Azole antifungal aromatase inhibitors - verifications and updates

Since the initial 1999-2001 publications came out, more laboratories replicate findings and extend them.

Jul 19, 2021

More than 20 years ago, I published the characterization of the Structure Activity Relationship (SAR), rank order, and potency profile of several commercial and pharmaceutical azole (imidazoles and triazoles) antifungal compounds in the inhibition of human placental aromatase. Teaming up with BD Biosciences colleagues, we extended the work using high-throughput fluorescent assays. Coincident with these data, was an indepth review of the clinical implications of aromatase enzyme inhibition, especially in primate pregnancy.

So… what happened in the ensuing two decades?

Structure Activity Relationship SAR

IN 2004, Swiss toxicologists involved in monitoring the safety of chemicals used in agriculture, tested a full panel of azoles in their aromatase assay. Rank order was similar and concentration for inhibition was within the order of magnitude. The most potent medicinal antifungals of their test set were bifonazole, miconazole, and clotrimazole.

In 2019, the Danish investigated endocrine disrupting potential of four commonly used azole antifungal drugs, clotrimazole, miconazole, ketoconazole and fluconazole in vitro, using the H295R cell assay and two recombinant CYP17A1 and CYP19A1 (aromatase) assays. Results showed that azole antifungal drugs inhibited steroidogenic cytochrome enzymes at concentrations within therapeutic levels. Their data were very consistent with my earlier publications.

Placenta and Primate Pregnancy

In 2016 JAMA published the clinical observation study from Denmark comparing use of oral fluconazole vs topical azoles therapies in pregnant women.

Among 3315 women exposed to oral fluconazole from 7 through 22 weeks’ gestation, 147 experienced a spontaneous abortion, compared with 563 among 13,246 unexposed matched women. There was a significantly increased risk of spontaneous abortion associated with fluconazole exposure (HR, 1.48; 95% CI, 1.23-1.77). http://jama.jamanetwork.com/article.aspx?articleid=2480487

There were significantly more miscarriages in the oral fluconazole group as compared to the topical drug comparator group. No data were presented comparing to those using nonazole topicals (eg nystatin) which have no aromatase inhibition potency. I am doubtful that such data will be allowed to be published. This was a comparison with a competitor, thus was driven by marketing incentives. Not safety.

A 2021 study of human relevant doses of clotrimazole, but in rodent pregnancy, also resulted in fetal losses:

https://www.researchgate.net/publication/351161721_Human-relevant_concentrations_of_the_antifungal_drug_clotrimazole_disrupt_maternal_and_fetal_steroid_hormone_profiles_in_rats

The clinical effects of aromatase inhibition in the placenta during pregnancy include a rise in the level of androgens. Primates studies from Albrecht’s lab indicated generational, inherited changes in gonadal and carbohydrate metabolism from that exposure (see Kragie 2002 review). More recently, polycystic ovary syndrome (PCOS) research examined effects of androgen fetal exposures:

"The placenta plays an important role in protecting the growing fetus, and we and others have shown that women with PCOS have disturbances in the function of the placenta which can affect the fetus and it's germ cells, and thereby theoretically contribute to the development of PCOS in the offspring through epigenetic processes," … Studies have also shown that newborn daughters have an increased so-called anogenital distance, a sign of fetus exposure to male sex hormone in utero during pregnancy. Even sons of women with PCOS and their brothers have metabolic disorders. - Elisabet Stener-Victorin

https://doi.org/10.1080/09513590.2018.1564742

Brain Aromatase

The effects of aromatase inhibition include the critical period of imprinting on the brain gonadal axis, to establish fertility hormonal signaling. Wisconsin Primate Research Institute published in 2013, their studies on rhesus monkeys’ hypothalamus:

… the research team infused letrazole, an aromatase inhibitor that blocks the synthesis of estrogen, resulting in a lack of estrogen as well as GnRH release from the brain. Together, these methods demonstrated how local synthesis of estrogen in the brain is important in regulating reproductive function. … "The discovery that the primate brain can make estrogen is key to a better understanding of hormonal changes observed during every phase of development, from prenatal to puberty, and throughout adulthood, including aging," - Kenealy, https://medicalxpress.com/news/2013-12-estrogen-ovaries.html

In adults, aromatase conversion of androgens to estrogen participates in response to brain trauma:

there's mounting evidence that in the healthy brain, aromatase and the estrogen it enables neurons to produce, helps keep our brains and us nimble. Now scientists are learning that with injury, aromatase and estrogen expression seem to shift to cells in the brain called astrocytes, aiding their support and nurturing of now-stressed neurons, …
Even in culture, neurons will connect and communicate, but when scientists add an aromatase inhibitor to the mix, connectivity is interrupted. Some of the first in vivo studies in zebra finches showed that aromatase levels increased following a brain injury, which also supports a protective role for the protein. More brain damage results when aromatase inhibitors are given. "There seemed to be more inflammation," - Dr. Darrell Brann, Medical College of Georgia, 2015

So obviously, we should be fully aware of any compounds that inhibit aromatase in humans. That is why we added aromatase inhibition potency to Tier 1 EDSTAC testing of new chemicals, and require it for reproductive toxicity testing in preclinical pharma development.

But will that information ever get out to the practitioner and patient who need it? Twenty years gone by … and still I wait.

Kragie L, Turner SD, Patten CJ, Crespi CL, Stresser DM. 2002 Assessing pregnancy risks of azole antifungals using a high throughput aromatase inhibition assay. Endocrine Research 28 (3): 129-140 https://www.researchgate.net/publication/10983695_Assessing_pregnancy_risks_of_azole_antifungals_using_a_high_throughput_aromatase_inhibition_assay

Kragie L. 2002 Aromatase in primate pregnancy: a review. Endocrine Research 28 (3): 121-128 https://www.researchgate.net/publication/10983694_Aromatase_in_primate_pregnancy_A_review

ER Trösken et al. Comparative Assessment of the Inhibition of Human Recombinant CYP19 (Aromatase) by Azoles Used in Agriculture and as Drugs for Humans. Endocr Res 2004 Aug;30(3):387-94. https://doi.org/10.1081/ERC-200035093

CH Munkboel, et al 2019 The classic azole antifungal drugs are highly potent endocrine disruptors in vitro inhibiting steroidogenic CYP enzymes at concentrations lower than therapeutic Cmax. Toxicology Volume 425, 1 September 2019, 152247 https://doi.org/10.1016/j.tox.2019.152247

MK Draskau et al 2021 Human-relevant concentrations of the antifungal drug clotrimazole disrupt maternal and fetal steroid hormone profiles in rats. Toxicology and Applied Pharmacology 422 (2021) 115554

Association Between Use of Oral Fluconazole During Pregnancy and Risk of Spontaneous Abortion and Stillbirth. JAMA. 2016;315(1):58-67. doi:10.1001/jama.2015.17844 https://jamanetwork.com/journals/jama/fullarticle/2480487

Preventing the development of polycystic ovary syndrome https://medicalxpress.com/news/2021-07-polycystic-ovary-syndrome.html

Elisabet Stener-Victorin et al, Epigenetic inheritance of polycystic ovary syndrome—challenges and opportunities for treatment, Nature Reviews Endocrinology (2021). DOI: 10.1038/s41574-021-00517-x

Estrogen: Not just produced by the ovaries https://medicalxpress.com/news/2013-12-estrogen-ovaries.html

Enzyme that converts testosterone to estrogen has big role in the healthy, injured brain https://medicalxpress.com/news/2015-05-enzyme-testosterone-estrogen-big-role.html

Peer Review Results for the Aromatase Assay U.S. Environmental Protection Agency Exposure Assessment Coordination and Policy; Division Office of Science Coordination and Policy. January 2008 DOI: 10.13140/2.1.4653.1369 Conference: Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC)

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