1 was modulated by 25 mM of D-glucose, showing putative competition with PGH2 at the catalytic site of the enzyme (
Because of the constitutive expression of COX-1 in most tissues and the induced release of AA and/or stimulation of COX-2 expression under many pathologic conditions, we herein propose that an increased expression of AKR1B1 induces an aberrant overproduction of PGF2α. In response, but depending on tissues, PGF2α overproduction can be compensated up to a certain point by the release of compensatory PGE2 through a FP receptor-dependent mechanism (
Low expression levels (basal) of AKR1B1 have been suggested to be involved in the protection against oxidative or electrophilic stresses (US 2006/0293265). In contrast, overexpression of AKR1B1 associated with COX is producing increased levels of PGF2α, which leads to pain via ischemia as previously shown with menstrual pain. However, since glucose is a poor substrate of aldose reductase, the PGFS activity of AKR1B1 therefore predominates in the whole organism since AKR1B1 is ubiquitously expressed, despite a greater expression in skeletal muscle, cardiac muscle, kidney, ovary, testis, prostate and small intestine (Jin et al, Annu Rev Pharmacol Toxicol, 47; 263-92, 2007). Activation or overexpression of AKR1B1 is achieved in response to primary signals such as osmotic shock, reactive oxygen species (ROS), and other localized stress agents. Depending on the physiological and toxicological context, the beneficial or detrimental effects associated with the expression level of AKR1B1 are related to the synthesis level of PGF2α from PGH2. Our laboratory has clearly shown that, in human endometrium, cytokines such as IL-1β and TNF-α (inflammatory and apoptotic cytokines) both increased the expression levels of COX-2 and AKR1B1 simultaneously (
Further, we showed that IL-1β stimulated AKR1B1 and COX-2 protein expression, as well as PGF2α production, in primary human umbilical artery smooth muscle cells (
In addition, a recent study demonstrated that AKR1B1 inhibitors such as Sorbinil™, Tolrestat™ and Zopolrestat™ were capable to reduce the production levels of PGE2 produced by macrophages treated with endotoxines or lipopolysaccharides (Ramana K. V. et al., 2006). In accordance with the present invention, this would result from a decrease in PGF2α production by AKR1B1, which is disrupting the equilibrium between the different PGs. Furthermore, ROS like hydrogen peroxide are capable of increasing both COXs and PGF2α expression levels in the endometrium, but to a lesser extent than cytokines.
In addition to demonstrating the PGFS activity of AKR1B1 and the regulation of its expression by IL-1β, we have previously characterized its gene promoter region. It showed an association between PGF2α production and gene regulatory signals acting on osmotic response elements (ORE), antioxidant response elements (ARE) and AP-1 sites, all of which were previously shown to increase the expression level of AKR1B1 (Jin Y et al., Annu Rev Pharmacol Toxicol 47; 263-92. 2007).
Moreover, we investigated if the cytosolic nature of AKR1B1 allowed for its coupling with either COX-1 or COX-2, or with a pool of COX-2 distinct from the one used by mPGES-1 and which would remain available in the presence of a COX-2-specific inhibitor such as rofecoxib (Vioxx™).
Therefore, under conditions of aberrant overexpression of AKR1B1, the use of NSAIDs, especially COX-2-specific inhibitors, to treat pain or inflammation may prevent the compensatory release of relaxing PGE2, thus leading to ischemic responses like those observed with Vioxx™ and Bextra™. Indeed, since PGs possess a compensation mechanism based on the expression of the different PG isotypes and receptors having antagonistic effects, undesirable side-effects of NSAIDs, COX-2-specific inhibitors and AKR1B1 inhibitors could originate from a perturbation in the equilibrium of the various PGs produced.
Consequently, the PGFS activity of AKR1B1 is a primary activity of this enzyme and represents a therapeutic target for the development and validation of modulators of its expression or activity. Moreover, FP receptor blockers and PGF2α agonists analogs acting on FP receptors are to be considered as efficient tools for controlling the aberrant PGFS activity of AKR1B1, or for compensating for the lack of PGFS activity of AKR1B1 that could result, for example, from a congenital disorder or from a pharmacological inhibition.
PGFM is a stable metabolite of PGF2α cleared in urine and that could be used in a diagnosis test to evaluate the metabolic status of any subjects if expressed relatively to PGEM levels. Normal subjects with normal PGF2α and PGE2 levels have an equilibrated PGFM/PGEM ratio with low absolute values. Subjects with insulin resistance should also have an equilibrated ratio, but with higher absolute levels of both metabolites. Subjects at risk of cardiovascular events would however have a higher PGFM/PGEM ratio, reflective of a higher concentration of PGFM relative to the concentration of PGEM.
Thus, the same ratio can be used as a safety measure before and during use of COX-2-specific inhibitors in a subject having insulin resistance, type 2 diabetes, or any other disorder or symptom motivating the administration of a COX-2-specific inhibitor. For example, a ratio PGF/PGE is used in order to monitor the safety of prescribing a COX-2-specific inhibitor to a subject having insulin resistance or type 2 diabetes. The determination of the PGF/PGE ratio is performed by measuring the concentration of PGF variants and the concentration of PGE variants in a biological sample, such as blood, urine or tissues, with an immunoassay such as a radioimmunoassay or an ELISA.
An ELISA test kit is developed with goat antimouse IgG antibody-coated microtiter plate wells. Controls and samples are introduced into the wells, and PGFM and/or PGEM tracers are added for example, in the case where the ratio to be observed is a PGFM/PGEM ratio. It will be understood that an ELISA kit can be developed using PGF20 and PGE2 tracers, or any other PGF20 and PGE2 variants, with the appropriate antibodies. The two tracers can be put together into a single well or in two separate wells, depending on the design of the ELISA test. Tracers can be conjugated with any kind of detection system, such as alkaline phosphatase or acetyl cholinesterase. The addition of a mouse monoclonal anti-PGFM and/or anti-PGEM (Cayman Chemical Company, MI USA) initiates the reaction. During incubation, there is a competition between the PGFM and/or PGEM present in the samples and the tracers for binding to the mouse anti-PGFM and/or anti-PGEM bound to the wells via the goat anti-mouse IgG antibody. Washing of the wells after the incubation period removes the unbound PGFM and/or PGEM, and addition of a substrate of the enzyme, such as p-nitrophenyl phosphate for alkaline phosphatase for example, allows for the plate to be optically read at a given wavelength, such as 405 nm. Addition of EDTA can be performed prior to reading to terminate the enzymatic reaction.
When the samples contain high levels of PGFM and/or PGEM, there is less tracers bound to the monoclonal antibodies, which results in lower optical density values. Lower levels of PGFM and/or PGEM do in turn produce higher optical density readings caused by the binding of a higher proportion of tracers to the monoclonal antibodies. The actual concentrations of PGFM and/or PGEM can therefore be calculated from the comparison of the optical densities of the samples with a reference curve established from the optical densities of the control wells having a known concentration of PGFM and/or PGEM.
If the measurement is performed in urine, urine samples are to be used in the test in order to normalize from urine dilution by obtaining the urinary creatinine values.
Immunoassays as described in example 4 can be used for predicting the predisposition or risk of a subject to develop a cardiac problem, such as cardiac ischemia or heart failure, before or after the occurrence of a metabolic disorder, such as obesity, type 2 diabetes or insulin resistance. Cardiac problems as used herein are also intended to encompass myocardial infarction and its complication, such as congestive heart failure, myocardial rupture, arrhythmia, cardiogenic shock and pericarditis. Additional examples of metabolic disorders includes, in a non-limitative manner, disorders of carbohydrate metabolism, disorder of amino acid metabolism, disorder of organic acid metabolism, disorder of fatty acid oxidation and mitochondrial metabolism, disorder of porphyrin metabolism, disorder of purin or pyrimidine metabolism, disorder of steroid metabolism, disorder of mitochondrial function, disorder of peroxisomal function, and disorder of lysosomal storage Non-limitative examples of metabolic disorder complications includes diabetes, osteoporosis, menstrual disorders, neuropathy, retinopathy, and cataracts.
Controls to be used for such a determination are to be reflective of the various stages or severity levels for the tested metabolic disorder or cardiac problem, that is with control values for various types of type 2 diabetes for example being reflective of the severity of the predisposition.
We believe that some COX-2-specific inhibitors, such as Vioxx™, do not target COX-2 activity alone, but rather the biosynthetic complex formed by the association of COX-2 and PGE2, making it highly efficient to block pain and inflammation, but also more prone to induce an aberrant PGF/PGE ratio. We therefore propose that the different prevalence of cardiovascular complications amongst NSAIDs and COX-2-specific inhibitors users depends on the relative ability of AKR1B1 to generate PGF variants in the presence of those drugs. This can be determined in vitro by comparing various PGF/PGE ratios obtained in presence of various doses of COX-2-specific inhibitors, and monitored in vivo by measuring PGFM and PGEM in urine and/or blood of the subjects. An immediate treatment in the case of a highly unbalanced PGF2α/PGE2 ratio for example could be the administration of a PGF2α receptor antagonist.
PGs-related compounds such as TXA2 and PGI2, are chemically unstable and strictly act locally at their site of biosynthesis. However, PGF2α and PGE2 have the chemical stability to allow action on cells and tissues adjacent to the site of production through a paracrine action limited only by the PG transport system that we have described in the bovine and human uterus. None of the PGs can exert a systemic response because after entering general circulation they are enzymatically degraded in the lungs.
COX-1 is a constitutive enzyme that reacts instantly to elevated concentrations of AA, transforming it into PGH2, which is next converted into PGF2α by AKR1C3 or AKR1B1 as needed. COX-2 is an inducible enzyme, reacting to lower concentrations of AA than COX-1, and transforming it into PGH2, which, most of the time, will be converted into PGE2 by mPGES-1. PGE2 can be converted into PGF2α by a 9 KPGR, in order to maintain a PGF2α/PGE2 ratio suitable for an equilibrium of the opposed effects of those two PGs (Farina, M G et all 2006 POLM 79; 260-270). During the menstrual cycle, if this ratio switches in favor of PGE2, the subject will present abundant bleeding. By opposition, if the ratio switches in favor of PGF2α, it will induce myometrial and vascular contractions, which can lead to myometrial ischemia and menstruation-related pain.
We examined the effect of a knock-down of the genes encoding the two terminal PG synthases mPGES-1 and AKR1B1 on PGs production in cells. A knock-down of mPGES-1 induced a decrease only in the synthesis PGE2, as expected, but surprisingly, the knock-down of AKR1B1 induced a decrease in the synthesis of both PGF2α and PGE2. Therefore, it appeared that when AKR1B1 was blocked, mPGES-1 activity but not expression was also blocked. Conversely, as verified by a knock-in of the AKR1B1 gene, an increase in AKR1B1 activity also induced an increase in mPGES-1 activity, leading to PGE2 synthesis. Therefore, it seems that there is a cross talk between the biosynthetic enzymes leading to PGE2 and PGF2α thus ensuring the balance between the two. Increased PGF2α resulting from excess AKR1B1 activity in response to osmotic stress, insulin resistance or else would then be compensated by increase mPGES-1 and PGE2. However, if this mechanism is blocked such as in presence of Vioxx™, excess PGF2α will eventually build up and combined vasoconstriction and increased left ventricle contraction generate ischemic responses and heart failure.
Glucose, along with ROS, induces AKR1B1 expression, which will convert glucose into sorbitol. Insulin also induces the expression of AKR1B1 gene through p38MAPK and PI3K. However, the conversion of glucose into sorbitol only occurs at high concentrations of glucose, such as in diabetic subjects. During normal glycemia, AKR1B1 does not convert glucose, but it will still continue to exert its other activities, such as PGFS and detoxification of peroxidized lipids. In subjects presenting insulin resistance, the glucose transport is affected, which results in a higher production of insulin for preserving the glycemia at a level close to normal. But because only the PI3K component of the insulin receptor is desensitized, the higher concentrations of insulin will likely induce an increase in the expression rate of insulin-responsive genes regulated by the p38MAPK transduction system, including the AKR1B1 gene (Kang E S et al Free radical Biology and Medicine 43: 535-545 2007).
AKR1B1 possesses two enzymatic pockets one rigid and one adaptative (Steuber et al., J Mol Biol 369(1); 186-97, 2007). Therefore, it is theoretically possible to interfere or regulate its activity without binding on the active rigid site, or compete directly with other substrates. This may explain how glucose exhibiting a molecular structure distinct from PGH2 can both be metabolized by AKR1B1 and interfere with its PGFS activity.
Aspirin™ blocks the expression of the AKR1B1 gene, but COX-2-specific inhibitors, such as Vioxx™, do not influence AKR1B1 gene expression. However, COX-2-specific inhibitors are likely to block preferentially the formation of PGE2, because mPGES-1, the most important inducible PGES, is strictly associated with COX-2 to produce PGE2. However, as previously mentioned, in cases of diabetes, oxidative stress and insulin resistance, the expression of AKR1B1 is increased. If any subject described previously experiences chronic pain symptoms, knowing that COX-2-specific inhibitors are amongst the most efficient anti-inflammatory and analgesic drug, they will be prescribed with COX-2-specific inhibitor or NSAIDs. However, since AKR1B1 expression is increased, preferred blocking of PGE2 synthesis will switch the equilibrium of PGs toward a high concentration of PGF2c, compared with a much lower concentration of PGE2 thus favoring ischemic responses.
Because of the promiscuity between mPGES-1 and COX-2, COX-2-specific inhibitors such as Vioxx™ are particularly efficient in blocking PGE2, they often represent the preferred option to block pain and inflammation. However, they are also more prone to induce aberrant PGF/PGE ratios. We claim that the different prevalence of cardiovascular complications amongst NSAIDs and COX-2-specific inhibitors depends on the relative ability of AKR1B1 to generate PGF2α in their presence. This can be determined in vitro by comparing the PGF/PGE ratios in presence of various doses of COX-2-specific inhibitors and monitored in vivo by measurement of PGFM and PGEM in urine and blood (
Further, we show that the PGFS activity of AKR1B1 can be used as a therapeutic target to decrease the risks of COX-2-specific inhibitor-associated cardio-vascular disorders in subjects exhibiting aberrant overexpresssion of AKR1B1, such as in subject having insulin resistance or type 2 diabetes for example.
Such compounds for targeting AKR1B1 as a therapeutic target can be identified by traditional methods of drug screening, mainly by exposing a cell to a COX inhibitor and studying the effect of those compounds on the AKR1B1 expression or activity, on the PGF2α expression or activity, or on the PGF/PGE ratio in the cell. The exposition of the cell to the COX inhibitor can be reflective of a short term exposure (from 3 to 6 hours exposition) or of a long term exposure (from 2 to 7 days exposition). While such testing can be performed in vitro on human endometrial epithelial cells, human endometrial stromal cells, adipocytes, endothelial cells, human umbilical vein endothelial cells, kidney cells, HEK293 cells, smooth muscle cells, myoblasts, heart cells and cardiomyocytes, in vivo testing can also be performed on traditional animal models such as mouse or rats. Preferably, all cells are human cells, and the animal models for in vivo tests are transgenic animals expressing or overexpressing human AKR1B1. Cell lines such as human endometrial stromal cell line HIESC-2 (IDAC number 301008-04) and human endometrial epithelial cell line HIEEC-22 (IDAC number 301008-05) can also be used for in vitro tests.
Use of a PGF/PGE ratio for the identification of a compound alleviating a COX inhibitor-associated side-effect, wherein said compound induces a decrease in the value of PGF/PGE ratio in a cell treated with said COX inhibitor.
Omega-3 and omega-6 fatty acids have been greatly publicized over the last few years. Since PGs possess a 20 carbon atoms structure derived from fatty acids, they can easily be synthesized from omega-3 and -6 fatty acids having 20 carbons, such as from the omega-3 fatty acid eicosapentaenoic acid (EPA), and from the omega-6 fatty acids dihomo-gamma-linolenic acid (DGLA) and arachidonic acid (AA). For example, under the action of COX, DGLA can be converted into PGH1, AA into PGH2 and EPA into PGH3, which, under the effect of the PGFS action of AKR1B1, can respectively be converted into series 1, 2 and 3 prostaglandin F variants, namely PGF1α, PGF2α and PGF3α.
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/CA08/02012 | 11/14/2008 | WO | 00 | 5/9/2011 |
| Number | Date | Country | |
|---|---|---|---|
| 60988220 | Nov 2007 | US |
