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MELCANGI 2014 Altered Levels of Neuroactive Steroids in Cerebrospinal Fluid and Plasm of PFS Patients Journal of Steroid Biochemistry and Molecular Biolo.pdf


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G Model
SBMB-4178; No. of Pages 6

ARTICLE IN PRESS
D. Caruso et al. / Journal of Steroid Biochemistry & Molecular Biology xxx (2014) xxx–xxx

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Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.
General data and self-reported frequency of the most symptoms reported by the patients at the moment of sampling . . . . . . . . . . . . . . . . . .
3.2.
Assessment of neuroactive steroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction
Observations obtained in multiple double-blind randomized
controlled trials for male pattern hair loss have indicated that
finasteride (i.e., a 5␣-reductase inhibitor used for the treatment
of human benign prostatic hyperplasia and androgenic alopecia)
treatment was associated with sexual dysfunction [1–3]. Similar
side effects were also reported in patients treated for benign prostatic hyperplasia [4–7]. Very important, observations performed
in a subset of patients for male pattern hair loss seem to indicate
that persistent sexual side effects (e.g., low libido, erectile dysfunction, decreased arousal and difficulty in reaching orgasm) have been
reported even after discontinuation of the treatment [8,9]. Patients
also developed depression during finasteride treatment [10,11]
that still persisted despite treatment withdrawal [12]. Depression
after finasteride treatment might be due to impairment in the
levels of neuroactive steroids. This steroid family, which includes
both steroid hormones produced in peripheral glands and steroids
directly synthesized in the nervous system (i.e., neurosteroids), has
an important role in the control of nervous function, affecting mood,
behavior, reproduction and cognition, as well as being protective
agents in models of injury and neurodegenerative diseases [13–16].
Indeed, finasteride is not only able to block 5␣-reductase (5␣-R)
enzyme, which converts testosterone (T) into dihydrotestosterone
(DHT), but also the conversion of progesterone (PROG) into dihydroprogesterone (DHP) [16]. In this context, it is also important to
highlight that these neuroactive steroids are then converted by the
action of the 3␣- or 3␤-hydroxysteroid dehydrogenase into 5␣androstane-3␣,17␤-diol (3␣-diol) or 5␣-androstane-3␤,17␤ diol
(3␤-diol) in case of DHT and into tetrahydroprogesterone (THP),
also known as allopregnanolone, or into isopregnanolone in case of
DHP [16]. It is interesting to note that THP, as well as the 3␣-diol
(i.e., a metabolite of DHT), are known as ligands of GABA-A receptor [17]. Moreover, isopregnanolone does not bind directly to the
GABA-A receptor [18], but it antagonizes the effect of THP on the
GABA-A receptor [19,20]. Changes in GABA as well as in neuroactive
steroid levels in plasma and cerebrospinal fluid (CSF) are associated
with depression in several human studies [21].
Interestingly, our recent preliminary observations obtained in
three male patients who received finasteride for the treatment of
androgenic alopecia and that after drug discontinuation still had
long-term sexual side effects as well as anxious/depressive symptomatology showed altered neuroactive steroid levels in plasma
and CSF vs. those assessed in 5 healthy patients [22]. A further link
with neuroactive steroids may be supported by recent observations. Indeed, as reported in a subset of post-finasteride patients
with persistent symptomatology, a decline in their alcohol consumption was also observed [23]. This is very interesting, because
a relationship between GABAergic neuroactive steroids and ethanol
consumption is well documented [24].
On the basis of this interesting finding, we here extend our
observations analyzing by liquid chromatography–tandem mass
spectrometry (LC–MS/MS) the levels of neuroactive steroids, such
as pregnenolone (PREG), PROG and its derivatives, DHP, THP and
isopregnanolone, dehydroepiandrosterone (DHEA), testosterone
(T) and its derivatives, DHT, 3␣-diol, 3␤-diol and 17␤-estradiol

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(17␤-E), in paired plasma and CSF samples obtained from seven
post-finasteride patients (i.e., patients who received the drug for
the treatment of androgenic alopecia and resulting in long-term
sexual side effects as well as anxious/depressive symptomatology
after finasteride discontinuation) and comparing these levels vs.
those assessed in twelve healthy controls.

2. Materials and methods
PREG, PROG, DHP, THP, isopregnanolone, T, DHT, 3␣-diol,
3␤-diol DHEA and 17␤-E were purchased from Sigma Aldrich.
17,21,21,21-D4 -PREG (D4 -PREG) was kindly synthesized by Dr.
P. Ferraboschi (Dept. of Med. Biotech. & Translational Medicine,
University of Milano, Italy); 2,2,4,6,6-17␣,21,21,21-D9 -PROG (D9 PROG) was obtained from Medical Isotopes (Pelham, NH, USA);
2,3,4-13 C3 -17␤-estradiol (13 C3 -17␤-E) was obtained from SigmaAldrich, Italy. SPE cartridges (Discovery DS-C18 500 mg) were from
Supelco, Italy. All solvents and reagents were HPLC grade (Sigma
Aldrich, Italy).

2.1. Study design and sample preparation
Patients were recruited through the “Italian network finasteride
side effects”, where the possibility to undergo CSF and plasma
examination in the context of an approved pilot study was made
available. Given the exploratory nature of the study no exclusion criteria were established, except the use of drugs known to
potentially interfere with neuroactive steroids levels. Symptoms
reported by the patients were collected using a standardized questionnaire prepared after consensus among the members of the
“Italian network on finasteride side effects” based on an extensive
collection of the reported symptoms. The presence of a representative pattern of these symptoms was necessary to be eligible for
neuroactive steroid assessment.
The questionnaire was used as a method to systematically collect information on patients conditions and not as a validated tool
to assess the features of post-finasteride syndrome. In order to limit
selection and recall bias it was filled in by patients only once before
they were made aware of the possibility to undergo neuroactive
steroid assessment.
The study procedure was approved by the Ethics Committee of
the S. Gerardo Hospital, Monza-Italy and the participating subjects
provided their written informed consent before enrollment.
In order to obtain reliable normal control values, CSF and plasma
were collected from 12 subjects who underwent spinal anesthesia
for orthopedic surgery at San Gerardo Hospital of Monza. These
subjects were otherwise healthy, were carefully screened for the
absence of any neurological or psychiatric disorder in their personal or family history and gave their written informed consent to
the use for scientific purpose of the aliquot (approx 100–200 ␮l)
of CFS drawn to verify the correct position of the spinal needle,
according to the procedure approved by the Ethics Committee of
the S. Gerardo Hospital in Monza.

Please cite this article in press as: D. Caruso, et al., Patients treated for male pattern hair with finasteride show, after discontinuation of the drug, altered levels of neuroactive steroids in cerebrospinal fluid and plasma, J. Steroid Biochem. Mol. Biol. (2014),
http://dx.doi.org/10.1016/j.jsbmb.2014.03.012