EXHIBIT 1014
`
`EXHIBIT 1014
`
`

`

`' 'i I
`
`f'ERTILJT\' AND STERILITY
`
`\'ol 5J. No. t. Jnnun.ry 1900
`Prrntt.·d 011 ctt;d.fr-.""f: /Xl~,. ;n U.S.A.
`
`Effect of low-dose oral contraceptive on gonadotropins,
`androgens, and sex hormone binding globulin
`in nonhirsute women*'t
`
`Ana Alvarez Murphy, M.D.:j:·§
`Craig S. Cropp, M.D.I
`Beverly S. Smith, B.S.
`
`Ronald T. Burkman, M.D.#
`Howard A. Zacur, M.D., PhD.V
`
`University of California, San Diego School of Medicine, San Diego, Californio and
`The Johns Hopl~ins Utiiuer;;ity School of Medicine, Baltimore, Maryland
`
`The effectiveness of a low-dose oral contraceptive (OC) in suppressing plasma levels
`of gonadotropins, ovarian, and adrenal androgens and stimulating sex hormone-binding
`globulin (SHBG) was evaluated prospectively in nonhirsute women. Thirty-three women
`ingested 35 pg of ethinyl estradiol and 1 mg of norethindrone beginning within day 1 to 5
`of the menstrual cycle. Baseline levels of luteinizing hormone, follicle-slimulnting hor(cid:173)
`mone, Lotal testosterone (T), androstenedione (A), dehydroepiandrosterone sulfate
`(DHEAS), and SHBG were obtained before ingestion of the OC and repeated after 3, 6
`and, 9 months of OC use on day 1 to 5 of the OC "cycle". A significant suppression of
`gonadotropin levels is seen in nonhirsute women. Sex hormone binding globulin is consis(cid:173)
`tently stimu lated by the low-dose OC. A significant suppression ofT and DHEAS is ob·
`served. No change was seen in levels of A. The demonstrated effects become eviclcntut ::1
`months and are maintained at 6 and 9 months.
`Pertil Steril 53:35, 1990
`
`The use of combined oral contraceptives (OCs) is
`associated with many metabolic changes and side
`effects. OC associated changes such as increased
`risk of peripheral arterial phenomena, myocardial
`infarction in smokers, liver tumors, hypertension,
`
`ncccived June 12, 1 989; revised and accepted August 9, 1989.
`• Supported in part by the General Research Center grant
`PHS RR·OOS27 and grant N0l·HD·32816 from the Notional
`Institutes of Health, Bethesda, Maryland.
`t Presented at the Thirty-finh Annual Meeting of the Soci·
`ely of Gynecologic Investigations, March 18 to 20, 1988, Bolli·
`more, Maryland.
`t Department of Reproductive Medicine, University of Cali·
`fornia, San Diego School of Medicine.
`§Reprint requests: Ana A. Murphy, M.D., UCSD Medical
`Center 1'·002, 225 Dickinson Street, San Diego, California
`92103.
`U Present add ress: National Cancer 1 nstitute, Bethesda,
`Maryland.
`~ Department of Gyneco iO!.'Y a nd Obstetri cs, Johns Hopkins
`University, School of Medicine.
`II Present address: Department of Obstetrics and Gynecol·
`ogy, Henry Ford Hospital, Detroit, Michigan.
`
`and changes in glucose metabolism appear to bees·
`t.rogen dependent.!-!• To reduce t.he magnitude of
`these changes, the dose of estrogens has been
`markedly reduced such thal most OCs in the
`United States contain 30 to 35 ~g of ethinyl estra(cid:173)
`diol (EE2). There has been a trend to decrease the
`content. of progestogens as well. Progestins de(cid:173)
`crease high-density lipoprotein (HOL)-cholesterol
`and increase low-density lipoprotein (LDL)-cho(cid:173)
`lesterol an effect that promotes heart. disease. The
`arleriosclerotic process responsible for heart at·
`tacks and strokes is att.ribut.ed to the progestin
`component. Oral contraceptive with high progestin
`content, unlike today's low-dose formulations, pro·
`duce the unfavorable lipoprotein levels.6
`Oral contraceptives containing 50 to 80 J.lg ofEE2
`or it's equivalent have been commonly used to treat
`both idiopathic hirsutism and h irsutism associated
`with hyperandrogenemia. Tradil.ionally, OCs have
`been used t.o suppress androgens of ovarian origin.
`However, several studies have indicated suppres(cid:173)
`sion of adrenal androgens such as dehydroepian·
`
`drosterone sulfate (DHEAS) with OC.7•8 Given the
`
`Vol. 53, No. 1, January 1990
`
`Murphy et al. Contracepliues, conadalropins, androgens
`
`35
`
`Petitioner Exhibit 1014
`Petition for Inter Partes Review of U.S. Patent No. 7,704,984
`Page 1
`
`

`

`combined decrease in the OC components, there is
`the possibility that suppression of pituitary-ovar(cid:173)
`ian/adrenal function may not be as "efrective" as
`with higher dose formulations. Additionally, little
`normative data is available on the long term effects
`of low dose OCs on these parameters. The present
`study was designed to evaluate the short and long
`term efl'ect of a low-dose OC on ovarian and adre(cid:173)
`nal function in normal women.
`
`MATERIALS AND METHODS
`
`Subjects
`
`Thirty-three healthy, normally menstruating,
`nonhi rsute women aged 18 to 35 years were stud(cid:173)
`ied. None had received steroid treatment within
`the last 3 months before the study. The study pro(cid:173)
`tocol was approved by the internal review board
`and informed consent was obtained from each vol(cid:173)
`unteer. An OC containing 35 J.lg ofEE2 and 1 mg of
`norethindrone (NET) was begun within day 1 to 5
`or the menstrual cycle. Serum samples were ob(cid:173)
`lained between 7 to 9 A.M. before beginning OCs
`and again at 3, 6, and, 9 months on day 1 to 5 of the
`OC "cycle". Day 1 of the OC cycle is the first day
`of OC ingestion. All sera were stored at -2o•c until
`analysis. All patient sera were included in t.he same
`assay t.o eliminat.e inLerassay variabilit.y.
`
`Assay Methods
`
`Luteinizing hormone (LH) and follicle-stimulat(cid:173)
`ing hormone (FSH) were quantified by direct ra(cid:173)
`dioimrmmoassay (RIA) using a second antibody
`precipitation method to separate botmd a nd free
`reactants.!J.ro LER-907 standards were used fo r
`both LH and FSH. Sera were supplied by National
`Institutes of Health (Bethesda, M D). Iodinated an(cid:173)
`tigens were purchased from New England Nuclear
`(Boston, MA). Second antibody was obtained from
`Pell Free?.e Inc. (Rogers, AK). The FSH assay sen(cid:173)
`sitivity is 25 J.lg/mL and 10 JJg/mL for the LH as(cid:173)
`say. The int.ra-assay coefficient of variation was
`2.1% for FSH and 3.2% for LH. Sex hormone-bind(cid:173)
`ing globulin (SHBG) values were determined by
`RIA using a commercial kit (Tech land S.A., Liege,
`Belgium). The sensitivity of the assay is 10 J.lmolf
`L. Androstenedione (A), DHEAS, and total T were
`also determined by RIA according to t he manufac(cid:173)
`turers recommendation (Diagnostic Products Cor(cid:173)
`poration, Los Angeles, CA). The sensitivity of t he
`A assay is 0.02 J.lg/mL, 2.1 J.lg/dL for DHEAS and
`
`so
`
`40
`
`..J 30
`
`X ' C) 20
`z
`
`10
`
`0
`500
`
`400
`
`..J
`X
`
`300
`' u 200
`z
`
`100
`
`0
`
`FSH
`
`0
`
`6
`3
`TI ME IN MONTHS
`
`g
`
`Fi.:urc 1 Effects of n low-close OC on mean (±SE) serum
`FSH and LH levels at baseline and 3, 6, 9 months. •Significant
`difl'erences determined by Dunnett's multiple range test
`(P < 0.005).
`
`11 J.Lg/dL forT. The intra-assay coefficient of varia(cid:173)
`tion was 3.7% for SHBG, 4.3% for A, 0.51% for
`DI-IEAS, and 1.5% forT.
`
`Statistical Methods
`
`Repeated measures analysis of variance and co(cid:173)
`variance was used for analysis of data (Biomedical
`Data Program [BMDP] Statistical Software, Inc.
`Los Angeles, CA). Once statistical significance was
`established with analysis of variance, Dunnett's
`mu ltiple range test was used to compare the base(cid:173)
`line data with that obtained at 3, 6, and, 9 months
`separately (BMDP Statistical Software, Inc.). Re(cid:173)
`sults are expressed as mean± standard error of the
`mean (SEM).
`
`RESULTS
`
`The changes observed for gonadotropins are
`shown in Figure 1. Follicle-stimulating hormone
`levels were significantly reduced from baseline at 3
`months, 6 months, and 9 months (P < 0.002). The
`maximum decrease in FSH occurred at 3 months
`(61 %). Similarly, LH was maximally suppressed
`from baseline by 30% at 3 months with continued
`suppression at 6 and 9 months (P < 0.0001).
`The mean sex hormone binding globulin level
`before treatment was 65.1 ± 4. 7 mmol/L as seen in
`
`36
`
`Murphy et al. Contraceptivl!$, gonadotropins. androgr!fls
`
`Fertility ond Sterility
`
`Petitioner Exhibit 1014
`Petition for Inter Partes Review of U.S. Patent No. 7,704,984
`Page 2
`
`I
`
`

`

`SHBG
`
`200
`
`ISO
`
`100
`
`so
`
`0
`
`...1
`
`' ...1
`
`0
`::£
`z
`
`0
`
`6
`3
`TI ME IN MONTHS
`
`9
`
`Figure 2 EH'ects of o low-dose OC on mean l±SE:) serum
`SHBG levels at baseline and 3, 6, 9 months. ~significanL
`diffe rences determined by DunneLL's mu ltip le range t.est
`lP <0.001).
`
`Figure 2. A statistically significa nt increase oc(cid:173)
`curred at 3 mont hs, which continued at 6 months
`and 9 months (P < 0.0001).
`Serum DHEAS was significantly reduced after 3
`months and 6 months of low-dose OC (P < 0.028) .
`At 9 months DHEAS remained suppressed, as seen
`in Figure 3.
`The plasma levels of A at 3 months, 6 months,
`and 9 months were not significantly different from
`baseline. Total testosterone was significantly de(cid:173)
`creased at 3 months. The effect was maintained at
`6 months and 9 months (P < 0.0001).
`
`DISCUSSION
`Our results are the first to demonstrate that a
`"low dose" monophasic OC s ignificantly sup(cid:173)
`presses serum immunoreactive gonadotropin levels
`in normally menstruaLing women when compared
`with basel ine in the early follicular phase. A
`smaller study by Carr et al. 11 in four women using
`a "higher dose" OC (1.0 mg of NET and 80 Jlg of
`mestranol) found a suppression of serum LH to un (cid:173)
`detectable levels ( <20 ng/mL) and a marked de(cid:173)
`crease of serum FSH that was independen: of the
`t ime before t he contraceptive induced withdrawal
`bleed. A study by Dericks-Tan et al. 12 using a com(cid:173)
`bined low-dose preparation, while showing a trend
`toward decreased gonadotropins, failed to demon(cid:173)
`strate a statistically significant difference. H ow(cid:173)
`ever, significance was not obtained possibly be(cid:173)
`cause of the small sample size (N = 6). While our
`study shows a significant suppression of immuno(cid:173)
`reactive levels of both gonadotropins on lew-dose
`OC, FSH showed a two fold greater decrease than
`LH (60% versus 30%). Such a result may have been
`anticipated since Franchimont et al. 13 reported
`that higher doses of EE2 and it.s derivatives (mes(cid:173)
`tranol and 1-hydroxyethinyl estradiol 1.3, diace-
`
`tate) depressed both LH and FSH, whereas lower
`doses of EE2 (20 ~Jg) primarily suppressed ser um
`FSH in postmenopausal women. Moreover, the
`less pronounced decline in LH seen in our study
`may explain t he failure of E lstein and associates 14
`to detect changes in urinary LH secretion, except
`fo r suppression of the ovulatory peak, in women
`taking 50 11g of mestranol. Taken together with the
`1
`previously published data, 11
`1 our results are con(cid:173)
`'
`:
`sistent with a dose dependent gonadotropin sup(cid:173)
`in normal cycling women adminis(cid:173)
`pression
`tered OCs.
`Because our daLa followed individual pat ients
`prospectively, it was possible to discern t hat a sig(cid:173)
`nificantly larger number of women fa iled to show
`suppression of immu noreactive LH (11 of 33) t han
`fa iled to s how suppression of FSH (3 of 33) (P
`< 0.02). All (three) subjects who showed no sup(cid:173)
`pression of FSH also showed no suppression ofL.H.
`It is widely postulated that differential control of
`gonadotropin secretion is regulated by inh ibin, ac(cid:173)
`tivin, and other ovarian secretory products. W hile
`
`400
`
`300
`
`200
`
`100
`
`0
`40
`
`30
`
`20
`
`10
`
`0
`3
`
`OH€AS
`
`' -
`
`TESTOSTERONE
`
`ANDROSTENEDIONE
`
`I
`
`...1
`
`0 ' l:l
`
`::>
`
`...1
`0
`
`' l:l
`a.
`
`...1
`;[
`
`' l:l :z
`
`0
`
`0
`
`3
`6
`TIME IN MONTHS
`
`9
`
`Figu r e 3 Effccu or a low-do~e OC on mean (±SE) serum T,
`A, DHEAS levels oL boselinc and 3, 6, 9 months. •SignificonL
`difrerences determined by DunneLL's multiple range test
`(P <0.05).
`
`Vol. 53, No. J, January 1990
`
`Murp h y cL ul. Contraceplives,gonadotropins, ondrogcru;
`
`3 7
`
`Petitioner Exhibit 1014
`Petition for Inter Partes Review of U.S. Patent No. 7,704,984
`Page 3
`
`

`

`lt , 5 £1! "" l Mijff1
`
`q
`
`' I
`I
`I I
`
`·I
`
`the effects of OCs on the secretion of these gonado(cid:173)
`tropin regulatory proteins is unknown, it is possi(cid:173)
`ble that relatively specific inhibitors of FSH are
`stimulated by either estrogens or progestins or that
`suppression of FSH stimulators occurs. Alterna(cid:173)
`tively it could be argued chat inabil ity to detect
`equal changes in LH is an artifact that results from
`the greater pulsatile excursions in human serum of
`LH resu lting from the much shorter half life of LH
`than FSH.
`Sex hormone-binding globulin is substantially
`increased by estrogens and somewhat reduced by
`p rogestogens. 1r,. u; The effect of NET containing OC
`on SHBG is predominantly estrogenic rather than
`progestogen ic. A levonorgestrel (LEV)-containing
`OC has been demonstrated to cause a decrease in
`SHBG capacity. 17 "Higher" dose OCs have been
`shown to increase SHBG in hirsute women. 111 Tal(cid:173)
`bert and Sloan I!J have shown " low dose" NET-con(cid:173)
`ta ining OCs significantly
`increase SHBG
`in
`women with polycystic ovarian d isease. A shor t
`term study (20 days) of normal women demon(cid:173)
`strated that low-dose EE2 (30 Jig) a lone signifi(cid:173)
`cantly increases S HBG.~u Our data showing a sig(cid:173)
`nificant rise in SHBG is consistent with this earlier
`work, and demonstrates for the first time that, in
`normal women on monophasic J)Ieparations, these
`a lterations persist throughout the course of ther(cid:173)
`apy when followed for as long as 9 months.
`Dehydroepiandrosterone su lfate has been shown
`to be an important marl<er for adrenal androgen
`secretion in women. 21 The administration of estro(cid:173)
`gen alone results in either no change or an increase
`in DHEAS levels.t2 .t~• Klove7 studied the efl:"ect of
`low-dose OCs o n serum concentration of DHEAS
`in six nonhirsute women and noted that OCs con (cid:173)
`taining NET but not LEV have a suppressive effect
`on adrenal androgen secretion. Our study confirms
`this finding with an NET-containing low-dose OC.
`A significant decline in DHEAS levels is seen and
`maintained for at least 9 months. The mechanism
`for this suppression is not established, although it
`has been suggested that plasma adrenocorti(cid:173)
`cotropic hormone (ACTH) levels a re decreased
`by 0Cs. 11
`Jung-Hotrman and Kuhl2·' noted a significant
`suppression of nonprotein bound T d uring treat(cid:173)
`ment with both a low-dose monophasic OC and a
`triphasil preparation in normal women. Low dose
`OCs have been shown to suppress T in hirsute
`2
`1 Our study is
`women with chronic anovulation.s· 18
`·
`·
`the first long term study in normal women showing
`a significant Sllppression of protein bound T with
`
`a low-close monophasic OC that is maintained to
`9 months. Taken with the significant decrease in
`SHBG, the suppression would appear to be clini(cid:173)
`cally significant.
`Wild et al.2r; studied 15 hirsute women before a nd
`after the completion of one cycle of a n OC contain (cid:173)
`ing 2 mg of NET and 100 1-1g of mestranol. Andro(cid:173)
`stenedione was reduced by 47%. A reduction in the
`magnitude of the diurnal change in A synchronous
`with cor tisol as well as a reduction in the A re(cid:173)
`sponse to ACTH was seen. As noted previously,
`adrenal suppression seems to be dependent on the
`type of progestin. The low-dose OC used in this
`study contains ha lf the progestin content that was
`used by Wild et al. 25 Our data in normal women
`show no change in A. Taken together with the pre(cid:173)
`viously published data2
`'' on hirsute women, we sug(cid:173)
`gest there may be a dose dependent suppression of
`A which fails to reach significance at the low dose
`used in our study. The differing results may be ex(cid:173)
`pla ined by the different doses and populations (hir(cid:173)
`sute versus normal).
`In conclusion, our data document that a low-dose
`OC containing 35 pg of EE2 and 1 mg of NET re(cid:173)
`duces gonadotropin levels in nonhirsute, normally
`cycling women when compared with the early fol(cid:173)
`licular phase. A differential effect is seen with FSH
`being more consistently suppressed than LH. Sex
`hormone-binding globulin is consistently stimu(cid:173)
`lated by the low-dose OC. Our results show a sig(cid:173)
`nificant decline in DHEAS levels that is sustained
`to 9 months. Serum total T is suppressed, but an(cid:173)
`drostenedione did not significantly vary from base(cid:173)
`line obtained in the early follicular phase. This
`large, prospective, longitudinal study provides im(cid:173)
`portant normative data for a commonly used
`monophasic low-dose oral contraceptive.
`
`Aclmowleclgmenls. We thank Edward E. Wallach, M.D. and
`Joseph Mortola, M.D. for their ki nd review of the manuscript
`and Mr. Paul Shragg of the General Clinical Research Center
`for the stm.istical analysis.
`
`REFERENCES
`
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`38
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`M u rph y ct a l.
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`Co11lrocl!pt.ivc.~. gonadotropins. ondro.qens
`
`Fertility and Sterility
`
`Petitioner Exhibit 1014
`Petition for Inter Partes Review of U.S. Patent No. 7,704,984
`Page 4
`
`

`

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`
`~ I
`'
`
`Vol. 53, No. 1, January 1990
`
`Murphy et al. Contraceptiu(!S, gonadotropins. androgens
`
`39
`
`Petitioner Exhibit 1014
`Petition for Inter Partes Review of U.S. Patent No. 7,704,984
`Page 5
`
`