The relationship between gender and pharmacology

Abstract

The part of sexuality in pharmacology research was not acknowledged, and it was not thought-out to be a determinant that could impact strength and disease. For decades research has mainly contained male, women and animals, leading to a lack of news about syndromes in females. Still, it is critical to guarantee equal likeness so that determine the security, influence, and resistance of healing agents for all individuals. The underrepresentation of female models in preclinical studies over various decades has surpassed to disparities in the understanding, disease, and treatment of ailments 'tween genders. The closeness of sexuality bias has happened recognized as a contributing determinant to the restricted interpretation and replicability of preclinical research. Many demands operation have stressed the significance of including sexuality as a organic changeable, and this view is acquire growing support. Regardless of important progress in incorporating more female models into preclinical studies, differences prevail contemporary. The current review focuses on the part of sexuality and common in biomedical research and, therefore, their potential function in pharmacology and analyze the potential risks guide.

1. Introduction

The development of therapeutic agents is primarily motivated by the medical necessity, the prevalence of the disease, and the probability of success. The selection of therapeutic agent candidates involves an ongoing process that combines chemistry and biology, with the objective of refining the molecular properties until a compound suitable for human use is discovered. This process is both costly and time-consuming, typically spanning a period of 10–15 years, and carries a high level of risk. In fact, a significant majority of research projects, around 80–90%, fail before reaching the stage of human testing. For every therapeutic agent that receives approval from the Food and Drug Administration (FDA), over 1000 others have been developed but ultimately failed. Prior to being administered to humans, the pharmacology and biochemistry of the therapeutic agent are established through a comprehensive range of in vitro and in vivo testing procedures during preclinical studies. These studies begin with the examination of the effects of the medicine on cell cultures, progress to animal experimentation, and ultimately culminate in clinical trials involving human subjects (Tamimi and Ellis, 2009). It is also a regulatory requirement by the FDA that the therapeutic agent be tested on animals to evaluate its safety. Additionally, later-stage animal testing is conducted to assess its potential carcinogenic and reproductive effects. The objective of preclinical studies is to accurately replicate, in animals, the desired biological effects of a medicine. This enables doctors to predict the treatment outcomes in patients and identify any toxicities associated with the therapeutic agent, with the ultimate goal of anticipating any adverse events in humans (Polson and Fuji, 2012) (see Fig. 3, Fig. 4).

Fig. 3.

Sex differences im drug processing.

Fig. 4.

Age, sex, drug trials in women.

A revolution has taken place in clinical trials to incorporate females into the research process. However, there has been minimal progress in the preclinical sector, even though preclinical research sets the foundation for subsequent clinical trials. In 1993, the Council for International Organizations of Medical Sciences introduced a regulation, which was approved by the FDA, that mandated the inclusion of women in clinical trials funded by the National Institutes of Healthik (NIH). Women of reproductive age were previously excluded from clinical trials until 1993, with no women ever participating in such trials. In 2014, the NIH implemented a directive to ensure that both male and female subjects are represented in preclinical studies (Clayton and Collins, 2014). A similar initiative by the Canadian Institute of Health, which required researchers to consider sex and gender in their research funding applications, led to a significant increase in applications that accounted for both male and female genders (from 26% to 48%). It was noted that scientists in the biological field were the least likely to acknowledge the importance of including sex in their studies. These findings underscore the potential for policy interventions to address gender bias. However, not all research is funded through these avenues, and the full inclusion of both male and female genders in research pipelines has not yet been achieved (Johnson et al., 2014).

Kim and her colleagues conducted a study on the representation of cell sex in relevant articles published in the same journal in 2018. Their aim was to assess the progress made in reporting cell sex since the previous examination in 2013. Out of the 107 papers describing cell experiments, 53 of them reported the sex of the cells. Among these, 12 studies exclusively used female cells, 18 studies used both male and female cells, and 23 studies solely used male cells. It was observed that cell lines were more commonly used than primary cells, leading to a higher frequency of sex omission. Interestingly, over half of the investigations involving mouse primary cells exclusively utilized male cells (Kim et al., 2021).

In addition to this, other studies focused on the 10-year outcomes and lessons learned from applicant forms, the development of resources for applicants and evaluators, and the requirements for grant reviews. The objective was to provide insights for the implementation of scientific policy. The study group consisted of all participants in 15 Canadian Institutes of Health Research contests initiated by investigators between 2011 and 2019, as well as grant evaluators between 2018 and 2019. A total of 39,390 applications were submitted since 2011. The percentage of reports incorporating gender and sex increased from 12% to 33% and 22%–83%, respectively. Notably, gender was given the most consideration in applications related to population health research (82%). Furthermore, applications led by female principal investigators were more likely to integrate sex (and gender) compared to those led by male principal investigators in every competition. Since 2018, applications with high scores for sex integration and gender integration have had a greater chance of receiving funding. Qualitative observations revealed that there was often confusion between sex and gender (Haverfield and Tannenbaum, 2021).

A series of publications have examined the current normative practices in preclinical research, the efforts to include women, and the underlying reasons for the existing discrepancies.1. The researchers explore organizational change theory to develop the necessary strategies at both institutional and individual levels in order to transform the current situation and create a scientific setting where sex-sensitive methods are integrated into preclinical research automatically (Karp and Reavey, 2019; Docherty et al., 2019; Gogos et al., 2019).

2. Methods and materials

We conducted a review by searching the Google Scholar, PubMed, and Directory Open access Journal databases for relevant information using keywords such as pharmacology, sex, gender, drugs, male, females, preclinical studies, pharmacodynamics, pharmacokinetics, phenotypes, aspirin, metabolism, to identify primary comparative studies on the relationship between gender and pharmacology. The quality and strength levels of the results were considered and when available meta-analyses and systematic reviews, large epidemiological studies and randomized control trials represented the main source of data.

To guarantee that the research search was multidisciplinary and inclusive, we combed databases from the fields of cure, nutrition (MEDLINE, PubMed, PsycInfo, PsycIndex, PsycArticle, SPORTDiscus, and Netting of Skill) utilizing a predefined orderly database search agreement grown all at once with a experimental research authority. The search blueprint organized both keywords and a regulated jargon and free-text search conditions. We likewise searched remark lists of appropriate review documents and retraced study protocols and colloquium performances to recognize other conceivably fit studies. Later duplicates are distant, articles were picked in a three-step process (visualize Fig. 1). Fundamentally, trained reviewers alone secluded titles and abstracts in accordance with the four eligibility tests, accompanying no critic screening two together the title and abstract of the unchanging paper (see Fig. 2).

Fig. 1.

PRISMA checklist (Flowchart of the study selection process).

Fig. 2.

Phyisiological difference in PK, PD of drugs.

3. Results

3.1. Gender difference in pharmacology

Women and men exhibit different responses to treatments due to variations in physiological, anatomical, and hormonal characteristics. The disparities in therapeutic agent pharmacokinetics and pharmacodynamics play a crucial role in determining the effectiveness of treatments (Beierle et al., 1999; Bies et al., 2003; Bigos et al., 2009; Chen, 2005; Dawkins and Potter, 1991; Dawkins et al., 1993; Gandhi et al., 2004; Marazziti et al., 2013; Regitz-Zagrosek, 2014; Zucker and Prendergast, 2020; Fletcher et al., 1994; Franconi and Campesi, 2017; Frost et al., 2015; Anderson, 2008; Anthony and Berg, 2002; Flores Pérez et al., 2003; Franconi et al., 2007, 2011a; Greenblatt et al., 2004, 2014; Gupta et al., 1995; Harris et al., 1995; Jiang et al., 2015; Krecic-Shepard et al., 2000; Lee et al., 1999; Luzier et al., 1999; Patrick et al., 2007; Soldin and Mattison, 2009; Song et al., 2018; Swan and Hursting, 2000; Tamargo et al., 2017; Thürmann and Hompesch, 1998; Ueno and Sato, 2012; Vachharajani et al., 2011; Yonkers et al., 1992; Yoon et al., 2021). Although the gender differences in pharmacology were recognized as early as 1932 with the study on barbiturates in rats, a comprehensive understanding of the significance of gender pharmacology only emerged towards the end of the last century (Gandhi et al., 2004; Franconi et al., 2007, 2011a, 2011b; Anderson, 2005). Pharmacokinetics involves the examination of absorption, distribution, metabolism, and elimination of medication in the body, with these processes being significantly influenced by age and hormones, leading to sex-related differences (Spoletini et al., 2012). Sex hormones can interfere with drug efficacy and metabolism through various mechanisms, such as absorption, transporter competition, regulation of drug-metabolizing enzymes, and interactions with pharmacodynamics (Moyer et al., 2019). In females, variations in endogenous sex steroid hormones during the menstrual cycle, pregnancy, and menopausal transition can impact drug effectiveness and adverse reactions (Mitchell et al., 2009). Additionally, women use exogenous hormones for contraception, hot flashes, and other conditions, which can act as both treatments and contributors to adverse drug reactions. Therefore, while drug metabolism can affect exogenous hormone therapy, these hormones can also influence the metabolism of other medications.

Variations in the response to exogenous hormones can be attributed to individual differences in metabolic pathway components (pharmacogenetics) (Moyer et al., 2019). Unlike female reproductive aging (menopause) or organic androgen deficiency in males, male reproductive aging does not lead to a complete cessation of testosterone production or spermatogenesis. In fact, the decrease in testosterone levels due to aging is generally moderate, with levels typically remaining in the low-normal range for men. However, a small percentage of aging men may experience testosterone deficiency, which can be influenced by the presence of other health conditions. Recent research suggests that elderly men who maintain their health and fitness tend to have regular serum testosterone levels. Various terms, such as andropause, viropause, partial androgen deficit in the aging male, and late-onset hypogonadism, have been used to describe age-related low testosterone in men (Figueiredo et al., 2023). Furthermore, certain medications, including glucocorticoids and opioids, can suppress the gonadal axis (Wu et al., 2008; Bawor et al., 2015; de Vries et al., 2020). In terms of pharmacogenetics, it has been observed that individuals carrying the UGT1A4*1a allele may have reduced clearance of testosterone compared to those with the *3a allele (Zhou et al., 2011). On the other hand, pharmacodynamics focuses on the effects of therapeutic agents on the body and studies their biochemical and physiological effects, as well as their mechanisms of action. There are several pharmacodynamic differences between sexes, primarily influenced by hormones, genes, and the environment. The kidney is the primary organ responsible for excreting drug metabolites or parent drug molecules, and it has been documented that all three main renal processes—glomerular filtration, tubular secretion, and tubular reabsorption—exhibit sex differences. Men generally have higher renal clearance than women.

The activity of hepatic enzymes is affected by elevated levels of estrogen and progesterone, resulting in either increased drug accumulation or decreased drug elimination in certain cases. Prolactin and female steroid hormones influence autoimmunity, leading to a higher incidence and severity of autoimmune/inflammatory diseases in females compared to males (Soldin and Mattison, 2009). This is due to the regulation of immunity by the hypothalamic-pituitary-adrenal and hypothalamic-pituitary-gonadal axes. Autoimmune illnesses are more commonly observed in females of reproductive age. Fluctuations in hormone levels during menstruation, oral contraceptive use, pregnancy, and menopause can also impact metabolic changes. For example, some asthmatic women may experience worsened symptoms before or during their menstrual periods. Increased oxidative stress has been associated with intense physical activity, and this stress has been linked to gender disparities, particularly as individuals age. Despite the belief that sex hormones play a significant role in modifying sex-based differences in pharmacokinetics, studies investigating this relationship have produced conflicting results (Kharasch et al., 1999; Shah et al., 2001). For instance, the clearance of midazolam, a measure of CYP3A4 metabolic activity, did not vary throughout the menstrual cycle. Similarly, studies on eletriptan, a medication used to treat migraines, also showed no changes in response based on sex or menstrual cycle. While pharmacokinetic differences are relatively easier to analyze, detecting pharmacodynamic differences is more challenging (Franconi et al., 2007).

3.2. Preclinical studies

The primary objective of preclinical studies is to determine the pharmacokinetics and pharmacodynamics of therapeutic agents, projecting the active level in the target compartment, in order to anticipate the safe initial dose and dose escalation plan for phase 1 clinical trials (Beery and Zucker, 2011; Prendergast et al., 2014). There is a prevalent and persistent sex bias in preclinical research, with a focus primarily on male animals, even when the disease being studied is more common in women. Yoon et al. found that only 12% of studies on diseases common in women included research on females or both male and female subjects (Yoon et al., 2014). This gender bias is not limited to in vivo studies; in vitro studies have historically disregarded the importance of the sex of the cells' origin, with female animal usage being relatively low during preclinical experimentation (Regitz-Zagrosek, 2012, 2014; Franconi et al., 2011a, 2011b; Maselli et al., 2009; Wang et al., 2012; Legato, 2016). Despite this, it is important to recognize that even cultured cells have a sex, particularly during the initial maintenance stages (Beery and Zucker, 2011; Taylor et al., 2011; Vallabhajosyula et al., 2020; Maselli et al., 2009). Pharmacodynamic differences related to significant pharmacological targets are increasingly being identified, with hormones playing a crucial role in modulating these reactions (Romano and Gorelick, 2018). Estrogens and androgens, for example, elicit distinct molecular responses. Moreover, hormonal changes are influenced by factors such as age, reproductive stage, pregnancy, postnatal period, and menopause in women. In addition to hormones, genetic factors also play a role in varying treatment responses between genders (Hernandez et al., 2009; Nugent and McCarthy, 2011; Trout et al., 2007; Bilik et al., 2010; Franconi et al., 2017).

However, a recent shift has been observed. In 2018, Cvitanovíc Tomas and his team made adjustments to the SteatoNet in silico model, resulting in the creation of the LiverSex computer model (Cvitanović Tomaš et al., 2018; Naik et al., 2014). This model takes into consideration sex variances in the liver, incorporating sex-related impacts on growth hormone release from estrogen and androgen receptor responses. While the model has been tested on mice, it has not yet been validated on humans. Moving forward to 2020, Thiele and colleagues introduced sex-specific whole-body metabolic models (Thiele et al., 2020), encompassing 20 organs, 6 sex organs, 6 different blood cell types, systemic blood circulation, the blood-brain barrier, and the gastrointestinal lumen, along with the microbiome, to depict the physiological variances between males and females.

3.3. Chromosome

Sex in humans is determined by sex chromosomes, specifically the X and Y chromosomes. These chromosomes carry different numbers and sets of genes. The X chromosome carries approximately 1000 genes, while the Y chromosome carries only a few dozen genes. The morphological differentiation of the sex chromosomes is a result of a series of recombination events followed by the loss of genetic material on the Y gene (Clayton and Collins, 2014). It has been traditionally believed that the development of male or female is primarily attributed to the presence of a single locus, the sex-determining region gene (SRY), on the Y chromosome. However, recent studies have shown that this concept is more variable than previously hypothesized, and alternative mechanisms can play a role in sexual development that is different from what is expected based on the karyotype. This suggests that the sex-limited chromosome in some systems evolved independently and is not connected to the X or Z chromosomes (Nokkala et al., 2000, 2003). For example, in certain species like Rhinocola aceris and Cacopsylla peregrina, B chromosomes serve as the Y chromosome, while in several Lake Malawi cichlids, they serve as the W chromosome. There is strong evidence to suggest that the Lepidoptera W chromosome evolved after the Z chromosome, possibly from a B chromosome. In the case of the pill bug (Armadillium vulgare), the integration of a Wolbachia feminizer into the nuclear genome gave rise to the W chromosome. This discovery opens up interesting possibilities for the creation of non-homologous W chromosomes through the transportation of cytoplasmic male sterility factors, which are common in both insects and plants, to the nuclear genome (Leclercq et al., 2016).

ik the significant genetic distinction between the sexes is also supported by earlier research. Mary Lyon proposed in 1961 that one of the two X chromosomes in females becomes genetically inactive at an early stage in the development of a female embryo. Moreover, this process occurs randomly from one cell to another, indicating that biological females possess remarkable genetic mosaicism (Lyon, 1961). A considerable number of exceptions, and consequently, a wide range of underlying mechanisms, are brought to light by the diversity of the sex chromosomes. This diversity demonstrates that the regulations governing the evolution of sex chromosomes are intricate and not universally applicable (Furman et al., 2020). The systems that undergo the most frequent divergence or turnover may offer the most valuable insights moving forward, as comparisons can help distinguish cause from effect. The International Mouse Phenotyping Consortium discovered that sex played a significant role in the variability within the control data and as a modifier of treatment effects by examining data from 10 institutions, 14,000 wild type mice, and 40 thousand knockout mice for 234 characteristics (Karp et al., 2017). Sexually dimorphic effects across various biological systems emphasize the importance of considering both male and female sexes and incorporating sex as a factor of variation. Enzymes involved in the metabolism of therapeutic agents exhibit sexually dimorphic expression patterns in various species, influencing their metabolism. In humans, measurements of mRNA and protein levels of cytochrome p450 (CYP) 3A4 in the liver indicate higher levels in females. Furthermore, research has shown that this enzyme is more active in females. Rat and mouse livers display a significant degree of sexually dimorphic gene expression. For example, rats exhibit a male-specific CYP2C11 pattern, while CYP2C12 is exclusive to females (Wolbold et al., 2003; Hunt et al., 1992; Clodfelter et al., 2006, 2007; Yang et al., 2006; Robertson et al., 1990; Wauthier and Waxman, 2008; Shapiro et al., 1995).

When comparing male and female mouse liver microsomes of the same age, it was consistently observed that CYP1A2 was more prevalent in males. In mice aged 3–4 weeks, both male and female mice had higher levels of hepatic expression of CYP2B9 compared to mice of other ages. Interestingly, pregnant mouse liver microsomes showed higher levels of CYP2B9 compared to age-matched females. It is worth noting that only the kidney exhibited sexually dimorphic expression of CYP2B9, 2D26, 2E1, and 4B1 (Hersman and Bumpus, 2014; Emanuele et al., 2002; Becker and Cha, 1989). The complexity of metabolic pathways highlights the importance of understanding drug exposure in each sex, at the appropriate time, and in the relevant tissue when conducting pharmacodynamic assessments. However, it should be noted that sex differences observed in rodents may not always translate into similar patterns in humans.

The exclusion of females from certain research studies is partly attributed to the female estrous cycle. Rats go through a 4–5 day estrous cycle, with progesterone levels rising rapidly during the metestrum phase on day 1 and falling quickly during the diestrum phase on day 2. Ovulation leads to a significant increase in progesterone release and estrogen levels during proestrum. When estrous occurs on day 4, hormone levels return to normal after a temporary rise in estradiol. The unpredictability introduced by the estrous cycle is believed to affect experimental procedures. Hormonal fluctuations during the menstrual cycle can also impact the responsiveness of therapeutic agents in females. To mitigate the effects of the estrous cycle, it has been suggested to administer pharmacological tests only during diestrum, use male models exclusively, or employ a counterbalanced design to average out any changes (Hersman and Bumpus, 2014; Emanuele et al., 2002; Becker and Cha, 1989; Hu and Becker, 2003; Hughes, 2007).

Ovariectomy and castration techniques can be employed to eliminate gonadal effects from female and male rats in order to investigate the impact of hormonal fluctuations on experimental results. To study the physiological implications of hormonal levels, the behavioral and neurochemical characteristics of these animals can be contrasted with those of normally cycling females and unaltered males. Additionally, exogenous hormone treatments like estradiol, progesterone, or a combination of both, as well as testosterone if necessary, can be administered to ovariectomized and castrated animals.

3.4. Drug use and abuse exhibit differences between men and women

Drug use and abuse exhibit differences between men and women. Females appear to have a higher susceptibility to various phases of drug use, such as acquisition, maintenance, regulation, and relapse. This increased vulnerability may be influenced by the ovarian cycle, as studies have shown a correlation between higher estrogen levels and increased illicit drug use among women. The impact of gender and gonadal hormones on responses to addictive substances has been better understood through the use of animal models (Becker et al., 2001; Becker and Hu, 2008; Berry et al., 2016; Anker and Carroll, 2011). Our summary of preclinical pharmacological studies, including gender assessment, can be found in Table 1.

Table 1.

Summary of the pharmacological studies which include gender evaluation.

Ref #	Year	Topic
[38]	2018	Inflammation
[102]	2022	Psychiatry
[103]	2013	Cannabinoid
[104]	2007	Cannabinoid
[105]	2018	Cannabinoid
[106]	2017	Cannabinoid
[107]	2013	Cannabinoid
[108]	2012	Cannabinoid
[109]	2011	Cannabinoid
[110]	2021	Cannabinoid
[111]	2006	Cannabinoid
[112]	2013	Cannabinoid
[113]	2012	Pain
[114]	1998	Pain
[115]	2005	Analgesia
[116]	2018	Cannabinoid
[117]	2021	Analgesia
[118]	2013	Endocrinology
[119]	2019	Endocrinology
[120]	2019	Endocrinology
[121]	2012	Endocrinology
[122]	2018	Endocrinology
[123]	1973	Endocrinology
[124]	2018	Endocrinology
[125]	2022	Endocrinology
[129]	2021	Endocrinology
[130]	2015	Cardiology
[131]	2020	Cancer
[132]	2015	Cancer
[133]	2020	Cancer
[134]	2020	Cancer
[135]	2009	Rheumatology
[136]	2008	Rheumatology
[137]	2008	Rheumatology
[138]	2014	Antiretroviral drugs
[139]	2015	Antiretroviral drugs
[140]	2016	Antiretroviral drugs
[141]	2017	Antiretroviral drugs
[142]	2019	Antiretroviral drugs

Psychedelics, also known as hallucinogens, affect sensory processing, perception, and cognition primarily through the serotonin 5-HT2A receptor (5-HT2AR). This class of psychoactive drugs, which includes substances like lysergic acid diethylamide, psilocybin, mescaline, and DOI, is gaining attention in relation to mental health conditions such as depression and substance use disorders. Unfortunately, research often overlooks the role of gender in studying the potential clinical effects of hallucinogenic drugs on individuals.

While rodent models have significantly contributed to our knowledge of psychedelic pharmacology, most preclinical studies have focused solely on male mice. Jaster and colleagues investigated the effects of DOI on head-twitch behavior in male and female mice, a behavioral model for assessing the potential impact of psychedelic drugs in humans. In C57BL/6 J mice, females exhibited more pronounced behavior changes following DOI administration compared to males, with this sex-specific response not observed in 129S6/SvEv animals. Interestingly, the 5-HT2AR antagonist volinanserin completely blocked the DOI-induced behavior changes in both male and female C57BL/6 J mice.

There was no gender-related disparity in the quantity of inositol monophosphate that accumulated in the frontal cortex following DOI treatment in C57BL/6 J mice. Nevertheless, the pharmacokinetic properties of DOI differed between genders; female C57BL/6 J mice exhibited lower brain and plasma levels of DOI 30 and 60 min post-treatment compared to male C57BL/6 J mice (Jaster et al., 2022). Understanding the impact of gender on cannabinoid pharmacology is essential due to the increasing popularity of cannabis edibles for medicinal purposes and the ongoing misuse of these products. Female rats seem to display greater sensitivity to various effects of cannabinoid use, including anti-nociception, discriminative stimulus, and reinforcing effects, based on studies investigating gender differences in the behavioral effects of cannabinoids (Craft et al., 2013a, 2013b; McGregor and Arnold, 2007; Cooper and Craft, 2018; Wiley et al., 2017). Intriguingly, for optimal acquisition and maintenance of stimulus control in delta-9-tetrahydrocannabinol (THC) discrimination protocols, female rats required a lower THC training dose than male rats. When rats of either gender were trained to differentiate between identical THC doses, THC was more effective at eliciting discriminative stimulus effects in female Sprague Dawley rats compared to males of the same strain. However, the potency of THC in producing discriminative stimulus effects was similar in male and female C57BL/6 J mice trained to differentiate 5.6 mg/kg of THC from the vehicle, as well as in mice trained to differentiate a higher THC dose (30 mg/kg) (Wiley et al., 2011, 2017; Craft et al., 2013b; Winsauer et al., 2012).

In 2020, Wiley et al. conducted a study to examine the impact of intraperitoneal administration of THC and its main psychoactive metabolite, 11-OH-THC, on psycho-activity and molecular assays of cannabinoid receptor type-1 pharmacology in rodent models. The study aimed to investigate the effects of age, sex, and rodent species on these effects. The researchers found that both 11-OH-THC and THC acted as partial agonists in guanosine triphosphate, labeled on the gamma phosphate group, with equal intensity in both species and both male and female sexes (Wiley et al., 2021).

Furthermore, cannabinoids have shown promise in treating various challenging pain conditions and may serve as a non-opioid alternative for long-term management of chronic inflammatory pain (Blake et al., 2006; Johnson et al., 2013). Women, compared to men, have a higher prevalence of chronic pain and experience higher levels of experimentally induced and postoperative pain (Nahin, 2015; Riley et al., 1998; Aubrun et al., 2005). In a mouse model of inflammatory pain, LaFleur et al. observed that female mice exhibited lower susceptibility to the effects of Δ-9-THC and a synthetic cannabinoid compared to male mice. The S426 A/S430A mutation exacerbated the attenuation of nociceptive behaviors for both agonists in both male and female mice. Female mice also showed a delayed tolerance to Δ-9-THC, while the S426 A/S430A mutation caused a delayed tolerance in both male and female mice. Additionally, male S426 A/S430A mutant mice displayed resistance to tolerance to the synthetic cannabinoid compared to wild-type controls (LaFleur et al., 2018).

In the evaluation of NKTR-181 in preclinical pharmacology, both female and male Sprague Dawley rats and male CD1 mice were studied. The results showed that NKTR-181, a novel mu-opioid receptor agonist with limited brain entry, exhibited dose- and time-related anti-nociception in the hot-water tail-flick test, with peak effects similar to morphine, and no differences based on sex or species were observed (Kopruszinski et al., 2021).

Animal models play a crucial role in preclinical diabetes research. Glucose tolerance tests (GTTs) are commonly used in metabolic research to assess new antidiabetic treatments in the presence of elevated blood glucose levels (Pacini et al., 2013). Female mice are often excluded from diabetes studies due to their reduced glucose intolerance and insulin resistance (Nyavor et al., 2019; Kaikaew et al., 2019; Pettersson et al., 2012; Rebolledo-Solleiro and Fernández-Guasti, 2018). This reduced phenotypic behavior is believed to diminish their usefulness in treatment efficacy trials, potentially introducing bias and hindering translation to different clinical populations. The perceived higher variability in blood glucose levels throughout the estrous cycle in female mice may also contribute to researchers' reluctance to include them in studies (Bartke et al., 1973; Beery, 2018). Despite the growing recognition of the importance of considering sex as a biological variable in preclinical research, the impact of sex and the estrous cycle on blood glucose fluctuations during GTT has not been extensively studied (Clayton and Collins, 2014; Docherty et al., 2019; Beery, 2018).

Kennard and colleagues conducted a study to investigate the effects of sex, dosage method, length of fasting, and acute habituation stress on glucose tolerance test (GTT) measures used in preclinical assessment of potential glucose-modulating therapies. The researchers observed that female mice were less responsive to human involvement when initiating a fast. After a 6-h fast, both male and female mice showed quicker stabilization of basal blood glucose levels when the bedding was kept intact while the cage base was changed. Continuous fasting for 16 h resulted in an exaggerated GTT response but significant basal hypoglycemia. Following protocol optimization, Exendin-4 and metformin had a similar effect, with female mice displaying a more moderate but consistent GTT response (Kennard et al., 2022).

In the field of obesity and diabetes treatment, unimolecular peptides that target the glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) receptors (known as GLP-1/GIP co-agonists) are currently undergoing clinical testing (Frias et al., 2017, 2018). These peptides have shown superior efficacy in enhancing body weight, glucose management, and lipid metabolism compared to the best-in-class GLP-1 monotherapies, as demonstrated in rodent models of obesity, non-human primates, and humans (Frias et al., 2017, 2018; Finan et al., 2013).

Sachs et al. aimed to discover biomarkers that could facilitate non-invasive metabolic monitoring of compound treatment effectiveness and explore additional treatment impacts on an individual level despite variations in sex-specific plasma proteome profiling. The GLP-1R/GIPR co-agonist, as opposed to mono-agonist therapies, notably decreased obesity, glucose intolerance, non-alcoholic fatty liver disease, and dyslipidemia in male and female mice alike. Contrary to mono-agonist treatments, the proteome profiling variances in male and female mice showed more significant changes in plasma proteins following the GLP-1/GIP co-agonist (Sachs et al., 2021).

In a research conducted by Kremer et al. on dog models, beagles were administered etilefrine, sotalol, and hydralazine and implanted with a miniature telemetry blood pressure transmitter to monitor their blood pressure. Both male and female beagles exhibited changes in blood pressure due to etilefrine. The effects of hydralazine and sotalol were similar in both male and female beagles and lasted for 19 h post-dose. The exposure levels were dose-dependent and measurable between 7 and 7.5 h post-dose in the beagles that received etilefrine, sotalol, and hydralazine. Male and female beagles exposed to 10 mg/kg of etilefrine showed varying concentrations (169 vs. 268 ng/mL or 69%), with generally minor differences observed (Kremer et al., 2015).

Bourdi et al. conducted safety evaluations of metarrestin in beagles through both in vitro and in vivo studies. They observed that there was an increase in systemic exposure in a dose-related manner, with no differences between male and female dogs on days 1 and 27. Over the course of 27 days, metarrestin accumulated in both male and female dogs at all dose levels. No adverse effects were reported during the 28-day dosage period, where the estimated level of metarrestin in the dogs was 0.25 mg/kg. On day 27, the mean maximum concentration in male and female dogs was 82.5 ng/mL, with an exposure of 2521 h ng/mL (Bourdi et al., 2020).

The use of oxaliplatin, a platinum-based chemotherapeutic, for treating colorectal cancer is associated with a significant adverse effect known as peripheral neuropathy (Ewertz et al., 2015). In a recent phase 2 clinical study, thrombomodulin alfa, a recombinant human soluble thrombomodulin, was shown to inhibit oxaliplatin-induced peripheral neuropathy (Kotaka et al., 2020). A preclinical pharmacology study was conducted on rats treated intravenously with oxaliplatin (6 mg/kg). It was found that thrombomodulin alfa, administered through a single intravenous infusion, inhibited treatment-induced mechanical hyperalgesia in a dose-dependent manner, with no differences in effectiveness based on sex (Minami et al., 2020).

Preclinical studies on rheumatoid arthritis often use collagen-induced arthritic rats. In the initial examination of sex differences in these rats, male rats exhibited 43% higher dexamethasone clearances (Earp et al., 2008a, 2008b, 2009). Female rats, on the other hand, displayed earlier development, peak times, and remission of paw edema. Both male and female rats with similar capacity values experienced reduced paw edema with dexamethasone, but females had lower dexamethasone potency (Song et al., 2018). Inadequate antiretroviral (ARV) penetration leading to low-level viral replication in tissues is a potential cause of viral rebound (Fletcher et al., 2014; Thompson et al., 2015; Cottrell et al., 2016). Research on therapeutic agent concentrations in colorectal and female genital tract tissues demonstrated significant variations in ARV penetration for HIV prophylaxis. Despite the importance of these tissues in HIV pathogenesis, there is limited data on medication exposure in the presumed lymph node reservoir. Preclinical models involving HIV-infected humanized mice and nonhuman primates (NHPs) with simian/HIV reverse transcriptase showed that sex did not affect ARV pharmacokinetics in the collected lymph (Dimopoulos et al., 2017; Burgunder et al., 2019).

3.5. The impact of females on pharmacology

A literature review revealed that the majority of preclinical research has focused on male subjects (80%). Surprisingly, 44% of the studies on preclinical research models for diseases that predominantly affect women failed to specify the gender of the animals used; among those that did, 88% exclusively studied male animals (Beery and Zucker, 2011; Yoon et al., 2014). This gender bias in preclinical investigations has been identified as a contributing factor to the challenge of translating animal findings to humans (Karp and Reavey, 2019). While regulatory toxicity studies are required to include both male and female subjects before human trials, researchers are only encouraged to “consider” gender when designing safety pharmacology studies to assess short-term side effects on physiological functions. In addition to gender bias, animal models have other limitations. It is important to note that there are more factors than just X and Y chromosomes that differentiate between males and females (Beery, 2018). There has been a misconception that male and female rodents exhibit similar characteristics in preclinical studies. In reality, males and females differ in various physiological traits, such as body composition, neuroendocrine functions, immune responses, and behaviors beyond reproduction (Karp et al., 2017; Hughes, 2007). Furthermore, many diseases affect men and women differently, leading to variations in treatment outcomes, symptom progression, and susceptibility to diseases. Conditions like cardiovascular diseases, autoimmune disorders, chronic pain, and neuropsychiatric disorders have well-documented gender differences, with females often experiencing higher rates of incidence compared to males (Regitz-Zagrosek, 2012).

Lack of accuracy in reporting data can be observed in various aspects such as study design, phenotypes, pharmacokinetics, pharmacodynamic measurements, and the interpretation of results. It is important to consider sex as a covariate in these areas (Docherty et al., 2019; Gogos et al., 2019; Beierle et al., 1999; Dawkins et al., 1993; Gandhi et al., 2004; Fletcher et al., 1994; Anderson, 2008; Franconi et al., 2017, Franconi et al., 2011b, Franconi et al., 2011a, Franconi et al., 2007; Greenblatt et al., 2004, Greenblatt et al., 2014; Soldin and Mattison, 2009; Thürmann and Hompesch, 1998; Spoletini et al., 2012; Hunt et al., 1992). In addition, sex distinctions have been identified in disease onset and development, particularly in relation to animal models used in pharmacological investigations. It is also beneficial to analyze disaggregated data by age, taking into account the different periods of life that can impact health outcomes and treatment response, especially for women. To ensure the transferability of a preclinical research model to human reality, the age variable should also be considered (Tannenbaum et al., 2017; Jackson et al., 2017; Sukoff Rizzo et al., 2020).

3.6. The risks of preclinical studies

Throughout history, the sex/gender debate has seen its fair share of challenges and advancements. For instance, in 1977, the FDA prohibited women who could potentially become pregnant from participating in phase 1 and phase 2 trials following concerns related to thalidomide (Contergan) and diethylstilbestrol. Subsequently, in 1993, revisions were made to these guidelines after studies highlighted the underrepresentation of women in clinical trials. Between 1997 and 2000, eight out of ten prescription drugs were withdrawn from the market due to posing a higher health risk to women (Liu and Mager, 2016; Carey et al., 2017).

Sex plays a critical role as a biological determinant with significant implications. The lack of representation of female cells and animals in preclinical research has resulted in a limited understanding of pathophysiological, physiological, and biochemical pathways in females compared to males. Without data from females, it is challenging to determine if findings from male cells and animals are applicable to females (Kim et al., 2021; Karp and Reavey, 2019; Buoncervello et al., 2017; Voelkl et al., 2018). Basic science serves as the foundation for developing new therapies and identifying novel molecules. The translation of these preclinical findings to the health of both men and women is influenced by the identification of sex differences in pathophysiologic pathways in animal models of diseases (Sandberg et al., 2015). The analysis of gender disparities in scientific findings poses challenges, and the field is burdened by inconsistent outcomes. Factors such as rat age and strain could impact the results, and variables like the time of day the data were collected and methodological differences between laboratories could also influence the data (Voelkl et al., 2018, 2020; Freedman et al., 2015). Accurate determination and evaluation of the estrus cycle are essential for advancing the exploration of gender differences, particularly when comparing with a male group. Furthermore, the continued utilization of studies on hormonal and neutering treatments contributes to the growing understanding and recognition of the importance of gender differences in pharmacology. However, excluding female animals from preclinical studies may result in wasted resources on treatments that ultimately do not gain approval. Studies that include females will require larger sample sizes, but the impact of gender on pathological processes and treatment responses highlights the critical role of gender differences in preclinical research. If the inclusion of female models in preclinical research is still uncertain, the study of diseases and drug response in the transgender model is almost non-existent. However, data from international scientific literature indicates that the transgender population accounts for approximately 0.5–1.2% of the total population. In Italy, for instance, there are approximately 400,000 transgender individuals (Istituto Superiore di Sanità). Therefore, there should be an increasing interest from the scientific community in the health of transgender people. In recent years, numerous studies have been published on this population segment, but the limited number of subjects studied prevents us from drawing definitive conclusions regarding susceptibility and risk factors for chronic-degenerative diseases, thus hindering the development of specific healthcare plans for these groups. Preclinical research must promptly adapt to this situation by proposing an experimental transgender model to investigate how a combination of hormones can potentially influence disease progression and therapy responses. Currently, there is a scarcity of scientific literature related to preclinical research in this field. Despite transgender individuals often receiving gender-affirming hormone therapy, no hormonal agents or clinical protocols for transgender medicine have been approved by international medical regulatory agencies such as the European Medicines Agency or the Food and Drug Administration. Hormone therapies used in transgender medicine are considered “off-label” and are based on recommendations from Endocrine Societies or similar organizations (T’Sjoen et al., 2019).

The primary objectives of hormone therapies in humans, which are often administered throughout one's life, are to decrease secondary sex characteristics and restore sex hormone levels to the normal range for cisgender individuals. For transgender men, hormone therapy involves the injection of testosterone (either intramuscularly or subcutaneously). Recently, there has been a suggestion to use transdermal administration (such as patches or gel) of a longer-acting form, as it is suitable for long-term use. On the other hand, hormone therapy for transgender women typically consists of β-estradiol, which can be given transdermally, orally, or intravenously, either alone or in combination with medications that suppress androgen levels. The dosage levels and administration windows vary depending on the method of β-estradiol administration. In Europe, the most commonly used anti-androgen medication is cyproterone acetate (50 mg daily). Currently, there is limited information available regarding the potential effects of hormone therapy on the health of transgender individuals and its long-term consequences. Risk assessment aims to evaluate the chemical risks in potentially sensitive sub-population groups and select appropriate safety measures. In this context, toxicological considerations should also be taken into account. Endocrine disruptors, which have similar targets and modes of action as hormone therapy, are among the environmental and food pollutants that transgender individuals, like the general population, are regularly exposed to. This makes transgender individuals a subgroup that is more susceptible and vulnerable to the effects of these disruptors. To accurately assess the risks for transgender individuals undergoing hormone therapy, specialized animal models should be established and utilized (Pettit, 2021).

Targeted animal models are essential in toxicological investigations to obtain reliable information for identifying chemical hazards (Rusyn et al., 2022). Individuals with TG who have undergone HT exhibit distinct characteristics that make them particularly susceptible and vulnerable to chemical contaminants. As a result, they require appropriate animal models that are based on relevant and innovative biomarkers (Tassinari et al., 2021).

One significant model for determining whether sex variations in phenotypes are caused by the complement of sex chromosomes (XX vs. XY), gonadal hormones, or both, is the “four core genotypes” (FCG) mouse model. In this model, a Sry transgene is inserted onto an autosome after the deletion of the testis-determining gene Sry from the Y chromosome. By breeding XX and XY mice with testes and XX and XY mice with ovaries, it becomes possible to compare mice with the same type of gonad (XX vs. XY) and determine the phenotypic consequences of sex chromosomal complement in cells and tissues (Arnold, 2020; Burgoyne and Arnold, 2016; Burgoyne et al., 1998; De Vries et al., 2002; Eicher et al., 1991; Mahadevaiah et al., 1998).

The number of X chromosomes (including X dose, X imprint, or indirect effects of X inactivation), the presence or absence of the Y chromosome, or both, may be the cause of a sex chromosome effect (XX not equal to XY) in FCG mice (Burgoyne and Arnold, 2016; Arnold, 2017). To distinguish between these possibilities, the XY* model proves to be helpful. In XY* mice, which were initially discovered by Eicher et al., a defective pseudo-autosomal region on the Y chromosome recombines improperly with the X chromosome (Burgoyne et al., 1998; Eicher et al., 1991). When XX females are mated with XY* fathers, it results in the production of mice that are comparable to XX and XO gonadal females, as well as XY and XXY gonadal males (Burgoyne and Arnold, 2016). When comparing females with one X chromosome (XO) to females with two X chromosomes (XX), or males with one Y chromosome (XY) to males with an extra X chromosome (XXY), the impact of having one X chromosome versus two can be determined. Similarly, comparing males with one Y chromosome to those with no Y chromosome (XX) can assess the impact of having a Y chromosome. According to the XY* paradigm, mice with a Y chromosome are considered gonadal males.

A recent study examined the effects of testosterone cessation on ovarian dynamics using a mouse model that simulated trans-masculine testosterone therapy. Over a period of six weeks, post-pubertal female C57BL/6 N mice aged 9–10 weeks were administered either testosterone enanthate or a control injection of 0.9 mg once a week. Within one week of starting the testosterone treatment, all the mice receiving testosterone ceased cycling and entered a state of chronic diestrus, while the control mice continued to cycle regularly. At the end of the study, the age-matched control mice in diestrus and a group of mice treated with testosterone for six weeks were sacrificed. Additionally, the age-matched control mice and another group of mice that had resumed cycling after receiving testosterone therapy were sacrificed during diestrus after four cycles.

Comparing the post-testosterone group to both the age-matched controls and the mice treated with testosterone for six weeks revealed stromal alterations characterized by clusters of large round cells. These clusters exhibited periodic acid-Schiff staining, which is typically observed in multinucleated macrophages in aging mouse ovaries. Furthermore, a significant number of these cells showed staining for the macrophage markers CD68 and CD11b. Analysis of ovarian ribonucleic acid-sequencing between the age-matched controls and the ovaries from mice treated with testosterone for six weeks indicated upregulation of immunological pathways in the post-testosterone group (Kinnear et al., 2023).

The incorporation of both male and female sexes is a legitimate strategy to enhance diversity. Failure to specify the sex used, allowing sex to remain uncontrolled, or neglecting to address sex as a significant source of variation can lead to replication challenges. Female cells function differently from male cells, impacting their sensitivity to stressors, susceptibility to disease, and response to medication. Therefore, investigating gender disparities and cell injury mechanisms is crucial for developing more effective therapeutic approaches. Scientists must adequately account for these differences in diseases and patient populations to ensure successful drug development. This, in turn, allows for the creation of new pharmaceuticals through tailored preclinical research and safety studies that align precisely with the characteristics of the new drug, while also identifying when sex-specific considerations are necessary.

4. Conclusion

It is crucial to consider the impact of sex on pharmacological studies for personalized treatment and precision medicine. Despite the presence of sexual dimorphism in many diseases, a large number of preclinical research studies overlook the importance of sex. Sex-based statistical analyses are not commonly conducted in preclinical investigations. Moreover, women are often underrepresented in therapeutic trials despite the well-known sexual dimorphism. Societal perceptions of certain diseases as either “male” or “female” may lead to delays in diagnosis and treatment for both sexes. Additionally, there are sex variations in the effectiveness and safety of various pharmacological classes due to differing pharmacokinetic characteristics, resulting in more adverse effects in women. To enhance the translation of research findings and promote tailored therapy, sex should be considered as a variable from the preclinical stage onwards. Further research focusing on sex differences in specific areas or diseases is necessary to explore diverse therapeutic approaches in depth.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

The authors are unable or have chosen not to specify which data has been used.