The role of purinergic P2X3 receptors and endometriosis-associated hyperalgesia

Endometriosis is a chronic gynecological condition characterized by endometrial tissue outside the uterine cavity. Recent research has focused on the relationship between the purinergic system and endometriosis. As a purinergic system component, extracellular adenosine triphosphate (ATP) has been involved as a crucial mediator of chronic pain associated with the disease, as well as P2X3 receptors, which play a key role in sensitization of nerve fibers and generation of nociceptive, neuropathic, and inflammatory pain. In addition, ectonucleotidases such as CD39 and CD73, which regulate ATP hydrolysis, have an altered expression in endometriotic lesions, contributing to extracellular ATP accumulation, intensifying inflammation and pain. Together, immune cells and their product are increased in endometriotic foci and promote the emergence of new lesions through their contribution to retrograde menstruation. Some studies have observed significant changes in mitochondrial respiration, notably decreased oxygen consumption in the affected endometrium. These various cellular processes that are mediated by ATP and adenosine in the uterine cavity and exert effects on immune defense will be herein reviewed in detail to investigate possible inhibitors, their activity, and potential exploration of the purinergic pathway as a therapeutic alternative. Graphical abstract

P2X3, Hyperalgesia, Inflammation, Purinergic receptors, Ectonucleotidases

Endometriosis is currently defined as a complex clinical syndrome that generates a chronic estrogen-dependent inflammatory process, mainly affecting pelvic tissues. It is generated from the deposition of ectopic endometrial tissue and stroma outside the uterine cavity [1]. The pathology affects approximately 6 to 10% of all women of reproductive age in the world, with an estimated time for diagnosis of 4 to 11 years after onset of the symptoms, which commonly include chronic pelvic pain, dyspareunia, dysmenorrhea, and infertility, substantially compromising the carriers’ quality of life [2, 3]. The macroscopic extent of the disease is highly variable (from presence of minimal peritoneal deposits to deep infiltration that can invade the intestines, bladder, ureter and, more rarely, extrapelvic sites [2, 3]). In order to systematize diagnosis and management, the revised American Society of Reproductive Medicine (rASRM) classifies endometriotic lesions into four stages based on severity, size, depth, location, and number of lesions. It also classifies them into three categories: superficial peritoneal disease, ovarian endometriosis, and deep infiltrating endometriosis [2, 4]. Recently, attention has been drawn to the purinergic system’s involvement in the pathophysiology of several pathologies, including endometriosis [5]. Comprised by nucleotides such as ATP and subfamilies of purinergic receptors (P1 and P2), the purinergic system is closely involved in pain modulation and has been identified as a possible mediator in chronic pain genesis and maintenance in several pathologies [6, 7]. Its involvement in inflammatory processes has also been described [6–8]. To this end, this review aims at exploring, through the current literature, the purinergic system mechanisms involved in the pathophysiology of endometriosis, especially P2X3 receptors; their action on endometriosis hyperalgesia; and the development of new treatment therapies. Inflammation and extracellular ATP in endometriosis Endometriosis is a chronic disease triggered by the development of cells comparable to those of the endometrium outside the uterus, causing tissue growth in various body areas. This condition promotes chronic systemic inflammation and inflammation of pelvic tissues, which can affect organ metabolism and result in complications such as debilitating pelvic pain and infertility [1, 9]. Recent studies such as the systematic review with meta-analysis carried out by Vercellini et al. demonstrate that, although endometriosis does not have a well-established cause in the literature, there are some important hypotheses currently discussed, such as that the disease would be caused by the retrograde menstruation/implantation theory or by the embryonic remnant/coelomic metaplasia theories. In this context, the embryonic remnant/coelomic metaplasia theories argue that endometriosis is triggered by coelomic metaplasia or by embryonic tissue remaining from embryonic formation that transforms into endometrial tissue outside the uterine cavity. In contrast, the retrograde menstruation theory argues that endometriosis would be the result of menstrual flow migration to the extrapelvic environment, causing the emergence of endometriotic lesions. Furthermore, the results of the research by Vercellini et al. provided data suggesting that the retrograde menstruation theory would be the most plausible one to explain the emergence of endometriotic lesions. However, according to the study, there is not sufficient scientific evidence to rule out other theories, such as the coelomic metaplasia/embryonic remnant hypothesis [10]. Although the etiology of endometriosis has not been completely unraveled, Sampson’s retrograde menstruation theory is widely described in the literature to explain the inflammatory process [11, 12]. In the inflammatory process characteristic of the pathology, an increase is observed in the number of immune cells resident in the uterine stroma, mainly during the menstrual period [12]. The interaction between immune cells (such as macrophages, mast cells, and neutrophils) and uterine stromal cells modulates the biosynthesis and release of pro-inflammatory cytokines such as interleukins (IL)−1β, IL-6, IL-8, chemokines, and prostaglandins (PGs), resulting in local vasoconstriction, which can cause menstrual material to retrogradely flow into the peritoneal cavity, causing lesions and chronicizing inflammation based on Sampson’s theory [11, 12]. Furthermore, for being a characteristic of the inflammatory process of the pathology, attention has turned to non-hormonal therapies focused on modulating the energy metabolism of damaged tissues [13]. In vivo studies employing mitochondrial respirometry assays have shown that, when compared to eutopic endometrium in a non-human primate (NHP) model, endometrial tissue affected by endometriosis presents reduced oxygen consumption rates, particularly those linked to complexes I and II. Targeted metabolomics assays of endometriotic endometrial tissue from NHPs demonstrated a significant reduction in carnitine, creatine phosphate, nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD), malic acid, and tryptophan when comparing healthy endometrium samples. These metabolites are involved in the metabolism of tryptophan, nitric oxide, and riboflavin, whose reductions (associated with other factors observed in the study such as decreased functionality of respiratory chain complexes I and II) indicate alterations in the mitochondrial respiratory pathway [13]. A recent study on cellular respiration in endometrial biopsies from 91 gynecological patients also found reduced oxygen absorption levels for women with genital endometriosis and those with extragenital endometriosis. Furthermore, the messenger RNA (mRNA) expressions for cytochrome C, cytochrome C oxidase, and ATP synthase were also lower in these groups. In contrast, the mRNA expression for pyruvate kinase M1/2 (PKM) was elevated for both groups when compared to the control group. In this study, PKM was associated both with the Warburg effect (a shift in metabolic dominance towards glycolysis) and with the risk of cell migration via pTyr-42 RhoA-mediated superoxide generation, which was also related to extragenital lesions. These results were different for the adenomyosis group, which showed increased oxygen uptake and ATP synthase when compared to the control group [14]. However, it is important to highlight that both the study by Atkins et al. and the one by Toniyan et al. approach the energy metabolism alteration from an investigative point of view focused on oxidative phosphorylation and ATP synthase, not directly researching intracellular or extracellular ATP levels, thus limiting the conclusions about ATP bioavailability and signaling roles in these conditions. A recent in vivo study in rats proved that extracellular adenosine triphosphate (ATP) is an essential inflammatory factor in endometriosis, demonstrating that the endometriotic tissue micro-environment had high extracellular ATP levels. These levels were related to apoptosis and pyroptosis induction of epithelial cells. In the presence of extracellular ATP, cell growth was mainly inhibited through the MAPK/JNK/Akt pathway, the NF-kb pathway, the TNF signaling pathway, and pathways related to calcium flux homeostasis. High extracellular ATP concentrations improved macrophage dysfunction, increasing their cytotoxic power on epithelial cells of the damaged tissue without affecting eutopic tissue [15]. The study by Trapero et al. concluded that extracellular ATP accumulation in the ectopic and eutopic endometrial micro-environment, especially in deep infiltrating endometriosis, promotes generation and maintenance of the inflammatory state, combined with its involvement with immune system components linked to female infertility in endometriosis, increasing the pregnancy loss rate and decreasing the quality of gametes and their implantation. In addition, it also concluded the direct involvement of ATP accumulation with pain production in endometriosis [5]. However, further studies are required to clarify the effects of extracellular ATP in this pathology (Fig. 1). Purinergic system and the relationship between P2X3 and endometriosis The studies by scientist G. Burnstock drew the attention in the 1970 s when they indicated certain potential for cell membrane excitation triggered by the stimulation of purine molecules located in the extracellular space [14, 16, 17]. Studies such as these showed that purines like adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine (ADO) might activate membrane receptors to modify cells’ chemical and internal characteristics, such as potassium conductivity [18–20]. Ectoenzyme CD39 (ectonucleoside triphosphate diphosphohydrolases) converts ATP into ADP. Subsequently, it also converts ADP into AMP. AMP is a precursor agent of ADO, formed by the action of ectoenzyme ecto-5′-nucleotidase (CD73) [14, 19, 20]. ATP, ADP, AMP, and ADO can activate purinergic receptors of the P1 and P2 families to stimulate different cellular functions, pathways, and activities [20]. The central purinergic receptors identified in mammals are divided into two families: P1 and P2. The P1 adenosine receptor family contains the A1, A2A, A2B, and A3 receptors, and the P2 family is subdivided into two subgroups: P2XR and P2YR, with the P2X subfamily consisting of ion channels, and the P2Y subfamily consisting in G protein-coupled receptors (GPCRs). The P2XR subgroup contains the ionotropic receptors included in the set of P2X1 to P2X7, and the P2YR subgroup is comprised by the following metabotropic receptors: P2YR1, P2YR2, P2YR4, P2YR6, P2YR11, P2YR12, P2YR13, and P2YR14 [21]. P2X3 is an ionotropic purinergic receptor in cell membranes, mainly activated by ATP. It is involved in mediating nociceptive, neuropathic, and inflammatory pain in sensory neurons, expressed in peripheral nerves that innervate tissues related to nociception, chemosensation, and functionality [14, 22]. P2X3 is present in organs and anatomical structures such as bladder, urethra, intestines, and uterus. It is predominantly located in primary sensory afferent neurons in small-to-medium diameter C and Aδ type fibers throughout the body, which carry nerve impulses of sensory stimuli towards the central nervous system [14]. The study by Ding et al., which analyzed endometrial tissue samples from women with or without endometriosis subjected to laparoscopic surgeries, demonstrated through immunohistochemical staining that the P2X3 expression levels in endometriotic lesions and endometriosis endometrium were higher than in control endometrium, confirmed by Western blotting analysis. Furthermore, the P2X3 expression levels in endometriotic lesions and endometriosis endometrium were both correlated with pain severity in the study [23]. Regarding the ATP hydrolysis byproducts capable of promoting P2X3 sensitization, such as ADP, it was observed that they have a lower capacity to sensitize P2X3 than ATP [14] (Fig. 2). Essential in this signaling process, ectonucleotidases present a peculiar expression in the endometrium throughout the menstrual cycle [5, 24, 25]. NTPDase2, NTPDase3, NPP1, NPP3, ALP, CD26, and CD73 are detected in endometrial epithelial cells, while NTPDase1, NTPDase2, and CD73 are detected in stromal cells [5]. Furthermore, there is a difference in the expression of these enzymes in the eutopic tissue of women with endometriosis when compared to those without the pathology, in addition to concentration variations in the different types of endometriotic lesions. In the study by Trapero et al. [5], alterations in the CD39-CD73 pathway were found in endometriotic lesions, indicating that changes in ATP hydrolysis resulting from CD39 and CD73 activity are related to endometriosis severity, as their expression is lost in deep infiltrating lesions. These expression-related changes may lead to extracellular ATP accumulation, promoting the secretion of cytokines and growth factors into the ectopic environment, with a concomitant increase in endometrial cells’ survival and growth rates. Furthermore, CD73 loss has been observed in the two most severe forms of endometriosis (ovarian and deep) and is hypothesized to play a role in ectopic cell migration and invasion [5]. The NPP3 (ectonucleotide pyrophosphatase/phosphodiesterase 3) expression in endometriosis was found in the epithelium of eutopic and ectopic endometrial tissues but also in the stroma, which is not typically observed in women without the pathology. Therefore, it has also been indicated as a promising histopathological marker of the disease [5]. No other studies addressing the topic were found; consequently, it is necessary to develop new research to corroborate the findings described above. Furthermore, in the study by Trapero et al., the authors suggest that, in addition to its function in controlling extracellular ATP levels, NPP3 may play a role in the invasive capacity of endometrial stromal cells in endometriosis, as it is known that NPP3 over-expression in murine fibroblasts stimulates motility and invasiveness of these cells [5]. Therefore, the purinergic system appears to be highly important for relevant processes in several pathologies, including cell migration, proliferation, and inflammation; mechanisms also observed in endometriosis [5, 23, 26]. Thus, it is essential to characterize the purinergic elements of eutopic endometrium and endometriotic lesions so as to clarify the role of purinergic signaling in pathogenesis and recognize key endometriosis markers and new therapeutic targets. Furthermore, recognizing the micro-environment of ectopic lesions may improve understanding of how pain signals are generated and neurotransmitted in endometriosis. Relationship of P2X3 in endometriosis hyperalgesia Hyperalgesia is defined as an exacerbated sensation of painful stimuli due to increased sensitization of neural circuits involved in nociception. Despite being a common manifestation, the pathophysiology of endometriosis-associated pain is still unclear according to Maddern et al. [14], but may be related to chronic inflammation and anomalous activation of pain signaling pathways. The following can be mentioned among the main painful symptoms: cyclical pelvic pain, dysmenorrhea, dyspareunia, dysuria, and dyschezia, and, as for the types, nociceptive, inflammatory, neuropathic, and associations have been described [23, 26, 27]. Thus, the purinergic system, especially P2X3 receptors, has been involved due to its central role in mediating nociceptive pain (caused by tissue damage), neuropathic pain (damage or dysfunction of nervous system pathways), and inflammatory pain [23, 26]. Therefore, understanding the relationship between P2X3 and hyperalgesia may provide new perspectives and therapeutic approaches to treat patients with endometriosis and improve their quality of life. Pain is initiated by a peripheral noxious stimulus detected by nociceptors in the Aδ and C fibers, which have their cell bodies in the dorsal root ganglia (DRG) and send this information to the spinal cord, which conducts it to the brain, where pain is perceived, mainly through the ATF3/AP-1 signaling pathway [26, 28]. The P2X3 expression in peripheral nerve endings suggests a possible role with nociception and inflammatory response, especially in chronic conditions such as endometriosis [23]. When part of a lesion ruptures, such as endometriotic foci, large ATP amounts are released into the micro-environment around the injured area, activating P2X3 receptors [23, 26]. According to Bashir et al. [29], a possible mechanism to explain chronic pelvic pain in patients with endometriosis is central sensitization, which involves glial cell activation and neuroinflammatory responses in the central nervous system (CNS). Continuous peripheral stimulation can modify the neuronal circuits of pain pathways, resulting in an amplified response to painful stimuli, called hyperalgesia. Peripheral sensitization is another factor that may exert an impact on hyperalgesia in patients with endometriosis. Peripheral sensitization is characterized by a reduction in the activation threshold of neurons, resulting in pain to harmless stimuli (allodynia) or in pain intensification (hyperalgesia). Sensitization acts as a protective mechanism against further damage during inflammation. However, if inflammation persists or the nociceptors become chronically sensitive, this hypersensitivity may continue even after inflammation has disappeared, contributing to allodynia and hyperalgesia in patients with endometriosis [14] and favoring continued activation of P2X3 receptors, which may explain the high incidence of severe pain in patients with endometriosis. Finally, purinergic P2X3 receptors play a central role in mediating hyperalgesia in inflammatory states such as endometriosis. Continued activation of these receptors by high ATP concentrations released in the inflammatory micro-environment of endometriotic foci contributes to peripheral and central sensitization, exacerbating the chronic pelvic pain associated with the disease (Fig. 3). In contrast to endometriosis, in several pathologies at the cellular level, it has been found that 17β-estradiol modulates nociceptive receptors—transient receptor potential vanilloid type 1 (TRPV1) and P2X3—on sensory neurons, influencing pain perception [14]. Estrogen can upregulate and downregulate TRPV1 expressions, leading to pro- and anti-nociceptive effects [30]. Prolonged exposure of rats to 17β-estradiol caused an “anti-nociceptive” effect on adult lumbosacral nociceptor neurons, reducing the TRPV1 response to capsaicin mediated by a non-classical estrogen signaling pathway involving intracellular Erβ [31]. However, the study by Yamagata et al. demonstrated that estrogen-dependent increases in TRPV1 are likely involved in modulating the nociceptive response in the trigeminal area in rats [32]. Furthermore, it increases TRPV1 single-channel activity and open probability, potentially protecting against oxidative stress-induced cell death [33]. The study by Ma et al. observed increased P2X3 mRNA in the dorsal root ganglia (DRG) of rats after ovariectomy when compared to the control and orchiectomy groups. This directly caused the mechanical hyperalgesia observed through the animals’ behavior. ATP application increased the pain threshold in rats subjected to ovariectomy. In the same study, neuron cultures with DRG were performed and subjected to 17β-estradiol applications, causing a decrease in the P2X3 receptor expression even at low doses. These results suggest that the female gonadal hormone (17β-estradiol) may participate in peripheral pain signal transduction control by modulating events mediated by P2X3 receptors in primary sensory neurons, probably through genomic mechanisms [34]. Considering that P2X3 receptors are ATP-dependent Ca2+ permeable channels, it has been shown in other pathologies that estrogen can also regulate P2X3 expression by attenuating ATP-induced Ca2+ influxes, decreasing P2X3 mRNA and protein expression levels in primary afferent neurons [35, 36]. This suggests that estrogen inhibits P2X3-mediated peripheral pain transduction when related to neuropathic pain. However, in endometriosis, blocking estradiol production either naturally (menopause) or pharmacologically (ovarian suppression or aromatase inhibition) causes regression of the disease and its symptoms, including pain [1]. Therefore, it is essential to further investigate the P2X3 selective blockade and how it can contribute new advances in hyperalgesia control, offering practical therapeutic approaches for patients that do not respond to conventional treatments and improving quality of life in women with endometriosis [23]. Therapeutic perspectives in endometriosis Endometriosis treatment involves a multi-pronged approach based on a complete health history assessment and efficient imaging tests, as discussed by Mechsner, which includes pharmacological and surgical interventions. Non-steroidal anti-inflammatory drugs (NSAIDs) can be used to relieve discomfort, and estrogen-progesterone hormonal contraceptives can control estrogen levels. In addition, drugs that inhibit ovarian function and other medications, such as gonadotropin-releasing hormone (GnRH) agonists and danazol, help control pain and disease progression [37]. According to the literature, the surgical option should not be disregarded in moderate to severe cases, which include endometriotic tissue resection, restoring pelvic anatomy, preserving fertility, and alleviating pain. However, when conservative options are not viable or do not produce the expected results, total abdominal hysterectomy (with or without bilateral salpingo-oophorectomy), removal of the ovaries (oophorectomy), and fallopian tubes (salpingectomy) are considered definitive treatments, especially in patients that no longer wish to become pregnant [38]. Some studies show the existence of innovative therapeutic perspectives to expand the possibilities of treating endometriosis-associated diseases. Pharmaceutical companies are currently studying the Eliapixant molecule. From this perspective, it initially proved to be capable of acting as a selective P2X3 receptor antagonist in the treatment of hyperalgesia and hypersensitivity exacerbated by nervous disorders in endometriosis. Studies in animals have shown that there may be an important relationship between blocking P2X3 receptors and controlling disorders caused by endometriotic invasions, indicating that these purinergic receptors may be an effective therapeutic alternative in the endometriosis context [39, 40]. Furthermore, the study in rats with neurogenic uterine inflammation showed that Eliapixant has the potential to combat this inflammation, as it is a pathological condition associated with the P2X3 receptor expression [39]. Another clinical complication related to endometriosis in humans is dyspareunia [39]. Given this condition, the researchers implanted uterine horn fragments in the colon and mesenteric arteries of rats to simulate lesions typical of endometriosis and study the therapeutic potential of Eliapixant in combating dyspareunia. This research concludes that Eliapixant may have significant potential to combat dyspareunia in rats [39]. Although preclinical studies show a positive result between P2X3 receptor blockade by Eliapixant and neurogenic inflammation control [39], in a clinical study with 215 participants that evaluated efficacy of the molecule in controlling endometriosis-associated symptoms, there was no statistically significant or clinically relevant effect of this drug, not meeting the primary objective of the study. The same Phase 2 study by Bayer AG demonstrated two moderate liver injury cases when receiving 150 mg twice daily, which was discontinued after evaluating risks and benefits [14]. Pre-clinical studies have shown the efficacy of P2X3 antagonists in several pain models, including bladder pain and osteoarthritis [22]. Gefapixant and BLU-5937 are among the most promising compounds in Phase III and II clinical trials for the treatment of chronic pain, respectively [41]. In addition, new studies are emerging to develop new pain-relieving drugs such as Sivopixant (S-600918), a selective P2X3 receptor antagonist. This compound demonstrated potent analgesic effects in allodynia rat models [42]. A systematic review and meta-analysis that analyzed 67 articles on P2X3 receptor blockade showed a robust general analgesic effect in animal pain models. The effect size depended on the type of pain: greater for visceral, muscular, and neuropathic pain but smaller for inflammatory pain. Efficacy also depended on the pain outcome assessed, the medication used, and its administration route [14]. Purinergic P2X3 receptor blockade may be a potent way to combat the undesirable effects observed in endometrial lesions. However, as of the development of this review, there is absence of data in the literature to elucidate knowledge gaps that still persist on this topic. In addition, research needs to be developed to prove the safety of therapeutic products aimed at blocking P2X3 in endometriosis. Recent studies have also explored alternative treatments, as evidenced in the literature review by Amaral et al., such as vitamin supplementation and GnRH antagonists like Linzagolix and Relugolix, which have shown promise in treating endometriosis-related pain and improving quality of life [43]. Furthermore, the relationship between P2X3 and estrogen has not yet been fully elucidated. Investigating the purinergic system might be beneficial to understand the pathogenesis of endometriosis and identify new therapeutic strategies for the disease. It is worth noting that, according to the data found in this review, it is possible to raise the hypothesis that research targeting treatment associated with nerve fibers would be more appropriate in a subgroup of patients that present increased density of nerve fibers, such as those described in deep infiltrating endometriosis. This is because the results of studies have already reported that P2X3 receptors play a considerable role in the sensitization of nerve fibers and actively act in the genesis of neuropathic, nociceptive and inflammatory pain. In addition, it is also important to highlight that purinergic P2X3 receptors play an important role in the pathogenesis of endometriosis; therefore, it is important that new research be conducted aimed at modulating these receptors to combat the disease. Although some recent studies, such as with Eliapixant [39, 40] and Gefapixant [14], have not yet resulted in a truly valid treatment for endometriosis control, the scientific evidence found in this review suggests that P2X3 may be associated with such control. Therefore, although the treatment is currently only speculative, further clinical studies may be valuable. Final considerations The evidence discussed highlights the interaction between the inflammatory process and purinergic signaling, revealing that extracellular ATP plays a crucial role in modulating the immune response, as well as the over-expression of purinergic receptors (especially P2X3) associated with high ATP levels in the endometriotic micro-environment, suggesting an inflammation/pain vicious cycle. In association, the mechanism of ectonucleotidases in the metabolism of purines in the endometrium of women with endometriosis highlights the relevance of investigating the cellular micro-environment dynamics, even as potential biomarkers for diagnosis and clinical management of the disease. In addition, identifying potential non-hormonal therapeutic pathways (including purinergic signaling manipulation) may, if practical, improve the patients’ quality of life due to the disease symptoms and the conventional treatment side effects. Thus, future research should continue to explore the role of purinergic signaling in endometriosis, especially P2X3 receptors, seeking to elucidate their interactions and effects on the system in the pathophysiology of endometriosis. Considerations on new studies Researching the relationship between purinergic P2X3 receptors and endometriosis may demonstrate new clinical perspectives for treating endometriosis-caused hyperalgesia. Therefore, further studies are required to explore the purinergic system’s involvement in pain and inflammation in endometriosis, especially to more thoroughly investigate the role of P2X3, possible antagonists, and their action on painful stimuli in endometriosis. Kailane Paula Pretto undergraduate nursing student at the Federal University Fronteira Sul (UFFS) - Campus Chapecó. Member of the research group of "Tumors of the Central Nervous System: Analysis of the Purinergic System and oxidative stress" and of the extension project "Health Education Program through Work (PET-Health)". Currently, she is the treasurer of the Independent Academic Center of Nursing (CAENF) of UFFS, management 2025, and of the Academic League of Nursing in Oncology and Palliative Care (LEON - UFFS). Author contributions K.P.P.; L.S.M.; B.D.O.; A.A S.; C.F.P.; Y.A.R.R.; Y.T.S. and D.T.R.S. contributed to the conception of the manuscript. K.P P.; L.S M.; B.D.O. and DTRS. Contributed to formal analysis, research, and methodology. D.T.R.S. supervised the research. K.P.P.; L.S.M.; B.D.O. and D.T.R.S contributed to writing and translating the original draft. K.P.P.. and D.T.R.S. revised the final work. Funding None. Data availability No datasets were generated or analysed during the current study. Code availability Not applicable. Declarations Ethics approval Not applicable. Consent to participate Not applicable. Consent for publication Not applicable. Competing interest The authors declare no competing interests. Footnotes Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Change history 7/30/2025 A Correction to this paper has been published: 10.1007/s11302-025-10106-6

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