WO2022117828A1

WO2022117828A1 - Specialized pro-resolving lipid mediators for treating pcos

Info

Publication number: WO2022117828A1
Application number: PCT/EP2021/084195
Authority: WO
Inventors: Pedro Antonio REGIDOR; José Miguel RIZO; Rocío GUTIERREZ
Original assignee: Chemo Research , S.L.
Priority date: 2020-12-04
Filing date: 2021-12-03
Publication date: 2022-06-09
Also published as: MX2023006157A; EP4255412A1

Abstract

The present invention concerns a treatment of polycystic ovary syndrome (PCOS) by using specialized pro-resolving lipid mediators (SPM) or their active precursors, optionally in combination with a non-steroidal anti-inflammatory drug.

Description

SPECIALIZED PRO-RESOLVING LIPID MEDIATORS FOR TREATING PCOS

Field of the invention

The present invention concerns the use of specialized pro-resolving lipid mediators or their active precursors in the treatment of polycystic ovary syndrome and/or inflammation associated with polycystic ovary syndrome.

Background of the invention

The Polycystic Ovary Syndrome (PCOS) is a disease that causes irregular bleeding, chronic anovulation, androgen excess, and a typical ovarian ultrasound feature [1]. It affects between 5 and 10 % of women in their reproductive age, thus representing one of the most frequent causes of infertility [2]. The reasons for the development of a PCOS have not been resolved yet. Genetic predisposition, together with the gestational environment and lifestyle factors seem to be key contributors. The European Society of Human Reproduction and Embryology and the American Society for Reproductive Medicine defined the criteria for the definition of this disease [3, 4] at a meeting in Rotterdam in 2003.

Apart from the symptoms mentioned above, 30-40% of women with PCOS show a reduced glucose tolerance [5], often accompanied by insulin resistance, which both are independent of body mass index (BMI). 80% of the obese women and 30-40% of the lean individuals with PCOS suffer from hyperinsulinemia [6,7]. Hence, these aspects have been addressed by several studies to reach a general agreement on diagnostic criteria, it has been found that hyperinsulinemia is a key factor in the clinical pathogenesis of PCOS with its characteristic symptoms such as hyperandrogenism, chronic anovulation, typical PCOS ultrasound images, and skin issues such as acne, hirsutism, and seborrhea and seems to be independent of weight [8]. Excess insulin may lead to enhanced androgen synthesis by direct stimulation of the androgen production on the one hand and by reducing the serum levels of sex hormone-binding globulin (SHBG) on the other side [9].

However, the manifestation of PCOS is heterogeneous, and its etiopathology is still unclear [10,11]. As proven by familial clustering of PCOS incidence, results from twin studies, and the fact that specific endocrine and metabolic features of PCOS are heritable, genetic factors play an important role in the pathogenesis [10, 11 , 12]. It was further shown that embryos, which are exposed to unusually high levels of androgens and anti-Muller-hormone (AMH), have an increased risk for developing a PCOS later in life [13]. Additional factors, such as obesity, further have a substantial impact on the severity of PCOS symptoms [14, 15]. A state of chronic systemic inflammation is characteristic of obesity and can be determined by measuring increased serum levels of inflammatory cytokines and altered frequencies and functions of peripheral blood lymphocytes [16, 17, 18]. These changes are manifested at the tissue level and in adipose-, liver- and other tissue beds [17, 18]. They might be responsible for comorbidities that are often related to obesity, such as atherosclerosis, diabetes, and steatohepatitis [19, 20, 21 , 22, 23]. This kind of inflammation is often attributed to irregularities in innate immunity. However, innate and adaptive immune systems are closely interlinked, and consequently, obesity-related inflammation is associated with both processes [24].

Inflammatory processes occur due to traumatic events or infections and are also crucial for the turnover of cells during aging [25]. In this context, it is involved in the regulation of essential processes associated with cellular homeostasis, such as proliferation, necrosis, and apoptosis. Consequently, also by-products of cell necrosis and apoptosis, i.e., endogenous stimuli, can trigger inflammatory responses that are necessary for the regulation of tissue turnover. In obese individuals, systemic levels of free fatty acids are elevated, for example. These molecules are primary ligands of Toll-like receptors, which themselves are critical regulators of the innate immune response [26, 27]. In this way, the systems, which regulate obesity and inflammation, are linked directly.

A further relationship between inflammation and the metabolic system is visible on the cellular level, since adipocytes and macrophages are closely related, and their evolution might be traced back to a conventional primordial precursor cell [28].

It has also been demonstrated that insulin resistance and inflammatory processes are closely linked and may stimulate each other [29]. Both subclinical inflammation and insulin resistance are essential markers for the development of cardiovascular disease [30] and for women with PCOS, whose cardiovascular risks are elevated, a connection between inflammation and their hormonal- metabolic features were also shown [31].

In many chronic diseases, including vascular and neurological disorders, as well as metabolic syndrome, excessive inflammatory processes are manifested, thus representing a public health concern. When a host experiences a trauma, barrier breakage, or microbial invasion, potential invaders must be eliminated, the location must be cleared, and affected tissue must be remodelled and regenerated. For the acute inflammatory response, several lipid mediators are crucial. They include eicosanoids (prostaglandins and leukotrienes), which derive from arachidonic acid (ARA), an essential fatty acid [32, 33], and different cytokines and chemokines [35, 35, 36]. These molecules interact with each other, thereby further intensifying the inflammatory process that may, in turn, be counteracted with pharmacological inhibitors and receptor antagonists. Historically, the inflammatory response used to be separated into an active initiation and a passive resolution process [37]. Recently, however, mediators were identified which have pro-resolving capacities and can be synthesized from omega-3 (n-3) essential fatty acids (EFA). Studies have shown, that the resolution process can be "switched on" in animal models and may thus rather be an active response in the self-limitation of acute inflammation than a passive dilution of chemoattractants [38, 39].

Molecules, which are supposed to act as mediators, must be supplied in sufficient amounts in order to lead to reactions in vivo. For EPA and DHA, anti-inflammatory properties have been proposed for many years. These omega-3 fatty acids compete with arachidonic acid in reducing pro-inflammatory eicosanoids [40]. However, the underlying molecular mechanisms had remained obscure until recent results emerged, and whether EPA or DHA is more relevant for human health or therapeutic options is still under debate [40].

It has been shown for resolving inflammatory exudates that omega-3 fatty acids serve as substrates for the synthesis of specific signalling molecules - the so-called specialized proresolving mediators (SPMs), which comprise resolvins, protectins, lipoxins and maresins. These findings triggered new studies concerning the resolution pathways and the immune mechanisms underlying homeostasis. It was shown in animal models that SPMs promote critical paths of the inflammatory resolution, as they limit the infiltration of polymorphonuclear neutrophils and the elimination of apoptotic cells by macrophages [41].

Inflammations may be resolved entirely or become a chronic state. Previously, resolution of active inflammation has been considered a passive event, upon which inflammatory mediators such as prostaglandins or cytokines were merely diluted, thus disappearing from the site of inflammation. This would finally lead to prevent leukocyte infiltration into the tissue. However, Serhan et al. [41] provided new evidence to revise this theory by demonstrating the existence of an active resolution process mediated by so-called selective pro-resolving mediators (SPMs) in several studies. The SPM molecular superfamily contains subgroups named resolvins (Rvs), protectins, maresins, and lipoxins. SPMs are crucial for sufficient resolution of inflammatory processes, and based on these new findings, Serhan et al. described three novel pathways for the potential development of acute inflammation. They include the action of the SPMs.

Importantly, within this new perception of inflammatory processes, the resolution is an active mechanism, which does not start with a delay, but at experimental timepoint Zero.

Alfa signals Omega throughout the course of inflammation, mainly SPMs were found to repress inflammatory signals by ending tissue infiltration of neutrophils and preventing further recruitment of immune cells to the site of inflammation. Subsequently, phagocytic macrophages are stimulated, which further leads to increased clearance and elimination of apoptotic polymorphonuclear neutrophils (PMNs) by efferocytosis and phagocytosis [44]

SPMs are biosynthesized from eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3). Both are omega-three polyunsaturated fatty acids (PUFAs) and serve as precursors in the biochemical pathways leading to SPMs via the metabolites 18- hydroxyeicosa pentaenoic acid (18-HEPE), 17-hydroxydocosahexaenoic acid (17-HDHA), or 14- hydroxydocosahexaenoic acid (14-HDHA).

SPMs or their active precursors, 18-HEPE, 17-HDHA and 14-HDHA, could therefore potentially be used to elicit dynamic resolution of inflammation. In contrast to traditionally applied antiinflammatory therapies, they do not act as immunosuppressors, and debris is cleared, thus being potentially useful for the treatment of chronic inflammation. Substances, which are currently applied, have distinct disadvantages: steroids may interfere with wound healing, can promote osteoporosis, and is immunosuppressive. NSAIDs may lead to stomach bleeding, are potentially toxic for the cardiovascular system and the kidneys and interfere with wound healing. Cyclooxygenase (COX)2 inhibitors constitute a risk factor for cardiovascular and thromboembolic events. Anti-TNF therapies for blocking cytokines lead to increased rates of infections and enhance the risk of lymphoma development.

SPMs, on the other hand, were shown to increase the killing of microbial invaders and their clearance by immunocytes. It was demonstrated that they down-regulate infiltration and recruitment of PMN, enhance phagocytosis, and efferocytosis (M1 to M2). Application of SPMs also decreased the level of pro-inflammatory chemical mediators, while increasing the number of anti-inflammatory mediators like IL-10, for example. Finally, they can reduce inflammatory pain, stimulate the regeneration of inflamed tissue, and promote wound healing.

In obesity, adipose tissue is characterized by high levels of pro-inflammatory eicosanoids and depleted levels of SPMs. DHA or EPA is required for the formation of SPMs, but enzymes are critical to the formation of the SPM circuit. In some inflammatory conditions, lack of SPMs has been related to the inability of immune cells for processing SPM substrate (EPA /DHA). Thus, supplementation with EPA and DHA would be ineffective in restoring SPM levels.

Basil et al. (Nature Reviews Immunology, 16, 2016, 51-67) refer to specialized pro-resolving mediators being endogenous regulators of infection and inflammation. WO 2012/080273 discloses N-acetyl-L-cysteine for use in the treatment of a mammal having PCOS. Claria et al. (Molecular Aspects of Medicine, 58, 2017, 83-92) report on SPMs and their role in inflammation in adipous tissue. Without being bound by a particular theory, the present inventors have found that PCOS is characterized by depleted levels of SPMs and that therefore administration of 17-HDHA, 14-HDHA, 18-HEPE, or SPMs perse are effective in restoring SPM levels in PCOS patients.

Summary of the invention

In one aspect, the present invention concerns a composition for use in the treatment of polycystic ovary syndrome (PCOS), wherein said composition comprises a specialized pro-resolving lipid mediator (SPM), an SPM precursor and/or a pharmaceutically acceptable salt thereof, as well as any stereoisomer thereof. The patients are typically characterized by low levels of SPMs. PCOS is associated with inflammation in general and chronic inflammation in particular. Accordingly, in another aspect, the present invention concerns a composition for use in the treatment of inflammation associated with polycystic ovary syndrome (PCOS), preferably for use in the treatment of chronic inflammation, wherein said composition comprises a specialized pro-resolving lipid mediator (SPM), an SPM precursor and/or a pharmaceutically acceptable salt thereof, as well as any stereoisomer thereof.

In another aspect, the present invention concerns a method of treating polycystic ovary syndrome (PCOS) by administration of a therapeutically effective amount of a composition comprising one or more of a specialized pro-resolving lipid mediator (SPM), an SPM precursor and/or a pharmaceutically acceptable salt thereof, as well as any stereoisomer thereof.

The treatment of PCOS may further comprise the administration of a non-steroidal antiinflammatory drug (NSAID), such as acetylsalicylic acid. Without being bound by a particular theory, the non-steroidal anti-inflammatory drug, such as acetyl salicylic acid, functions as a coenzyme for lipoxygenase and thus contributes to the bio-synthesis of SPMs.

Brief description of the figures

Figure 1 : Results of the ARA-derived proinflammatory mediators of healthy and PCOS patients.

Figure 2: Results of quantified free fatty-acid-precursors of resolving mediators,

Figure 3: Results of the ratio pro-inflammatory parameters / SPMs including the monohydroxylates in serum compared to plasma. Detailed description of the invention

Definitions

In the present context, the term "SPM" or “specialized pro-resolving lipid mediator” is intended to mean a compound which is a resolvin, a protectin, a lipoxin, or a maresin.

In the context of the present invention, the term "resolvin" is intended to cover resolvins resulting from the cascade of EPA, also referred to as “E series resolvins”, and from the cascade of DHA, also referred to as “D series resolvins”. Examples of E series resolvins include the compounds RvE1 , RvE2, and RvE3. Examples of D series resolvins include the compounds RvD1 , RvD2, RvD3, RvD4, RvD5, and RvD6.

In the context of the present invention, the term "protectin'¹ is intended to cover protectins resulting from the cascade of DHA. Protectins include the compound PD1.

In the context of the present invention, the term "maresin" is intended to cover maresins resulting from the cascade of DHA. Maresins include the compounds MaR1 , MaR2, MCTR1 , MCTR2, and MCTR3.

In the context of the present invention, the term “SPM precursor” or “SPM precursors” refers to 18- hydroxyeicosa pentaenoic acid (18-HEPE), 17-hydroxydocosahexaenoic acid (17-HDHA), and/or 14-hydroxydocosa hexaenoic acid (14-HDHA).

The compounds of the present invention can be in a free form or in the form of a pharmaceutically acceptable salt. In the context of the present invention, the term “pharmaceutically acceptable salt” is to be understood as a salt formed with the acid group of the poly-unsaturated fatty acids (PUFAs) comprised by the SPMs and SPM precursors, wherein the resulting counter-ion does not significantly add to the toxicity of the compound of the present invention. Examples of pharmaceutically acceptable salts include alkali metal salts, such as sodium salts, potassium salts, etc. and alkaline earth metal salts, such as calcium salts, magnesium salts, etc..

Treatment of PCOS

In one aspect, the present invention concerns a composition for use in the treatment of polycystic ovary syndrome (PCOS), wherein said composition comprises one or more of a specialized proresolving lipid mediator (SPM), an SPM precursor and/or a pharmaceutically acceptable salt thereof, as well as any stereoisomer thereof. The depleted levels of SPMs observed in PCOS patients are an indication of deficient conversion of the substrates, EPA and DHA in the cascade leading to the SPMs. The active SPM precursors or the SPMs perse administered in accordance with the present invention may therefore alleviate this deficiency by re-establishing the deficient cascade. Administration of the SPM precursors is an interesting alternative to administration of the SPMs perse since the number of active precursors is limited compared to the number of SPMs per se. Furthermore, the resulting amounts of the individual SPMs are regulated by the physiological response to the active precursors.

Accordingly, in one embodiment, the composition of the invention comprises an SPM precursor and/or a pharmaceutically acceptable salt thereof, as well as any stereoisomer thereof. In a further embodiment, the composition comprises one or more compounds selected from the group consisting of 18-hydroxyeicosapentaenoic acid (18-HEPE), 17-hydroxydocosahexaenoic acid (17- HDHA), 14-hydroxydocosahexaenoic acid (14-HDHA) and pharmaceutically acceptable salts thereof, as well as any stereoisomers thereof. In another embodiment, the composition comprises two or more compounds selected from the group consisting of 18-hydroxyeicosapentaenoic acid (18-HEPE), 17-hydroxydocosahexaenoic acid (17-HDHA), 14-hydroxydocosahexaenoic acid (14- HDHA) and pharmaceutically acceptable salts thereof, as well as any stereoisomers thereof. In still another embodiment, the composition comprises three or more compounds selected from the group consisting of 18-hydroxyeicosapentaenoic acid (18-HEPE), 17-hydroxydocosahexaenoic acid (17-HDHA), 14-hydroxydocosahexaenoic acid (14-HDHA) and pharmaceutically acceptable salts thereof, as well as any stereoisomers thereof. In still a further embodiment, the composition further comprises EPA and/or DHA.

In an alternative embodiment, the composition according to the invention comprises an SPM and/or a pharmaceutically acceptable salt thereof, as well as any stereoisomer thereof. In another embodiment, the composition comprises one or more compounds selected from the group consisting of 5S,6R,15S-trihydroxy-7E,9E,11Z,13E-eicosatetraenoic acid (LxA4), 5S,14R,15S- trihydroxy-6E,8Z,10E,12E-eicosatetraenoic acid (LxB4), 5S,6R,15R-trihydroxy-7E,9E,11Z,13E- eicosatetraenoic acid (15-epi-LxA4), 5S,14R,15R-trihydroxy-6E,8Z,10E,12E-eicosatetraenoic acid (15-epi-LxB4), 5S, 12R, 18R-trihydroxy-6Z,8E, 10E, 14Z, 16E-eicosapentaenoic acid (RvE1 ), 5S,12R,18S-trihydroxy-6Z,8E,10E,14Z,16E-eicosapentaenoic acid (18S-RvE1), 5S,18R-dihydroxy- 6Z,8E, 10E, 14Z, 16E-ei cosa pentaenoi c acid (RvE2) , 17R, 18R/S-d i hyd roxy-5Z, 8Z, 11 Z, 13E, 15E- eicosapentaenoic acid (RvE3), 7S,8R, 17 S-trihydroxy-4Z,9E, 11 E, 13Z, 15E, 19Z-docosahexaenoic acid (RvD1), 7S,16R,17S-trihydroxy-4Z,8E,10Z,12E,14E,19Z-docosahexaenoic acid (RvD2), 4S,11R,17S-trihydroxy-5Z,7E,9E,13Z,15E,19Z-docosahexaenoic acid (RvD3), 4S,5R,17S- trihydroxy-6E,8E,10Z,13Z,15E,19Z-docosahexaenoic acid (RvD4), 7S,17S-dihydroxy- 4Z,8E,10Z,13Z,15E,19Z-docosahexaenoic acid (RvD5), 4S,17S-dihydroxy- 4Z,8E,10Z,13Z,15E,19Z-docosahexaenoic acid (RvD6), 7S,8R,17R-trihydroxy- 4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid (17R-RvD1), 7S,16R,17R-trihydroxy- 4Z,8E,10Z,12E,14E,19Z-docosahexaenoic acid (17R-RvD2), 4S,11 R,17R-trihydroxy- 5Z,7E,9E,13Z,15E,19Z-docosahexaenoic acid (17R-RvD3), 4S,5R,17R-trihydroxy- 6E,8E, 10Z, 13Z, 15E, 19Z-docosahexaenoic acid (17R-RvD4), 7S, 17R-dihydroxy- 4Z,8E,10Z,13Z,15E,19Z-docosahexaenoic acid (17R-RvD5), 4S,17R-dihydroxy- 4Z,8E,10Z,13Z,15E,19Z-docosahexaenoic acid (17R-RvD6), 7,13R,20-trihydroxy- 8E, 10Z, 14E, 16Z, 18E-docosa pentaenoi c acid (RvT 1 ) , 7 ,8, 13 E?-t r i hyd roxy-9E, 1 1 E, 14E, 16Z, 19Z- docosapentaenoic acid (RvT2), 7,12,13R-trihydroxy-8Z,10E,14E,16Z,19Z-docosapentaenoic acid (RvT3), 7,13R-dihydroxy-8E,10Z,14E,16Z,19Z-docosapentaenoic acid (RvT4), 7,8,17-trihydroxy- 8,10,13,15,19-docosapentaenoic acid (RvD1_n-3), 7, 16, 17-trihydroxy-8, 10, 12, 14, 19- docosapentaenoic acid (RvD2_n-3), 7,17-dihydroxy-8,10,13,15,19-docosapentaenoic acid (RvD5_n-3), 10R, 17 S-dihydroxy-4Z,7Z, 11 E, 13E, 15Z, 19Z-docosahexaenoic acid (PD1 ), 10S, 17S-dihydroxy- 4Z,7Z,11E,13E,15Z,19Z-docosahexaenoic acid (PDX), 10R,17S,22-trihydroxy-

4Z,7Z,11E,13E,15Z,19Z-docosahexaenoic acid (22-hydroxy-PD1 ), 10R,17R-dihydroxy-

4Z,7Z,11E,13E,15Z,19Z-docosahexaenoic acid (17-epi-PD1 ), 10S,17S-dihydroxy- Z Z., 11 E, 13E, 15Z, 19Z-docosahexaenoic acid (10-epi-PD 1 ), 10,17-di hydroxy-7, 11 ,13,15,19- docosapentaenoic acid (PD1_n-3), 16,17-dihydroxy-7,10,12,14,19-docosapentaenoic acid (PD2_n-3), 7R,14S-dihydroxy-4Z,8E,10E,12Z,16Z,19Z-docosahexaenoic acid (MaR1 ), 13R,14S-dihydroxy- 4Z,7Z,9E,11 E,16Z,19Z-docosahexaenoic acid (MaR2), 7S,14S-dihydroxy-4Z,8E,10E,12Z,16Z,19Z- docosahexaenoic acid (7-epi-MaR1 ), 14S,22-dihydroxy-4Z,7Z,10Z,12E,16Z,19Z-docosahexaenoic acid (MaR-L1 ), 14R,22-dihydroxy-4Z,7Z,10Z,12E,16Z,19Z-docosahexaenoic acid (MaR-L2), and IS, 14S-dihydroxy-8E, 10E, 12Z, 16Z, 19Z-docosapentaenoic acid (MaR1 _n-3).

Preparation of SPMs and their active precursors is known in the art, including WO 2013/170006.

Obesity affects the severity of POOS symptoms. Accordingly, obese POOS patients particularly benefit from the invention. Hence, in one embodiment, the POOS treatment is carried out with patients having a body mass index of at least 25 kg/m². In a further embodiment, the treatment is carried out with patients having a body mass index of at least 30 kg/m². In still a further embodiment, the treatment is carried out with patients having a body mass index of at least 35 kg/m². In yet a further embodiment, the treatment is carried out with patients having a body mass index of at least 40 kg/m².

Treatment of inflammation associated with PCOS

POOS is associated with inflammation and SPMs are known to play a role in the active resolution of inflammation. Accordingly, in one aspect, the invention concerns a composition for use in the treatment of inflammation associated with polycystic ovary syndrome (PCOS), wherein said composition comprises a specialized pro-resolving lipid mediator (SPM), an SPM precursor and/or a pharmaceutically acceptable salt thereof, as well as any stereoisomer thereof. In one embodiment, the inflammation is chronic inflammation.

Combination with a non-steroidal anti-inflammatory drug (NSAID)

Acetylsalicylic acid and other non-steroidal anti-inflammatory drugs function as co-enzymes for lipoxygenase and/or cyclooxygenase and thus contribute to the bio-synthesis of SPMs in the cascade having EPA and/or DHA as the substrate. Suitable non-steroidal anti-inflammatory drugs include ibuprofen, flurbiprofen, naproxen, indomethacin, diclofenac, celecoxib, and acetylsalicylic acid. Accordingly, in one embodiment the composition for use in treating PCOS and/or inflammation associated with PCOS according to the invention further comprises a non-steroidal anti-inflammatory drug. In a further embodiment, said non-steroidal anti-inflammatory drug is selected from ibuprofen, flurbiprofen, naproxen, indomethacin, diclofenac, celecoxib, and acetylsalicylic acid. In still a further embodiment, said non-steroidal anti-inflammatory drug is acetylsalicylic acid. In another embodiment, the acetylsalicylic acid is administered at a dose of 50 to 150 mg. In yet another embodiment, the acetylsalicylic acid is administered at a dose of 75 to 125 mg. In still a further embodiment, the acetylsalicylic acid is administered at a dose of approximately 100 mg.

Pharmaceutical formulation

The compounds comprised in the compositions of the present invention are intended for use as a medicament. The compounds of the invention may in principle be applied on their own, but they are preferably formulated with a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier is an inert carrier suitable for each administration method and can be formulated into conventional pharmaceutical preparation (tablets, granules, capsules, powder, solution, suspension, emulsion, injection, infusion, etc.). One particular example of a pharmaceutical formulation is a soft capsule containing the SPMs and/or active precursors as an oil. As an inert carrier there may be mentioned, for example, a filler, a binder, an excipient, a lubricant, a disintegrant and the like, which are pharmaceutically acceptable. When they are used as an injection solution or an infusion solution, they can be formulated by using distilled water for injection, physiological saline, or an aqueous glucose solution.

Administration

The administration method of the compositions of the present invention is not particularly limited, and a usual oral or parenteral administration method (intravenous, intramuscular, subcutaneous, percutaneous, intranasal, transmucosal, enteral, etc.) can be applied. Examples

Example 1 - Study of plasma and serum parameters in PCOS patients

Objective

Evaluation of inflammatory and resolutive plasma and serum parameters in PCOS patients.

Study design

Non-interventional study to detect inflammatory and pro-resolutive factors in plasma and serum in patients with PCOS. The analytical profile includes the following compounds:

• Fatty acids: EPA, DHA, ARA, DPA (docosapentaenoic acid)

• SPM precursors: 17-HDHA, 18-HEPE, 14-HDHA

• Resolvins: RvEl, RvDl, RvD2, RvD3, RvD4, RvD5

• Maresins: Marl, Mar2

• Protectins: PD1, PDX

• Lipoxins: LXA4, LXB4

• Prostaglandins: PGE2, PGD2, PGF2a

• Tromboxanes: TxB2

• Leukotrienes: LTB4

Study population

15 patients with PCOS were examined. There was a control collective of 5 healthy patients.

Inclusion criteria

1. Female patients, ages between 18 and 45 years

2. PCO syndrome according to the Rotterdam criteria

3. Written Consent Statement

Exclusion criteria

1. Current BMI <20 kg/m²

2. Patients with severe acute or chronic diseases (e.g. pancreatitis, hypertriglyceridemia, liver disease, benign or malignant liver tumor, malignant sex hormone-dependent diseases of genitals or breasts)

3. Migraines 4. Ing of herbal medicinal products that induce microsomal enzymes, in particular cytochrome P450 enzyme, e.g. phenytoin, phenobarbital, primidone, bosentan, carbamazepine, rifampicin, topiramate, felbamate, griseofulvin, some HIV protease inhibitors (e.g. ritonavir) and nonnucleoside reverse transcriptase inhibitors (e.g. efavirenz) as well as preparations of Aaron.

5. History of Cardiovascular Events

6.Advanced hypertension or diabetes

7. Presence or known risk of venous or arterial thromboembolism

8. Undiagnosed abnormal vaginal bleeding

9. Use of medicinal products containing ombitasvir/paritaprevir/ritonavir and dasabuvir during and two weeks prior to the start of the study

10. Pregnancy and lactation throughout the study period

11 .Menopausal women

Patient selection

A total of 15 patients were examined. 5 with a BMI <25, 5 with a BMI between 25 and 30 and another 5 with a BMI > 30. The control group is 5 patients who are healthy.

Data collection

Before participating in the study, the patient was informed by the doctor about the content of the study and received a paper version of the patient information brochure. As soon as the informed consent had been obtained in writing, the blood collection was carried out. During the visit, demographic data, medical history with the recording of menstrual bleeding characteristics, accompanying and possible medications (AE) were consulted and documented.

No follow-up phases are planned.

The data was collected during the single visit and documented in the source data and in the validated Case Report Form (CRF). This data was transferred from the doctor to the CRF.

Visit 1 (V1) included the following parameters, which were documented in the source data as well as in the CRF:

1. Written consent in the knowledge of the facts

2. Inclusion/exclusion criteria

3. Exclusion of pregnancy

4. Height, Weight

5. Demographic data

6. Medical history

7. Accompanying medicaments

8. Non-steroidal anti-inflammatory drugs 9. Vital signs

Blood sample analyses.

Blood samples of plasma and serum were obtained for each patient. Each of these samples was considered a mono-replicate.

After standard preliminary treatment, samples were stored at -80 degrees Celsius until they were processed in the laboratory. They were all analyzed separately.

Lipid mediator extraction and profiling (LC-MS/MS)

Lipid mediators were extracted from human plasma and serum samples following the SPE (Solid Phase Extraction) method described below. Internal labelled standards ds-5-HETE, d5-RvD2, ds- LXA4, d4-LTB4, d4-PGE2 (500 pg each, Cayman Chemical Company) in 4 mL of methanol (Methanol Optima LC/MS Grade, Fisher Chemical) were added to each sample (plasma or serum, 1 mL) previously thawed on ice. These labelled standards were used for the amount determined and the calculations of the recovery of the lipid mediators. Next, the samples were placed at -80°C for 30 minutes to allow the precipitation of proteins. The probes were centrifugated in the following working step (2000 g, 10 min, 4°C). The supernatants were obtained from each sample, and SPE was carried out according to optimized and reported methods [32, 51]. Samples were rapidly acidified to pH=3.5 with 9 mL of acidic water (HCI) before loading onto SPE columns (100mg, 10 mL, Biotage) and pH neutralized with 4 mL of MiliQ water, followed by 4 mL of n-hexane wash step. After, compounds were eluted with 9 mL of methyl format. Extracts from the SPE were brought to dryness under a gentle stream of nitrogen and immediately resuspended in methanol/water (50:50 vol/vol) (MeOH/Water Optima LC/MS Grade, Fisher Chemical, both) before injection into an LC-MS/MS system.

Targeted LC-MS/MS Acquisition Parameters

LC-MS/MS system consisting of a Qtrap 5500 (Sciex) equipped with a Shimadzu LC-20AD HPLC pump. A Kinetex Core-Shell LC-18 column (100 mm x 4.6 mm x 2.6 pm, Phenomenex) was kept in a column oven maintained at 50 °C. A binary eluent system of LC-MS/MS grade water (A) (Fisher Chemical) and LC-MS/MS grade methanol (Fisher Chemical) (B), both with 0.01 % (v/v) of acetic acid, was used as mobile phase. LMs were eluted in a gradient program with respect to the composition of B as follows: 0-2 min, 50 %; 2-14.5 min, 80 %; 14.6-25 min; 98 %. The flow rate was 0.5 mL/min.

The QTRAP 5500 was operated in negative ionization mode, using scheduled Multiple Reaction Monitoring (MRM) coupled with the information-dependent acquisition (IDA) and an Enhanced Product Ion scan (EPI). Each LM parameter (CE, target retention time (RT), and specific Q1 and Q3 mass) was optimized according to reported methods [51 , 52]. To monitor and quantify LMs of interest, quantities were taken as areas under the peak. We used MRM with MS/MS matching signature ion fragments for each molecule (at least six diagnostic ions; <0.1 picograms was considered below the limit of detection) using published criteria [52]. The laboratory analyses were performed at Solutex GC SL.

Statistical analysis

Quantitative values are represented as mean and standard deviation, minimum and maximum, and quartiles. 95% confidence intervals are calculated for the mean values of the primary endpoints to evaluate the accuracy of the estimates. If further statistical tests are to be carried out on these parameters, normal distribution tests are performed first either with the Kolmogorov-Smirnov test or in small case numbers of the Shapiro-Wilk test. With regard to distribution, appropriate methods are used for statistical calculations.

Ordinal and nominally scaled values are displayed in absolute and percentage frequencies. Two of these values can be compared in emergency tables to indicate either the change over time or a possible association with other parameters. Chi-square tests, including symmetry and, if necessary, accurate tests, can be used to verify that changes or associations are statistically significant.

All tests are performed on two sides with a significance level of 5%.

Results

In this observational study, we observed that the quantity of each parameter was detectable in the sera but not in the same way in the participants' plasma.

After quantitation, summation of total ARA-derived pro-inflammatory mediators resulted in a statistically significant increase (P<0.05) when comparing sera from PCOS patients with healthy subjects. These pro-inflammatory mediators include LTB4, PGD2, PGE2, PGF2oc, and TXB2, and values altogether were 100 times higher compared to those of healthy subjects (see Figure 1 ). Measured prostanoids, including PGD2, PGE2, and PGF2, all together were increased by 600% in serum from patients with PCOS compared to healthy subjects (Figure 1 ). Thromboxane TXB2 was also statistically significantly (P<0.05) higher in the serum from patients diagnosed with PCOS as compared to healthy subjects, which may reflect that these patients could suffer from coagulopathies (Figure 1).

We next studied whether these patients might have a disbalance in SPM formation. Specifically, comparing the ratio of total pro-inflammatory lipid mediators LTB4, PGD2, PGE2, PGF2oc, and TXB2 vs. total SPMs formed were statistically significantly different between the test groups (Figure 1). We observed in serum that the ratio of complete pro-inflammatory lipid mediators to the summation of SPMs, including 14-HDHA, 17-HDHA, and 18-HEPE, was statistically higher for patients with PCOS than those observed in the serum of healthy subjects (P <0.05). This finding suggested that infections could impair resolution mechanism(s) due to exacerbated inflammation.

We quantified the free fatty-acid-precursors of resolving mediators. We observed that DHA, DPA, EPA, and ARA were not statistically significantly higher in PCOS patients’ plasma and serum (Figure 2) as compared to those of healthy subjects. Interestingly, we observed that PCOS patients presented statistically more elevated amounts of the ratio pro-inflammatory parameters I SPMs, including the monohydroxylates in the serum compared to the plasma. (Figure 3). Evaluated parameters were:

• Fatty acids: EPA, DHA, ARA, DPA

• SPM monohydroxylated precursors: 17-HDHA, 18-HEPE, 14-HDHA

• SPM's:

Resolvins: RvE1 , RvD1 , RvD2, RvD3, RvD4, RvD5

Maresins: MaR1 , MaR2

Protectins: PD1 , PDX

Lipoxins: LXA4, LXB4

Prostaglandins: PGE2, PGD2, PGF2a.

Thromboxanes: TxB2

Leukotrienes: LTB4

As shown in Figure 1 , the mean value of total prostanoids in the serum was 30,000 pg/ml in healthy subjects and 60,000 pg/ml in PCOS patients (see Figure 1). When comparing the differences for the thromboxane values between the healthy subjects and the PCOS patients, statistically significant differences could also be observed. PCOS patients expressed significantly higher values than the healthy controls (see Figure 1 ).

Human plasma or serum was extracted using SPE and subject to targeted LC-MS/MS (see method above). Targeted LM and pathway markers were profiled. Each mediator was identified using published criteria obtained on their structure, including identification criteria of at least six characteristic diagnostic ions present in their MS-MS spectra.

Table 1 depicts all the values in a tabular form. Table 1

In the present example, an association with the lipid mediator profile in PCOS patients was demonstrated that was significantly shifted towards the pro-inflammatory axis compared to healthy women. Therefore, supplementation with DHA- or EPA-derived SPMs and their corresponding hydroxylated precursor metabolites, 18-HEPA, 17-HDHA, and 14-HDHA, represent an approach to treating the pathologic features associated with PCOS, in particular the inflammatory processes associated with PCOS. The greatly increased level of thromboxane TXB2 in PCOS patients compared to the test group is considered significant, as its precursor, TXA2, plays an essential role in platelet activation and aggregation. PCOS-affected women are known to have a 2-fold increased risk for venous thromboembolic events compared to healthy women. The present example therefore demonstrates that supplementation of SPMs and/or 18-HEPA, 17-HDHA, and 14-HDHA is likely to treat PCOS and the inflammation associated with PCOS.

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Claims

1. A composition for use in the treatment of polycystic ovary syndrome (PCOS), wherein said composition comprises one or more of a specialized pro-resolving lipid mediator (SPM), an SPM precursor and/or a pharmaceutically acceptable salt thereof, as well as any stereoisomer thereof.

2. A composition for use in the treatment of inflammation associated with polycystic ovary syndrome (PCOS), preferably for use in the treatment of chronic inflammation, wherein said composition comprises a specialized pro-resolving lipid mediator (SPM), an SPM precursor and/or a pharmaceutically acceptable salt thereof, as well as any stereoisomer thereof.

3. The composition for use according to claim 1 or 2, wherein said composition comprises an SPM precursor and/or a pharmaceutically acceptable salt thereof, as well as any stereoisomer thereof.

4. The composition for use according to claim 3, wherein said composition comprises one or more compounds selected from the group consisting of 18-hydroxyeicosapentaenoic acid (18-HEPE), 17-hydroxydocosa hexaenoic acid (17-HDHA), 14-hydroxydocosahexaenoic acid (14-HDHA) and pharmaceutically acceptable salts thereof, as well as any stereoisomers thereof.

5. The composition for use according to claim 1 or 2, wherein said composition comprises an SPM and/or a pharmaceutically acceptable salt thereof, as well as any stereoisomer thereof.

6. The composition for use according to claim 5, wherein said composition comprises one or more compounds selected from the group consisting of 5S,6R,15S-trihydroxy-7E,9E,11Z,13E- eicosatetraenoic acid (LxA4), 5S,14R,15S-trihydroxy-6E,8Z,10E,12E-eicosatetraenoic acid (LxB4), 5S,6R,15R-trihydroxy-7E,9E,11Z,13E-eicosatetraenoic acid (15-epi-LxA4), 5S,14R,15R-trihydroxy- 6E,8Z,10E,12E-eicosatetraenoic acid (15-epi-LxB4), 5S,12R,18R-trihydroxy-6Z,8E,10E,14Z,16E- eicosapentaenoic acid (RvE1), 5S,12R,18S-trihydroxy-6Z,8E,10E,14Z,16E-eicosapentaenoic acid (18S-RvE1), 5S,18R-dihydroxy-6Z,8E,10E,14Z,16E-eicosapentaenoic acid (RvE2), 17R.18R/S- dihydroxy-5Z,8Z,11Z,13E,15E-eicosapentaenoic acid (RvE3), 7S,8R,17S-trihydroxy-

4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid (RvD1), 7S,16R,17S-trihydroxy- 4Z,8E,10Z,12E,14E,19Z-docosahexaenoic acid (RvD2), 4S,11R,17S-trihydroxy- 5Z,7E,9E,13Z,15E,19Z-docosahexaenoic acid (RvD3), 4S,5R,17S-trihydroxy- 6E,8E,10Z,13Z,15E,19Z-docosahexaenoic acid (RvD4), 7S,17S-dihydroxy- 4Z,8E,10Z,13Z,15E,19Z-docosahexaenoic acid (RvD5), 4S,17S-dihydroxy- 4Z,8E,10Z,13Z,15E,19Z-docosahexaenoic acid (RvD6), 7S,8R,17R-trihydroxy- 4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid (17R-RvD1), 7S,16R,17R-trihydroxy- 4Z,8E,10Z,12E,14E,19Z-docosahexaenoic acid (17R-RvD2), 4S,11R,17R-trihydroxy- 5Z,7E,9E,13Z,15E,19Z-docosahexaenoic acid (17R-RvD3), 4S,5R,17R-trihydroxy- 6E,8E, 10Z, 13Z, 15E, 19Z-docosahexaenoic acid (17R-RvD4), 7S, 17R-dihydroxy- 4Z,8E,10Z,13Z,15E,19Z-docosahexaenoic acid (17R-RvD5), 4S,17R-dihydroxy- 4Z,8E,10Z,13Z,15E,19Z-docosahexaenoic acid (17R-RvD6), 7,13R,20-trihydroxy- 8E, 10Z, 14E, 16Z, 18E-docosa pentaenoi c acid (RvT 1 ) , 7 ,8, 13R-tri hyd roxy-9E, 1 1 E, 14E, 16Z, 19Z- docosapentaenoic acid (RvT2), 7,12,13R-trihydroxy-8Z,10E,14E,16Z,19Z-docosapentaenoic acid (RvT3), 7,13R-dihydroxy-8E,10Z,14E,16Z,19Z-docosapentaenoic acid (RvT4), 7,8,17-trihydroxy- 8,10,13,15,19-docosapentaenoic acid (RvD1_n-3), 7,16,17-trihydroxy-8,10,12,14,19- docosapentaenoic acid (RvD2_n-3), 7,17-dihydroxy-8,10,13,15,19-docosapentaenoic acid (RvD5_n-3), 10R, 17 S-dihydroxy-4Z,7Z, 11 E, 13E, 15Z, 19Z-docosahexaenoic acid (PD1 ), 10S, 17S-dihydroxy- 4Z,7Z,11E,13E,15Z,19Z-docosahexaenoic acid (PDX), 10R,17S,22-trihydroxy-

4Z,7Z,11E,13E,15Z,19Z-docosahexaenoic acid (17-epi-PD1 ), 10S,17S-dihydroxy-

4Z,7Z, 11 E, 13E, 15Z, 19Z-docosahexaenoic acid (10-epi-PD 1 ), 10,17-di hydroxy-7, 11 ,13,15,19- docosapentaenoic acid (PD1_n-3), 16,17-dihydroxy-7,10,12,14,19-docosapentaenoic acid (PD2_n-3), 7R,14S-dihydroxy-4Z,8E,10E,12Z,16Z,19Z-docosahexaenoic acid (MaR1 ), 13R,14S-dihydroxy- 4Z,7Z,9E,11 E,16Z,19Z-docosahexaenoic acid (MaR2), 7S,14S-dihydroxy-4Z,8E,10E,12Z,16Z,19Z- docosahexaenoic acid (7-epi-MaR1 ), 14S,22-dihydroxy-4Z,7Z,10Z,12E,16Z,19Z-docosahexaenoic acid (MaR-L1 ), 14R,22-dihydroxy-4Z,7Z,10Z,12E,16Z,19Z-docosahexaenoic acid (MaR-L2), and 7S, 14S-dihydroxy-8E, 10E, 12Z, 16Z, 19Z-docosapentaenoic acid (MaR1 _n-3).

7. The composition for use according to any one of the preceding claims, wherein said composition further comprises EPA and/or DHA.

8. The composition for use according to any one of the preceding claims, wherein the treatment is carried out with patients having a body mass index of at least 25 kg/m².

9. The composition for use according to claim 8, wherein the treatment is carried out with patients having a body mass index of at least 30 kg/m².

10. The composition for use according to anyone of the preceding claims further comprising a nonsteroidal anti-inflammatory drug.

11 . The composition for use according to claim 10, wherein said non-steroidal anti-inflammatory drug is acetylsalicylic acid.

12. The composition for use according to claim 1 1 , wherein the acetylsalicylic acid is administered at a dose of 50 to 150 mg.

13. The composition for use according to claim 12, wherein the acetylsalicylic acid is administered at a dose of 75 to 125 mg.

14. The composition for use according to claim 13, wherein the acetylsalicylic acid is administered at a dose of approximately 100 mg.

15. A method of treating polycystic ovary syndrome (PCOS) by administration of a therapeutically effective amount of a composition comprising one or more of a specialized pro-resolving lipid mediator (SPM), an SPM precursor and/or a pharmaceutically acceptable salt thereof, as well as any stereoisomer thereof.