Corcept Therapeutics, Inc.
`Exhibit 2012
`Neptune Generics, LLC v. Corcept Therapeutics, Inc.
`Case IPR2018-01494
`
`Ex. 2012-0001
`
`

`

`The antiprogestr'n. untiglucocorticoid RU 486: Spitz and Hardin
`
`and currently known by the generic name mifcpristone. The original
`studies showed that RU 486 displayed a relative binding activity five
`times that of progesterone to the rabbit uterine progestin receptor and
`three times greater than dexamethasone to the rat thymus glucocorticoid
`receptor. Its relative binding affinity for the rat prostate androgen receptor
`was only 25% that of testosterone, and there was no binding to the
`mouse uterine estrogen or rat kidney mineralocorticoid receptors. Further
`biological tests in animals showed that the compound behaved as a potent
`antagonist for progestins and glucocorticoids.“ The affinity of RU 486 for
`both the progestin and glucocorticoid receptor was not unexpected in
`view of the numerous biological studies showing that progestins have
`weak glueocorticoid activity and vice versa. Moreover, the predicted
`amino acid sequence of the glucocorticoid and mineralocorticoid receptor
`steroid binding domains shows a greater similarity to that of the progester-
`one receptor than to any of the other receptors in this family.“
`
`II. Structure of RU 486 and Other Antiprogestins
`
`Il-[4-{dimethylaminoiphenyl— I 7-hydroxy- 1 7-1 1 -propynyl}-[1 lB,
`RU 486,
`l7Bl-estta-4,9-dien-3-one, is a derivative of norethindrone that lacks the
`C i 9 methyl group and the 2—carhon side chain at C17 of progesterone and
`glucocorticoids [Figure ll. RU 486 differs from norethindrone due to a 4-
`{dimethylamino} phenyl group at the 1 its position and a l~propynyl chain
`
`
`
`mc’
`
`"has“
`
`NORE‘I’HINDRONE
`
`on
`
`”CID“
`
`emu"
`
`HIJ I" (MIFEPRIETDNE}
`
`2K OI ’3‘ {ULOWIGTDHE}
`
`2K 93 I” (ONIFRISTONEJ
`
`FIGURE 1. Chemical structure of progesterone. norelhindrone [1?-hydroxy-[17n}-1Q—norpregn-4-en-20-yn—3—
`one}. tamoxifen ((ZJ-E-[4—(1.2-dipheny—1—butenyl)phenoxy]—N‘N-dimethylethanamine]. RU 486 (1t—[4-dimelhy-
`Iamlno)phenyl] -1?-hydroxy-17A[1 -propynyl]-[11[5.17fl1-estra-4.Q-dien-S—one). 2K 983’34 (11-{4-[dimeihyiamino)-
`phenyI1—I7—hydroxy-17-[3-hydroxy—1-propenyr]- [115, 1m. 17::{2}]-estra-4.Q-dien-3-one] and 2K 98299 11—[4—
`{dimethylarnino)phenyl]-17—hydroxy-17-[3-hydroxypropyi-[HB. 130:. 17n]-estraA,Q—dien-3-one).
`
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`The antiprogestin, antigfueocortieoid RU 486: Spitz and Bardin
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`at the 170: position. The conjugated C9-C10 double bond in RU 486 should
`also be noted. The 170: substitution probably imparts higher binding affin-
`ity for the receptor. By analogy with antiestrogens, it is likely that the
`substitution at the [18 position is responsible for its antagonistic action
`by inducing or stabilizing a biologically inert receptor conformation}-In
`Numerous additional antiprogcstins have been synthesized and many
`are undergoing preliminary evaluation.“ ZK 98734 ililopristone} has a
`cis {Zl-configuration in the 3-hydroxy-1-propenyl side chain at the 170:
`position and is structurally very similar to RU 486 {Figure l}. The two-
`dirnensional structure of ZK 9829910napristone1is similar to RU 486 and
`ZK 98734 [Figure 1]. However, ZK 98299 has a different molecular shape
`due to configurational inversions at the C13 and C17 positions.“
`ZK 98734 and ZK 98199 also bind with high affinity to glucocorticoid
`and progesterone receptors. Although there are some species differences,
`their antiprogestational and antiglucoeorticoid activities are compara-
`ble.“ ZK 98299 exerts stronger synergistic effects when given with prosta—
`glandins and oxytocin, and it has the most petent effect on the cervix in
`the guinea pig.“ ZK 98734 and ZK 98299 have less antiglucocorticoid
`action than RU 486.“ At the present time, only RU 486 has been exten-
`sively studied in humans and, as a consequence, will be the major focus
`of this review.
`
`III. Mechanism of Action
`
`The aetion of progesterone in target tissues is mediated by the progester—
`one receptor [PRL which like the other members of this nuclear receptor
`family is a ligand-activated transcription factor with domains for DNA
`binding, hormone binding, and transactivation,” The PR of most species
`has a large hydrophobic pocket which can accommodate subsritutions
`at position 118 like those on RU 486.1“ Spontaneous mutations of the
`progesterone receptor have been difficult to identify since the phenotype
`of women with such a mutation would be unexplained sterility and the
`defect could not be inherited. Therefore, an analysis of how the amino
`acid sequence of the receptor relates to proge5terone binding must depend
`upon site-direcred mutations made in the laboratory. Nonetheless, an
`experiment of nature showed that a glycine in the hormone binding do—
`main of the human PR at position 722 {Gly’fll and at the comparable
`position of the PR of most Other species is critical for RU 486 binding
`and action lFigure 2].12 The hamster and the chicken PR that have a
`cysteine residue at this position” hind progesterone but not RU 486.
`These species are, therefore, insensitive to this antagonist? Substitution
`of this cysteine by glycine converts the hormone binding domain of the
`chicken PR to one that binds RU 486 and facilitates the antagonistic
`action of this compound.11 Substitution of Sly”?- with cysteine in the
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`The ontiprogestin, antigiucOcorricoid RU 486: Spitz and Bardr'n
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`
`
`FIGURE 2. Schematic diagram of the primary structure of the human progesterone receptor
`(hPFI) showing: [1) transactivation domain; (2) DNA binding domain: and (3] hormone binding
`domain. The amino acid glycine (Glymt in the hormone binding domain is critical for RU 485
`[but not progesterone) binding. Amino acids corresponding to Gly’” in the rabbit PR {erFl},
`chicken PR tcPFt). hamster PFI the PH). human glucocorticoid receptor {hGR}, human androgen
`receptor (hAR), and human mineralocortlcoid receptOr [hMFlJ are also shown. Fleceptors with
`glycine at this position bind and respond to RU 486; those with other amino acids do not. G ——
`glycine; C —- cysteine: A = alanine.
`
`human PR generates a receptor that behaves like the chicken and hamster
`PRsi Introduction of the same cysteine substitution at the corresponding
`position in the human glucocorticoid receptor resulted in a loss of binding
`not only to RU 486 but also to dexamethasone.” In fact,
`the human
`glucocorticoid and androgen receptors, which bind RU 486, also have a
`glycine in this corresponding position [Figure ll. Because glycine is the
`only amino acid without a side chain, these results suggest that Glyn" in
`the human PR is at a critical position in the 1 lB—poeket and the presence
`of amino acid side chains in this position may sterically impede RU 486
`binding.” The precise molecular mechanisms whereby progesterone and
`RU 486 produce their agonistie and antagonistic activities, respecrively,
`via the PR are under active investigation."-"' The most recent in vivo and
`in vitro data support the actions summarized in Figure 3. Progesterone
`and other progestins produce a dramatic change in conformation of the
`PR that is associated with transforming [activating] PR from a non-DNA
`binding form to a form that will bind to DNA. This transformation is
`accompanied by a loss of associated heat-shock proteins and dimerization.
`The activated receptor dimer can bind to progesterone response elements
`in the promoter region of progesterone-responsive genes and, in the pres-
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`The antiprogestr‘n, antigiucocorticoid RU 486: Spi'tz and Bardin
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`
`
`partial agonlst
`
`FIGURE 3. Proposed mechanisms of action of progesterone (P) and the antiprogestin (AP) RU
`485. In the absence of ligand. the progesterone receptor {PR} is associated with heat-shock
`proteins (HSP). Association of PR with either P or AP induces different conformational changes
`in PFI. both of which result in dissociation of HSP. dimenzetion of PH, and association of PR
`with the specific progesterone response elements in the promoter regions of progesterone-
`responsive genes. when bound. the P—PFt complex is transcriptionally active resulting in agonistic
`eflects {upper diagram).
`in the absence of P. the AP-PR complex behaves as a partial P
`agonist {lower diagram]. The AP-PH complex is inactive (antagonistic) in the presence of P.
`The mechanism by which RU 436 can be antagonistic and agonistic in the presence and absence
`of progesterone is not known. One possibility is that agonistic effects result when both binding
`sites on the receptor dimer bind the antiprogestin and that antagonistic effects result when one
`monomer binds antiprogestin and the other binds progesterone (not shown).
`
`ence of other nuclear transcription factors, increases the rate of transcrip-
`tion of these genes, thus producing agonistic effects at the cellular and
`tissue levels [Figure 3]. When RU 486 binds to the inactive progesterone
`receptor, it induces an equally dramatic change in receptor conformation,
`loss of heat-shock proteins, and dimerization. The steroid receptor antago-
`nist complex also will bind tightly to progesterone response elements.
`but these DNA hound receptors are transcriptionally inacrive if progester-
`one is present. This is the basis for the abortifacient action of antiprogcs-
`tins. However, in the absence of progesterone, the steroid receptor RU
`486 complex may be transcriptionally active on some genes and RU 486
`thus acts as a ”partial agonist". This term is used bccausc RU 486 does
`not duplicate all of the aetions of progestins {see below}. It is possible
`that antincoplastic effects of antiprogestins relate to these agonistic cf-
`fccts. The precise molecular mechanism whereby RU 486 [and possibly
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`The antiprogestin, antiglucocorticoid RU 486: Spitz and Bardin
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`other antiprogestins] can be agonistic and antagonistic on cells in the
`absence and presence of progesterone, respectively, is not known at this
`time.
`
`IV. Pharmacology
`
`RU 486 is administered by the oral route and is readily absorbed in all
`species, but metabolism in the splanchnic circulation reduces its bioavail-
`ability to 40% in man and rats and 15% in monkeys." When given by
`the intramuscular route to monkeys, there is a depOt effeCt and the com-
`pound is released slowly into the circulation.” Vaginal administration in
`humans is net an effective means of delivering the doses usually necessary
`for pregnancy termination.”
`The metabolic clearance rates were 72, 36, and 0.55 L/kg/day in rats,
`monkeys, and humans, respectively.H The clearance rate for an average
`woman would therefore he 30 L/day, which is considerably slower than
`cortisol [200 L/day} and estrone sulfate [160 L/dayl, two natural steroids
`that are considered to be cleared at slow rates. The differences in the
`
`clearance rates between man and animals result in part from an or, acid
`glyeoprotein, orosomucoid, in serum that binds RU 486 in humans (HI)
`'1 uM] but not in other species?“ RU 486 does not bind to cortisol
`binding globulin or sex-steroid binding globulin.l
`In women, following oral administration of single doses ranging from
`50—800 mg, serum RU 486 levels reached a maximum in 1 hour. Dc-
`pending on the dose administered, the pharmacokinctics displayed two
`distinct patterns.” After a low dose {50 mg], the disappearance of RU 486
`follows first order kinetics with a half—life of .1045 hours {Figure 4]. After
`ingestion of doses of 100-800 mg, there is an initial redistribution phase
`of 6—10 hours follmved by a plateau in serum levels for 24 hours or more.
`With these larger doses, there is no significant dose—dependent difference
`in serum concentrations within the first 48 hours [Figure 5].” During this
`period, serum RU 486 levels are in the micromolar range [i.e., 2.5 umol/
`Ll. One of the possible explanations for this unusual pattern of metabolism
`is in fact that orosomueoid binding sites are saturated at doses of RU
`486 above 100 trig}9 Following oral administration, detectable levels of
`unmetabolized RU 486 have been found in the circulation for up to 10
`days. ‘9 The major excretory pathway for RU 486 is fecal, with less than
`10% being recovered in the urine.”
`Animal studies have shown that the first steps of RU 486 metabolism
`involve a two—step demcthylation of the [dimethylaminolphenyl ring at
`the C11 position and hydroxylation of the l7-propyny] chain [Figure 5].
`The demethylated metabolites are further hydroxylated or acetylated.“
`In the human, micromolar concentrations of monodemethylated, dide-
`methylated, and the non-demethylated hydroxylated metabolite were ob-
`
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`The antiprogestin, antiglucocortieoid RU 486: Spitz and Bardin
`
`RU486(uglml)
`
`h
`
`.8
`
`.6
`
`.4
`
`.2
`
`1
`
`2
`
`4
`
`6
`Hours
`
`f--—-'—f
`10
`
`24
`
`43
`
`FIGURE 4. Hal! disappearance time of RU 486 following administration of 50 mg in the mid—
`luteal phase. Results show moan : SEM in 5 subjects. Modified from Shoupe at at?“
`
`served within 1 hour after oral administration of RU 486 {Figure 5]. In
`contrast to the parent compound, circulating concentrations of metabo-
`Iitcs increase in a dose—dependent fashion and with higher doses, the
`metabolite concentrations, especially the monodcmethylatcd derivative,
`are in excess of the parent compound.”
`The monodemethylated and hydroxvlated derivatives interrupt preg~
`nancy in the rat With a potency one-third that of RU 486. The antiglucocor-
`tieoid activity of these compounds are also one-third that of RU 486. The
`didemcthylated compound, however, is clearly less potent than the other
`metabolites in the rat.” The biological effects of each of these metabolites
`have nor been evaluated direcrly in humans, however, they do bind to
`the human progesterone and glucocorricoicl receptors and even though
`the affinity is less than that of RU 486, these metabolites may contribute
`to the overall cffecrs of the drug in view of their high concentrations in
`serum}I
`
`Although RU 486 is distributed into all tissues of the rat,H the tissue
`distribution in humans is less well studied. In women, RU 486 and its
`dcrncthylated metabolites were detected in the myometrium and abdomi—
`nal adipose tissue collected during hysrerectomy 12 -15 hours after oral
`administration of 2.00 mg of the antiprogestin.21 This dose resulted in
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`

`The antipmgestin, antiglucocorticoid R U 486: Spitz and Hardin
`
`-<-c—a‘
`
`O
`
`22>-
`
`.l
`"cm—M
`
`mu m I
`
`II
`
`“\
`“‘3’”
`
`in am
`
`II
`
`\
`I)
`
`at
`Awe—o—qg
`
`m
`-<-c—cn.
`
`—._
`
`0
`
`I!” «m
`
`RU mo
`
`
`
`
`
`O—Qnu ates
`0—- mnodemethyiated (RU 42633)
`H Didemathylatad (PM 42648)
`a": Hyfioxylaled (RU 42696)
`
` 10"molrL
`
`1
`
`4
`
`IO
`HOURS
`
`24
`
`' 43
`
`Freune 5. The upper panel depicts the metabolism at RU 4386 Into its monodemethylated (FIU
`42633). diclemethylated {RU 42848). and alcoholic name-methylated (RU 42693) derivatives.
`The mean t + SEM) concentration 01‘ RU 486 and these three metabotites after oral administralion
`of 100 mg and 600 mg in five subjects is shown in the middle and lower panels. respectively.
`Fledrawn from Léhteenmaki et al.” The concentrations of Ru 436 were similar after these closes.
`but the concentrations of metabolites were higher after 600 mg RU 486.
`
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`The antiprogestr’n. antigincocorticoid RU 486: Spitz and Bardin
`
`myometrial concentrations similar to those required to produce maximal
`stimulation of prostaglandin synthesis of human endometrial tissue in
`vitrofiu‘ As noted below, this action is believed to be one of the major
`reasons why this agent induces abortion. Lower than expected levels of
`RU 486 in fat are believed to be due to its avid binding to serum protein.12
`Studies have also shown that RU 486 and its monodemethylated metabo-
`lite cross the placental barrier during the second trirnesterfiiits In monkeys,
`it has been suggested that the efficacy of placental transfer may decrease
`with advancing pregnancy.”
`
`V. Maior Biological Effects
`
`A. Antightcocorticor‘d/Glucocorticor’d Actions
`
`RU 486 has been shown to have antiglucocorticoid activity by a broad
`array of in vivo and in vitro studies-“v3" In view of its use as an antiprogestin
`in normal women. its antiglucoeorticoid effects on the hypothalamic-
`pituitary-adrenal axis are of particular interest. RU 486 was found to have
`a dose-dependent inhibitory effect on the negative feedback of cortisol
`on ACTH secretionsW -" Single RU 486 doses of i and 2.2 lug/kg in women
`and men. respectively, were insufficient to produce this effect, but 4 mg}
`kg in women and 4.5 and 6 mg/kg in men led to a significant rise in
`ACTH and cortisol)“ The inhibition of the morning rise of ACTH, B-
`endorphin, and cortisol levels by 1 mg dexamethsone administered at
`midnight was abolished by a single dose of RU 486 [6 mgfkgl administered
`at the same time.” In humans, it was estimated that the optimal antigluco-
`corticoid effect occurs when the RU 486zcortisol ratio in serum is approxi-
`mately 10:1.”
`The central antiglucocorticoid effect of RU 486 is particularly evident
`during the morning hours when cortisol levels are rapidly increasing.
`Thus, the midnight intake of RU 486 [6 mg/kg] by inhibiting the negative
`feedback of cortisol upon the hypothalamic pituitary axis enhanced the
`increase of serum ACTH, B-endorphin, and cortisol during the subsequent
`morning, i.e., 8—12 hours after drug ingestion. There were no changes in
`these parameters within 8—14 hours of ingestion of the same dose of RU
`486 when it was given in the morning, however, on the following morning,
`the 8:00 a.rn. values were again higher than in controls.” Thus, the effect
`of RU 486 on the pituitary-adrenal system occurs only at a specific time
`of day. Since this enhanced cortisol response presumably represents an
`attempt to compensate for possible peripheral glucocorticoid antagonism
`and resultant hypoadrenalism, it appeared logical to administer RU 486
`in the evening to permit the subsequent increase in endogenous glucocor-
`ticoids the next morning to compensate for any possible antiglucocorti-
`coid action. It is of note, however, that these findings were derived from
`studies using lower doses of RU 486 [6 mg/Kg] than the single 600 mg
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`Contraception 1993:48, November
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`

`The antiprogestin. antiglucocorticoid RU 486: Spitz and Bardin
`
`[[0 mg/Kg] dose currently used in women for abortion induction. The
`latter dose results in elevated cortisol levels throughout the day. There-
`fore, it remained important to determine whether the rise in serum corti-
`sol levels was sufficient to compensate for peripheral receptor blockade.
`Accordingly, dogs were treated daily with 4-fold the usual human dose.‘3
`After 10 days of continuous treatment, no obiective evidence of cortisol
`deficiency was observed. These conclusions were based on observations
`that signs of giucocorticoid deficiency developed over 2—10 days in un-
`treated adrenalecromized dogs. Thus, the rise in serum cortisol can com-
`pensate for peripheral receptor blockade.
`It should be pointed out that higher doses of RU 486 are required to
`produce an antiglucocorticoid as opposed to an antiprogestin Bffectfml‘:
`For example, at the endometrium, bleeding ensues following RU 486
`administration of l mg/kgl" and changes in endometrial morphology occur
`with a dose as low as 0.1 mg/kg.“ The fact that higher doses are required
`for antiglucocorticoid effects is probably related to the high concentrations
`of cortisol in blood, due to the inhibition of the feedback regulation of
`the pituitary-adrenal axis and to the fact that many glucocorticoid effects
`are permissive.” Nonetheless,
`the RU 486 dose customarily given to
`women to induce abortion {600 mg] routinely stimulates the hypothal—
`amo-pituitary adrenal axis.
`To date, no objective clinical or laboratory manifestations of overt
`glucocorticoid deficiency have been reported as a result of short—term
`administration of RU 486 as an antiprogestin in humans. Furthermore,
`the regulation of the pituitary—adrenal axis is not compromised following
`three months of treatment with RU 486 in doses of 100 mg/day.“ Even
`though persistent elevations of ACTH and cortisol were observed, the
`acute stimulating effect of corticotropin releasing hormone remained un-
`changed and the diurnal rhythm was maintained.“ This finding implies
`that the central regulatory control mechanisms remain intact during long—
`term treatment. There are no known consequences of the elevated ACTH
`and cortisol levels in subjects receiving low doses. In some subjects with
`breast carcinoma or inoperable meningioma receiving higher doses
`{200—400 mgfdayi for many months, symptoms compatible with cortisol
`deficiency developed which appeared to reverse with dexamethasone.—"‘ -‘“
`The symptoms, which were worse at the onset of therapy, included weak-
`ness, nausea, and vomiting.‘“--N Other signs of cortisol deficiency found
`in adrenalccromized patients, such as fever, change in blood pressure,
`water intolerance, or persistent elevation of total eosinophils Were not
`observed. When high doses of RU 486 110 mg/kgfday] were administered
`to normal men for l4 days, one developed symptoms compatible with
`cortisol deficiency.“ However, in this patient and in those reviewed above,
`there have not been obiective signs of cortisol deficiency and,
`in fact,
`adverse reacrions to the drug were a plausible alternative explanation for
`
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`The antiprogestin, antigiucocorticoid RU 486: Spitz and Bardin
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`the symptoms observed. Apparent adrenal insufficiency did, however,
`develop in two subjects with fixed cortisol secretion treated with doses
`of RU 486 ranging from 10-22. mgjkg/day for inoperable Cushing’s syn-
`drome.-32 Since measurement of serum cortisol cannot be used to establish
`
`the diagnosis of functional hypoeortisolism during receptor blockade,
`clinicians will have to resort to seemingly antiquated in vivo end points
`that develop in the absence of glucocorticoids, such as eosinophilia and
`water intolerance, and focus on how such parameters change with gluco-
`corticoid treatment of patients taking large dose of RU 486. For the time
`being, it is clear that most patients with an intact pituitary-adrenal axis
`compensate for glucocorticoid receptor blockade by elevating serum corti-
`sol.
`
`long-term RU 486
`In addition to increasing ACTH and cortisol,
`treatment also increased the serum levels of the adrenal androgens
`androsrenedione and DHEA.-“‘-‘"»”" Serum estradiol concentrations were
`
`also increased in subjects treated with 200—400 mg RU 486 daily.-“‘--"-“’
`As the estradiol levels were correlated with both androstenedione and
`
`levelsfiiv‘” it is highly likely that the increments in estradiol
`cortisol
`were due to aromatization of adrenal androgens in nonendocrine tis-
`sues-“W“ lt should be noted that not all studies have reported a rise
`in serum estradiol levels with RU 486. We failed to observe estradiol
`
`elevation following long-term administration of 200 mg daily to borh
`men and women with inoperable meningioma lI.M. Spitz, manuscript
`in preparation}, and Kettel ct al.-'H reported no change in serum estradiol
`following administration of 100 mg daily for three months in patients
`with endometriosis.
`
`There is also some evidence that RU 486 may act as a partial glucocorti—
`coid agonist. Thus, it suppressed the ACTH response to ovine corricotro-
`pin releasing hormone in subjects with adrenal insufficiency, although to a
`far lesser degree than cortisol. On a weight basis, its relative glucocorticoid
`agonist activity was estimated to be approximately 1/250 that of cortisol.“
`Significantly, however, RU 486 was unable to support life in the adrenalee-
`tomized monkey.”
`
`B. Antiprogesrin Actions
`
`1. Effects on the endometrium. Administration of RU 486 in single doses
`of 50—800 mg on days 6-43 of the luteal phase to normal women induced
`profound changes in endometrial histology consisrent with progesterone
`wi'tl'ldratvalg““-‘-"'1 and mensrrual bleeding invariably ensued within 72
`hours.*”-"‘-‘“ When a single dose of RU 486 [200 mgl was given 2 days after
`the LH peak, a retardation of luteal phase development of the endome-
`trium began as early as 12 hours after RU 486 intake.“ In another study
`a single dose ranging from 5-200 mg given at various times during the
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`The antiprogestin. antiglucocorticoid RU 486: Spitz and Hardin
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`first half of the luteal phase inhibited endometrial glandular secretory
`activity, accelerated degenerative changes,
`induced various vascular
`changes, and increased stromal but not glandular mitotic activity.“ Inter-
`estingly, a significant endometrial response of one or more of l 7 morpho—
`logic features was observed at all doses studied, whereas induction of
`menstruation occurred only with the higher doses. This finding indicates
`that the antiprogestational action of RU 486 on endomctrial morphology
`occurs at much lower doses than those that will induce endomctrial
`
`bleeding.
`In posnnenopausal women receiving esrrogen replacement, RU 486
`unexpectedly produced agonistie effecrs on the endometrium, including
`induction of secretory changes, increased cstradiol dehydrogenase, and
`reduced DNA polymerase activity—effects that were also produced when
`progesterone was administered to esrrogcn-treated women.“ However,
`in estradiol plus progeSterone-treated postmenopausal women, RU 486
`antagonized the action of progesterone. "‘ Similar findings were observed
`in ovariectomized monkeys.“-"‘ That is, in estradiol plus progesterone-
`treated monkeys, RU 486 behaved as a classical progestin antagonist
`preventing the endometrial secretory transformation. By contrast, in cs-
`tradiol-treated Ovaricctomized monkeys,
`low doses of RU 486 ll mg]
`kg/dayi acted as a progestin agonist and induced endometrial secretory
`transformanon, whereas a higher dose [5 mg/kg/dayi inhibited both endo-
`metrial proliferation and secretory activity. Thus, in the absence of proges-
`terone, the high dose of RU 486 has anti-proliferative [anti-estrogeniel
`actions. This effect of RU 486 does nor depend on the estrogen receptor
`since neither RU 486 nor its principal metabolites hind to this rcceptor.‘ 4“
`Since high doses of progestins produce similar anti-mitogenic effects”
`and lead to endometrial atrophy, this action of RU 486 should be regarded
`as agonistic. In view of this anti-estrogcnic action, one possible use for
`RU 486 could he in the control of estrogen-dependent tissue growrh such
`as endometriosis and breast carcinoma. However,
`if this were the only
`action of RU 486 on endometrium and mammary epithelium, then its
`use for these diseases may not be practical in view of the lower cost of
`progeStins and the fact that RU 486 may elevate eStradiol irt postmeno-
`pausal women by peripheral aromatization of adrenal androgens.-‘-"--”‘“’
`in pregnant monkeys, RU 486 stimulated a rise of estradiol receptors
`in the decidua and myometriumfi” These results were interpreted as show-
`ing an antagonistic action of RU 486 since progesterone decreases estrogen
`receptor synthesis.WM Berthois et al.“ reached a similar conclusion from
`observatioris in women treated with RU 486:10 mg/dayl for4 days starting
`at the time of ovulation in that the concentrations of immunoStainahle
`
`estrogen and progesterone receptors were higher in endometrial glands
`and stroma from treated women compared to the control subjects,
`in
`which the receptor concentration showed the expected luteal phase dc-
`
`414
`
`Contraception 1993:48, November
`
`EX. 2012-0012
`
`Ex. 2012-0012
`
`

`

`The antiprogestin, antigiucocorticor‘d RU 486: Spitz and Bardi'n
`
`crease-'1' While this may be the correct interpretation of the action of this
`drug during pregnancy and the luteal period when progesterone levels are
`elevated, a study in ovariectomized, estradiol-treated monkeys showed
`that RU 486 produced a dose-dependent increase in endometrial receptors
`even in the absence of progesterone.‘1 These latter findings were unex-
`pected and suggest that RU 486 may directly stimulate a progesterone
`receptor-mediated rise in estrogen receptors independent of its ability to
`block the aetion of progesterone. Such observations suggest that some of
`the agonistic actions of RU 486 do not mimic the actions of progesterone.
`The above observations on the actions of RU 486 on the uterus suggest
`that binding of RU 486 to the PR in the presence of progesrerone results
`in a steroid-receptor complex that is transcriptionally inacrive on many
`progesrerone-responsive genes {Figure 3] {i.e., RU 486 is an antagonist of
`progesterone]. However, in the absence of progesterone, RU 486 may form
`a receptor complex that is active on some promoters {i.e., the potential
`agonistic effects of RU 486}. It
`is interesting that some of these latter
`effects may or may not mimic the effects of progesterone. It is the diverse
`agonistic effects of RU 486 that may give it some of its unique antineoplas-
`tic properties in breast cancer and meningioma as noted below.
`The quesrion arises as to whether the menstrual bleeding consequent
`to RU 486 administration results because ofa direct action on the endomo-
`
`tritim or whether this is an effect on the hyporhalamie-pituitary—ovarian
`axis. This issue was addressed by administering human chorionic gonado-
`tropin thG] during the luteal phase of the reproductive cycle in women
`to simulate early pregnancy by increasing estradiol and progesterone levels
`and delaying the onset of bleeding. This treatment did not prevent bleeding
`induced by an adequate dose of RU 486.“55 Thus, by maintaining corpus
`luteum function with exogenous liCG, RU 486 induced endometrial
`bleeding despite high circulating progesterone and estradiol levels.“ 5‘
`This indicates that RU 486 acts directly at the level of the endometrium.
`
`In a rat pituitary cell culture system
`2. Effects on gonadotropin secretion.
`primed with cstradiol, RU 486 inhibited GnRH-induced LH and FSH
`secretion in a dose-dependent manner without affecting basal gonadotro—
`pin release. The inhibition was specific and antagOnized by the addition
`of progesterone.“ Progesterone itself stimulated GnRH-indueed gonado-
`tropin secretion.“ A separate line of studies suggested that gonadotropin
`inhibition by RU 486 could operate at the hypothalamie level.” Preovula-
`tory RU 486 administration blocked the LH surge in monkeys, an effect
`which could not be reversed by simultaneous treatment with dcxametha-
`some.“ These results suggest that the antigonadotropic effects of RU 486
`are due to its antiprogestational rather than its antiglucocorticoid proper—
`ties. Such obscrvations also support the postulate that the small rise in
`progesterone in the late follicular phase facilitates the LH surge.
`
`Contraception 1993 :48, November
`
`415
`
`EX. 2012-0013
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`Ex. 2012-0013
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`

`The antiprogestin. antiglucocorricord RU 486: Spitz and Hardin
`
`Many Studies also indicate that RU 486 reduces gonadotropin secretion
`in humans. When 100 mg RU 486 was given from days 10—1 7 of the cycle,
`the LH surge was attenuated?" A single dose of 10 mg or 100 mg RU 486
`produced an immediate reduction in LH and FSH concentrations when
`administered in the mid and late follicular phase. The decrease in mean
`LH concentration was consequent to a reduction in amplitude of the
`LH pulses released by the pituitary without any alteration in LH pulse
`frequency. The reduction in mean LH concentration was greater in those
`women with higher plasma eatradiol concentrations.m Not all investiga-
`tors have observed such dramatic changes during the follicular phase."’-"?-
`During the mid-luteal phase, RU 486 also decreased mean LH scererion
`and LH pulse amplitude and blunted the pituitary rcspOnse to Can-l. In
`the late luteal phase, both LH pulse frequency and amplitude decreased.“
`In contrast to these short-term studies showing that RU 486 inhibits
`pituitary gonadotropin secretion,
`long-term treatment {50—100 mg/day
`for 3 months! increased mean LH and LH pulse amplitude without a
`change in FSI—l.—‘“»“-‘ These elevated Lli values may be transient and. after
`4 weeks of treatment, levels are reported to return to baseline.M In view
`of the fact that RU 486 could act both on the hypothalamus as well as
`the pituitary and due to its antagonistic as well as agonistic actions, it is
`not surprising that its effects on LH and FSH secretion are diverse.
`In addition to its activity on LH, FSH and ACTH, RU 486 also infl