Cilostazol and Its Effects on Resumption of Meiosis in the Human Ovary

Combined oral contraceptive pills (COCs) are the most commonly used hormonal form of birth
control in the United States with at least 87% of women of reproductive age reporting oral
contraceptive use at some point in their lives (9). Despite their frequent use, the six and
twelve month discontinuation rates for oral contraceptive pills are 31 and 47 % respectively
(17), with common reasons for discontinuation attributed to the side effects of abnormal
bleeding, headache, and weight gain.

Additionally, COCs are contraindicated in certain groups of women as outlined by The Centers
for Disease Control Medical Eligibility Criteria (11). Given the high prevalence of oral
contraceptive users who commonly discontinue use secondary to side effects or who are not
eligible for use as a result of underlying health conditions, the development of novel oral
non-hormonal methods that are equally effective at pregnancy prevention are warranted.

The potential for the development of non-hormonal contraception has been present in the
scientific literature since the 1980s at which point the critical components of gametogenesis
of the mammalian oocyte were being classified. Bornslaeger and colleagues discovered that
high concentrations of intracellular cAMP, an important second messenger for many biological
processes, inhibited resumption of meiosis I in mouse oocytes (3). Later studies demonstrated
that phosphodiesterase 3 inhibitors, capable of preventing the breakdown of cAMP, were able
to prevent resumption of meiosis in mouse oocytes cultured in vitro (6).

Since the 1980s, additional studies have examined phosphodiesterase inhibitors and their role
in prevention of oocyte maturation. Tsafriri and colleagues showed that phosphodiesterase
inhibitors were able to exert their effects selectively via type specific isoforms. This
study revealed that granulosa cells exert effects via type 4 phosphodiesterase inhibitors and
oocytes respond to signals via type 3 phosphodiesterase inhibitors such that oocyte
maturation was prevented in the presence of PDE3 inhibitors yet ovulation was unaffected
(15). Most recently, Jensen and colleagues examined both in vitro and in vivo effects of a
phosphodiesterase 3 inhibitor ORG 9935 on oocyte maturation in rhesus monkeys. In these
animal studies, it was found that in vitro, ORG 9935 at a concentration of 1.0umol/l was able
to completely inhibit oocyte maturation (7).

In vivo models demonstrated a similar reduction in oocyte maturation, with the most dramatic
reduction in monkeys treated with an extended dose regimen of ORG 9935 (200mg/kg/d) prior to
ovulation (8). Additionally, pregnancy rates in macaques treated with ORG 9935 was studied
and compared to controls. Overall, there was not a statistically significant decrease in
pregnancy rates in those macaques treated with PDE3 inhibitor; however, there did appear to
be evidence of a dose response as no animal became pregnant who had serum ORG 9935 levels
above 300nM/L (9). Despite a clear dose response, authors of this study acknowledge that
PDE3-Is have systemic effects that are also dose-limiting. At higher doses, adverse effects
such as tachycardia and hypotension were observed.

Phosphodiesterase 3 inhibitors have additionally been studied in vitro to assess oocyte
maturation in humans. Human oocytes cultured in the presence of ORG 9935 (1um) for 24 hours
demonstrated a statistically significant reduction in germinal vesicle breakdown (an early
marker of oocyte maturation) in comparison to control oocytes (12). However, no human in vivo
models assessing oocyte maturation following treatment with a phosphodiesterase 3 inhibitor
exist.

Cilostazol, an FDA approved drug for the treatment of intermittent claudication, is a class
IIIa phosphodiesterase inhibitor. Its systemic effects in humans include vasodilation,
decrease in platelet aggregation, reduction in triglyceride levels, and increase in HDL
cholesterol levels (4). Human clinical trials did not address fertility and pregnancy rates;
however, manufacturer product labeling reports that no fertility effects occurred in rats.
Most recently, however, two animal in vivo studies examining pregnancy rates of mice treated
with Cilostazol were conducted (1,10). In both of these studies, mice treated with Cilostazol
were shown to be completely infertile at follow up; however, once the drug was discontinued,
all mice that were unable to conceive during treatment with cilostazol were subsequently able
to have pregnancies of normal litter sizes. It is important to mention that in the study by
Albarzanchi, mice received PO cilostazol at a dose of 7.5 or 15 mg PO for three days (1). In
the study by Li conducted in China, the maxium dose was higher (300mg/kg) (10). Pregnancy
outcomes for both studies were the same.

These studies demonstrate two important aspects of a contraceptive method: efficacy and
reversibility. Additionally, the investigators who published Cilostazol's fertility effects
in mice published an abstract demonstrating a similar effect in swine (2). When a human dose
of 100mg bid of Cilostazol was given prior to ovulation, no documented pregnancies occurred.
Following cessation of study drug, all swine were able to conceive.

Given the success of phosphodiesterase inhibitors in both in vivo and in vitro models for
prevention of oocyte maturation and pregnancy in animal models, it is reasonable that we
further examine this potential non-hormonal oral contraceptive method. Given the many FDA
clinical trials that need to be conducted, new contraceptive methods take decades to develop.
However, since Cilostazol is an approved FDA medication with an acceptable side effect
profile, we believe that it is appropriate to complete initial studies of this drug in humans
using an in vivo technique validated in macaques, a model with the same timing of
peri-ovulatory events as women, to ascertain its possible use as a non-hormonal oral
contraceptive.

The primary objective of the research study is to determine if women taking Cilostazol
demonstrate impairment of oocyte maturation in comparison to paired historic controls
following ovarian follicle stimulation. For the purposes of our study, oocyte maturation will
be assessed using the oocyte maturational stages of prophase I, metaphase I, and metaphase
II. Thus, we will be assessing the percent of oocytes aspirated from follicles during
controlled ovarian stimulation that progress beyond the germinal vesicle stage (e.g. GVBD) in
those women receiving cilostazol and compare this to the maturation of oocytes obtained from
the same women who underwent ovarian follicle stimulation previously while not on study drug.

We propose that women undergoing treatment with the FDA approved dose of 100mg PO BID of
Cilostazol will demonstrate an impairment of oocyte maturation in comparison to paired
historic controls following ovarian follicle stimulation.

Inclusion Criteria:

- previous oocyte donor in USC IVF clinic within past 3 years

- age 18-33

- willingness and ability to commit to the time requirements of the study

- willingness to donate oocytes for research purposes

- willingness to discontinue current hormonal contraception

- otherwise healthy subjects

Exclusion Criteria:

- contraindications to combined hormonal contraceptive use (CDC MEC class 3 or 4)

- pregnancy

- history of cardiac arrhythmias

- history of heart failure

- history of bleeding disorder

- concomitant use of anti-platelet therapy such as aspirin

- current use of drugs that inhibit cytochrome P450 CYP 3A4 (erythromycin, diltiazem,
ketoconazole, itraconazole) or CYP 2C19 (omeprazole) as they may lead to increased
serum levels of cilostazol.