Attorney Docket No.: 85178-1006972
`Family ID No.: 008610US
`
`OPTIMIZING MIFEPRISTONE LEVELS FOR CUSHING'S PATIENTS
`
`PATENT APPLICATION
`
`Inventors:
`
`Joseph Belanoff, residing in Woodside, CA
`
`Coleman Gross, residing in Menlo Park, CA
`
`Assignee:
`
`Corcept Therapeutics, Inc.
`149 Commonwealth Drive
`Menlo Park, California 94025 United States of America
`
`Entity:
`
`Small
`
`KILPATRICK TOWNSEND & STOCKTON LLP
`
`1
`
`TEVA1042
`
`

`

`PATENT
`Attorney Docket No.: 85178-1006972
`Family ID No.: 008610US
`
`OPTIMIZING MIFEPRISTONE LEVELS FOR CUSHING'S PATIENTS
`
`CROSS-REFERENCES TO RELATED APPLICATIONS
`
`[0001] This application claims priority to U.S. Provisional Application No. 62/150,757, filed
`
`5 April21, 2015, which is incorporated in its entirety herein for all purposes.
`
`BACKGROUND OF THE INVENTION
`
`[0002]
`
`It has been reported previously that administration of the same dose of mifepristone can
`
`produce widely varying blood serum levels in different patients. The varied blood serum levels
`
`10
`
`can result in some patients not receiving an efficacious dose ofmifepristone. For patients
`
`suffering from a mental disorder, the blood serum levels need to be maintained at about
`
`1300 ng/mL. For patients suffering from Cushing's syndrome, it was surprisingly discovered
`
`that blood serum levels need to be maintained at a level of at least about 1631 ng/mL for a
`
`therapeutic response. Thus, a method for ensuring that the blood serum levels of mifepristone
`
`15
`
`remain in an efficacious and safe range is needed for patients suffering from Cushing's
`
`syndrome.
`
`BRIEF SUMMARY OF THE INVENTION
`
`[0003]
`
`In one embodiment, the present invention provides a method for improving efficacy of
`
`20 mifepristone treatment in a patient suffering from Cushing's syndrome. The method includes
`
`treating the patient with seven or more daily doses of mifepristone over a period of seven or
`
`more days; testing the serum levels of the patient to determine whether the blood levels of
`
`mifepristone are greater than 1631 ng/mL; and adjusting the daily dose of the patient to achieve
`
`mifepristone blood levels greater than 1631 ng/mL. The patient of the present invention is not
`
`25
`
`already suffering from a condition indicated for treatment with mifepristone. Thus, the method
`
`thereby improves the efficacy of mifepristone treatment for patients suffering from Cushing's
`
`syndrome for the patient suffering from Cushing's syndrome.
`
`1
`
`2
`
`

`

`DETAILED DESCRIPTION OF THE INVENTION
`
`I.
`
`INTRODUCTION
`
`[0004] Administration of the same dose of mifepristone can produce widely varying
`
`mifepristone blood serum levels in different patients. For the same dose, the blood serum levels
`
`5
`
`can differ by as much as 800% from one patient to another. For those patients with lower blood
`
`serum levels, the effectiveness of mifepristone treatment can suffer significantly. The present
`
`invention provides a method for optimizing the blood serum levels of mifepristone so that the
`
`blood serum levels remain in an efficacious range and the patient receives the necessary
`
`treatment.
`
`10
`
`[0005] The method of the present invention optimizes blood serum levels of mifepristone in a
`
`patient suffering from Cushing's syndrome by first treating the patient with mifepristone. The
`
`treatment can be for any appropriate period of time, such as seven or more daily doses over a
`
`period of seven or more days. Fallowing treatment for an appropriate period of time, the serum
`
`levels of the patient are tested to determine whether the blood levels of mifepristone are greater
`
`15
`
`than 1631 ng/mL. The daily dose of the patient is then adjusted in order to achieve mifepristone
`
`blood levels of greater than 1631 ng/mL.
`
`[0006] Previous methods of optimizing mifepristone levels are known for patients suffering
`
`from mental disorders. But the earlier methods describe a minimum mifepristone blood level of
`
`only 1300 ng/mL. While patients with Cushing's syndrome are known to have higher cortisol
`
`20
`
`levels, it is surprising that higher mifepristone blood level of 1631 ng/mL would be necessary to
`
`achieve optimal efficacy in treating Cushing's syndrome.
`
`II.
`
`DEFINITIONS
`
`[0007]
`
`"Mifepristone" refers to a compound having the following structure:
`
`2
`
`3
`
`

`

`[0008] The term mifepristone also refers to a family of compositions also known as: RU486 or
`
`RU38.486; 17-beta-hydroxy-11-beta-(4-dimethyl-aminophenyl)-17-alpha-(1-propynyl)-estra-
`
`4,9-dien-3-one); ll-beta-(4dimethylaminophenyl)-17-beta-hydroxy-17-alpha-(1-propynyl)-estra-
`
`4,9-dien-3-one); 11B-[p-(Dimethylamino)phenyl]-17B-hydroxy-17- (1-propynyl)-estra-4,9-dien-
`
`5
`
`3-one; 11B-(4-dimethyl-aminophenyl)-17B-hydroxy-17 A-(prop-1-ynyl)-estra-4,9-dien-3-one;
`
`17B-hydroxy-11B- ( 4-dimethylaminophenyl-1)-17 A-(propynyl-1)-estra-4,9-diene-3-one; 17B(cid:173)
`
`hydroxy-11B-(4-dimethylaminophenyl-l)-17A-(propynyl-l)-E; (11B,l7B)-11- [4-
`
`dimethylamino)- phenyl]-17-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one; and liB- [ 4-(N,N(cid:173)
`
`dimethy lamino) pheny 1 ]-1 7 A -(prop-1-yny 1 )-D-4, 9-estradi ene-1 7B-ol-3 -one. Salts, hydrates and
`
`10
`
`prodrug forms of mifepristone are also useful in the formulations of the present invention.
`
`[0009] Mifepristone and its analogs bind to the glucocorticoid receptor (GR), typically with
`
`high affinity, and inhibit the biological effects initiated/mediated by the binding of any cortisol
`
`or cortisol analogue to the GR. As such, mifepristone has been used to treat conditions
`
`associated with elevated cortisol levels including, for example, hyperadrenocorticism, also
`
`15
`
`known as Cushing's syndrome (Chrousos, pp 273-284, In: Baulieu, ed. The Antiprogestin Steroid
`
`RU 486 and Human Fertility Control. Plenum Press, New York (1989), Sartor (1996) Clin.
`
`Obstetrics and Gynecol. 39:506-510). Patients with some forms of psychiatric illnesses can be
`
`responsive to treatments which block the effect of cortisol, as by administering GR antagonists
`
`(Van Look (1995) Human Reproduction Update 1: 19-34). In one study, a patient with
`
`20
`
`depression associated with Cushing's Syndrome was responsive to a high dose, up to 1400 mg
`
`per day, ofmifepristone (Nieman (1985) J Clin Endocrinol. Metab. 61:536). Due to its
`
`antiprogestogenic activity, mifepristone has also been employed in emergency contraception,
`
`medical abortion, and treatment of uterine fibroids and meningioma (Healy (2009) Australian
`
`Prescriber 32: 152-154).
`
`25
`
`[0010]
`
`"Patient" refers to animals such as mammals, including, but not limited to, primates
`
`(e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice and the like. The patient
`
`can have a condition known to be treated by glucocorticoid antagonists such as mifepristone.
`
`Such conditions include, but are not limited to, psychiatric illnesses and hormonal disorders. In
`
`certain embodiments, the patient is a human. The patient can be male or female.
`
`3
`
`4
`
`

`

`[0011]
`
`"Cushing's syndrome" refers to an endocrine disease with an estimated incidence of
`
`approximately 10-15 per 1 million persons (Meier and Biller (1997) Endocrinol Metab Clin
`
`North Am 26:741-762), and is associated with an increased blood concentration of cortisol
`
`(hypercortisolism) over a long period of time. Cushing's syndrome is classified as either ACTH
`
`5
`
`dependent or non ACTH dependent. ACTH dependent Cushing's syndrome is characterized by a
`
`chronic ACTH hypersecretion which stimulates the growth of the adrenal glands and the
`
`hypersecretion of corticosteroids. The most common underlying cause of ACTH dependent
`
`Cushing's syndrome is excessive production of ACTH by pituitary adenomas known as
`
`Cushing's disease. Cushing's syndrome resulting from the production of ACTH in another
`
`10
`
`location than the pituitary gland is known as ectopic Cushing's syndrome. Examples of ectopic
`
`sites include thymoma, medullary carcinoma of the thyroid, pheochromocytoma, islet cell tumors
`
`of the pancreas and small cell carcinoma of the lung. ACTH independent Cushing's syndromes
`
`are caused by adrenal tumors that can be either adenomas or carcinomas. Both adrenal adenomas
`
`and carcinomas are characterized by chronic cortisol hypersecretion.
`
`15
`
`[0012]
`
`"Optimizing" refers to the process of testing mifepristone blood levels and adjusting the
`
`dosage of mifepristone administered to the patient in need in order to achieve mifepristone blood
`
`levels above 1631 ng/mL.
`
`[0013]
`
`"Treat", "treating" and "treatment" collectively refer to any indicia of success in the
`
`treatment or amelioration of an injury, pathology or condition, including any objective or
`
`20
`
`subjective parameter such as abatement; remission; diminishing of symptoms or making the
`
`injury, pathology or condition more tolerable to the patient; slowing in the rate of degeneration
`
`or decline; making the final point of degeneration less debilitating; improving a patient's
`
`physical or mental well-being; or, in some situations, preventing the onset of dementia. The
`
`treatment or amelioration of symptoms can be based on objective or subjective parameters;
`
`25
`
`including the results of a physical examination, neuropsychiatric exams, and/or a psychiatric
`
`evaluation.
`
`[0014]
`
`"Testing" refers to determining the mifepristone blood levels in a patient. The testing
`
`can be performed by any suitable instrument, such as a plasma sampling collection device
`
`capable of detecting mifepristone serum levels.
`
`4
`
`5
`
`

`

`[0015] A patient "not already suffering from a condition indicated for treatment with
`
`mifepristone" is a patient who is not suffering from a condition which is known in the art to be
`
`effectively treatable with mifepristone. Conditions known in the art to be effectively treatable
`
`with mifepristone include drug withdrawal, psychosis, dementia, stress disorders, and psychotic
`
`5 major depression.
`
`III. METHOD OF OPTIMIZING MIFEPRISTONE LEVELS
`
`[0016] The present invention provides a method of optimizing mifepristone levels in patients
`
`with Cushing's syndrome such that the blood serum levels remain at efficacious levels. The
`
`method involves administering mifepristone for a week, testing the blood serum levels of the
`
`10
`
`Cushing's patient, and adjusting the mifepristone dose to maintain the mifepristone blood serum
`
`levels of at least 1631 ng/mL.
`
`[0017] The present invention provides a method for improving efficacy of mifepristone
`
`treatment in a patient suffering from Cushing's syndrome. The method includes treating the
`
`patient with seven or more daily doses of mifepristone over a period of seven or more days;
`
`15
`
`testing the serum levels of the patient to determine whether the blood levels of mifepristone are
`
`greater than 1631 ng/mL; and adjusting the daily dose of the patient to achieve mifepristone
`
`blood levels greater than 1631 ng/mL. The patient treated in this method is not already suffering
`
`from a condition indicated for treatment with mifepristone, thereby improving the efficacy of
`
`mifepristone treatment for the patient suffering from Cushing's syndrome.
`
`20
`
`[0018] The seven or more daily doses of mifepristone can each be administered by any means
`
`suitable, as described in more detail below. In some embodiments, each of the seven or more
`
`daily doses of mifepristone are administered orally.
`
`[0019] The seven or more daily doses of mifepristone can each be administered in any suitable
`
`dose. For example, the mifepristone can be administered in an amount of at least about 100 mg.
`
`25
`
`The mifepristone can also be administered in an amount of about 300, 600, 900 or about
`
`1200 mg. In some embodiments, the daily dose can be at least 300 mg. In some embodiments,
`
`the daily dose can be at least 600 mg. In some embodiments, the daily dose can be at least 900
`
`mg. In some embodiments, the daily dose can be at least 1200 mg. Other daily doses are useful
`
`in the method of the present invention.
`
`5
`
`6
`
`

`

`[0020] The daily doses can be administered for any suitable period of time that is at least 7
`
`days in length. For example, the daily doses can be for 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
`
`19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days. The mifepristone can be administered for longer
`
`periods as required by the patient being treated. In some embodiments, the patient can be treated
`
`5 with 28 or more daily doses over a period of 28 or more days.
`
`[0021] The mifepristone blood levels can be tested by any means known to one of skill in the
`
`art. For example, the the testing can be performed by a plasma sampling collection device
`
`suitable for detecting mifepristone serum levels.
`
`[0022] The mifepristone blood levels can be at any suitable level to treat Cushing's syndrome.
`
`10
`
`For example, the mifepristone blood levels can be greater than about 1400 ng/mL, 1450, 1500,
`
`1550,1600,1650,1700,1750,1800,1850,1900,2100,2200,2300,2400,2500,2600,2700,
`
`2800 or great than about 2900 ng/mL. In some embodiments, the mifepristone blood level can
`
`be greater than 1450 ng/mL. In some embodiments, the mifepristone blood level can be greater
`
`than 1469 ng/mL. In some embodiments, the mifepristone blood level can be greater than 1600
`
`15
`
`ng/mL. In some embodiments, the mifepristone blood level can be greater than 1631 ng/mL. In
`
`some embodiments, the mifepristone blood level can be greater than 1662 ng/mL. In some
`
`embodiments, the mifepristone blood level can be greater than 1666 ng/mL. In some
`
`embodiments, the mifepristone blood level can be greater than 1700 ng/mL. In some
`
`embodiments, the mifepristone blood level can be greater than 1800 ng/mL. In some
`
`20
`
`embodiments, the mifepristone blood level can be greater than 1820 ng/mL. In some
`
`embodiments, the mifepristone blood level can be greater than 2000 ng/mL. In some
`
`embodiments, the mifepristone blood level can be greater than 2022 ng/mL.
`
`[0023] The daily dose can be adjusted to any suitable dose to maintain the mifepristone blood
`
`level above the necessary level. For example, if the mifepristone blood level is below 1631
`
`25
`
`ng/mL, the daily dose can be increased to 600 mg from 300 mg, to 900 mg from 600 mg, to 900
`
`mg from 300 mg, to 1200 mg from 900 mg, to 1200 mg from 600 mg, or to 1200 mg from 300
`
`mg. If after another seven daily doses, the mifepristone blood level is still not above the
`
`necessary level, the mifepristone daily can again be increased. For example, the mifepristone
`
`daily dose can be increased to 900 mg from 600 mg, to 1200 mg from 900 mg, or to 1200 mg
`
`30
`
`from 600 mg. In some embodiments, the adjusting step comprises increasing the daily dose of
`
`6
`
`7
`
`

`

`the patient to achieve mifepristone blood levels greater than 1631 ng/mL. Additional
`
`adjustments in the daily doses can be made to maintain the mifepristone blood level above
`
`1631 ng/mL.
`
`[0024] Any suitable percentage of the patient population can have the optimal response to
`
`5
`
`administration of the mifepristone. For example, at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or
`
`100% of the patient population can achieve the optimal response to the mifepristone treatment.
`
`In some embodiments, at least about 20% of the patient population can have the optimal
`
`response to administration of the mifepristone. In some embodiments, at least about 40% of the
`
`patient population can have the optimal response to administration of the mifepristone. In some
`
`10
`
`embodiments, at least about 60% of the patient population can have the optimal response to
`
`administration of the mifepristone.
`
`A.
`
`Patients in Need
`
`[0025] Patients amenable to treatment with mifepristone according to the method of the
`
`present invention suffer from Cushing's syndrome. Cushing's syndrome is a disorder resulting
`
`15
`
`from increased adrenocortical secretion of corticosteroid. Hyperfunction of the adrenal cortex
`
`may be adrenocorticotropic hormone (ACTH)-dependent or it may be independent of ACTH
`
`regulation, e.g. production of corticosteroid by an adrenocortical adenoma or carcinoma. A
`
`common cause of Cushing's syndrome is excessive production of ACTH by the pituitary gland.
`
`This elevated level of ACTH in the bloodstream typically is produced by a pituitary adenoma
`
`20
`
`(Cushing's disease), but in rare instances has a different etiology. Cushing's syndrome resulting
`
`from the production of ACTH in a location other than the pituitary gland is known as ectopic
`
`Cushing's syndrome. Examples of ectopic sites include thymoma, medullary carcinoma of the
`
`thyroid, pheochromocytoma, islet cell tumors of the pancreas and oat cell carcinoma of the lung.
`
`The overwhelming majority of Cushing's syndrome cases in humans, however, trace their
`
`25
`
`etiology to a pituitary adenoma. Symptoms of Cushing's syndrome include weight gain, central
`
`obesity, steroid hypersecretion, elevated urinary cortisol excretion, moon face, weakness, fatigue,
`
`backache, headache, impotence, mental status changes, muscle atrophy, and increased thirst and
`
`urination compared to mammals not suffering from this disease. Diagnosis and treatment of
`
`Cushing's syndrome remains a challenge (see Oldfield, E. W. et al., N. Engl. J. Med., 325:897-
`
`30
`
`905 (1991); Findling, J. W. et al., "Diagnosis and differential diagnosis of Cushing's syndrome,"
`
`7
`
`8
`
`

`

`Endocrinol. Metab. Clin. North Am., 30:729-47 (2001); Orth, D. N., "Cushing's syndrome," N
`
`Engl J. Med., 332:791-803 (1995)). In experienced specialized centers, surgical resection of
`
`ACTH-secreting pituitary microadenomas offers an overall cure rate of about 70-80%, but for
`
`macroadenomas cure rates only approximate 30%, and the extensive surgical resection required
`
`5
`
`portends significant risk to surrounding normal pituitary tissue, leading to partial or total
`
`hypopituitarism in about 80% of cases (Simmons, N. E. et al., "Serum Cortisol response to
`
`transphenoidal surgery for Cushing disease," J. Neurosurg., 95:1-8 (2001); Mampalam, T. J. et
`
`al., "Transsphenoidal microsurgery for Cushing's disease: A report of 216 cases," Ann. Intern.
`
`Med., 109:487-93 (1988); and Trainer, P. J. et al., "Transsphenoidal resection in Cushing's
`
`10
`
`disease: undetectable serum cortisol as the definition of successful treatment," Clin. Endocrinol.,
`
`38:73-8 (1993)).
`
`B.
`
`Formulations of Mifepristone
`
`[0026] Formulations of the present invention include mifepristone in combination with
`
`pharmaceutical excipients. Mifepristone is commercially available from a variety of sources
`
`15
`
`such as Eurolabs Ltd. (London, England). Mifepristone can also be synthesized by one of skill
`
`in the art using known synthetic procedures.
`
`[0027] Mifepristone refers to a family of compositions also referred to as RU486, or
`
`RU38.486, or 17-beta-hydroxy-11-beta-( 4-dimethyl-aminophenyl)-17-alpha-(1-propynyl)-estra-
`
`4,9-dien-3-one), or ll-beta-(4dimethylaminophenyl)-17-beta-hydroxy-17-alpha-(1-propynyl)-
`
`20
`
`estra-4,9-dien-3-one), or analogs thereof, which bind to the GR, typically with high affinity, and
`
`inhibit the biological effects initiated/ mediated by the binding of any cortisol or cortisol
`
`analogue to a GR receptor. Chemical names for RU-486 vary; for example, RU486 has also
`
`been termed: 11B-[p-(Dimethylamino)phenyl]-17B-hydroxy-17- (1-propynyl)-estra-4,9-dien-3-
`
`one; 11 B-( 4-dimethy 1-aminopheny 1 )-1 7B-hydroxy -1 7 A -(prop-1-yny 1 )-estra -4, 9-di en-3 -one;
`
`25
`
`17B-hydroxy-11B- ( 4-dimethylaminophenyl-1)-17 A-(propynyl-1)-estra-4,9-diene-3-one; 17B(cid:173)
`
`hydroxy- liB-( 4-dimethylaminophenyl-1)-17 A-(propynyl-1)-E; (liB, 17B)-li- [ 4-
`
`dimethylamino)- phenyl]-17-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one; and liB- [ 4-(N,N(cid:173)
`
`dimethy lamino) pheny 1 ]-1 7 A -(prop-1-yny 1 )-D-4, 9-estradi ene-1 7B-ol-3 -one. Salts, hydrates and
`
`prodrug forms of mifepristone are also useful in the formulations of the present invention.
`
`8
`
`9
`
`

`

`[0028] Formulations suitable for oral administration can consist of (a) liquid solutions, such as
`
`an effective amount of mifepristone suspended in diluents, such as water, saline or PEG 400; (b)
`
`capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as
`
`liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable
`
`5
`
`emulsions. Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium
`
`phosphates, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon
`
`dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders,
`
`diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes,
`
`disintegrating agents, and pharmaceutically compatible carriers. Lozenge forms can comprise
`
`10
`
`the active ingredient in a flavor, e.g., sucrose, as well as pastilles comprising the active
`
`ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels,
`
`and the like containing, in addition to the active ingredient, carriers known in the art.
`
`[0029] The pharmaceutical preparation is preferably in unit dosage form. In such form the
`
`preparation is subdivided into unit doses containing appropriate quantities of the active
`
`15
`
`component. The unit dosage form can be a packaged preparation, the package containing
`
`discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or
`
`ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can
`
`be the appropriate number of any of these in packaged form. The composition can, if desired,
`
`also contain other compatible therapeutic agents. Preferred pharmaceutical preparations can
`
`20
`
`deliver the compounds of the invention in a sustained release formulation.
`
`C.
`
`Administration of Mifepristone
`
`[0030] The formulations of the present invention provide serum levels of mifepristone of at
`
`least 1631 ng/mL. The mifepristone utilized in the pharmaceutical method of the invention is
`
`administered at the initial dosage of about 0.001 mg/kg to about 1000 mg/kg daily. A daily dose
`
`25
`
`range of about 0.01 mg/kg to about 500 mg/kg, or about 0.1 mg/kg to about 200 mg/kg, or about
`
`1 mg/kg to about 100 mg/kg, or about 10 mg/kg to about 50 mg/kg, can be used. The dosages,
`
`however, may be varied depending upon the requirements of the patient and the condition being
`
`treated. The dose administered to a patient, in the context of the present invention, should be
`
`sufficient to effect a beneficial therapeutic response in the patient over time. The size of the dose
`
`30
`
`also will be determined by the existence, nature, and extent of any adverse side-effects that
`
`9
`
`10
`
`

`

`accompany the administration of a particular compound in a particular patient. Determination of
`
`the proper dosage for a particular situation is within the skill of the practitioner.
`
`[0031] Generally, treatment is initiated with six daily doses, with the blood levels tested on the
`
`day of the seventh daily dose in order to determine whether the dose used is providing a
`
`5 mifepristone blood level of at least 1631 ng/mL. The testing is also performed to ensure the
`
`blood levels are below those afforded by an LD50 dose of about 1 OOOmg/kg. If the mifepristone
`
`blood level is lower than 1631 ng/mL. Additional testing of mifepristone blood levels can be
`
`necessary in order to confirm a mifepristone blood level of at least 1631 ng/mL or to adjust the
`
`mifepristone daily dose higher. For convenience, the total daily dosage may be divided and
`
`10
`
`administered in portions during the day, if desired. In addition, the interval from initiation of
`
`treatment and testing for mifepristone blood levels can be as short as 1 daily dose, or up to 28
`
`daily doses and longer.
`
`[0032] Mifepristone can be administered for any period of time, such as 7 daily doses over a
`
`period of seven days. Mifepristone can also be administered using more daily doses over a
`
`15
`
`longer period of time, such as via 28 daily doses over a period of 28 days. Longer times for
`
`administration of mifepristone are also within the scope of the present invention.
`
`D.
`
`Assay for Testing Mifepristone Levels
`
`[0033] Mifepristone levels can be determined by any method known in the art. Methods for
`
`detecting mifepristone levels include, but are not limited to, radio-immuno assay and mass
`
`20
`
`spectrometry (MALDI, SELDI, LS/MS, LS/MS/MS, among others). Liquid chromatography
`
`mass spectrometry (LC/MS or LC-MS) separates compounds chromatographically before they
`
`are introduced to the ion source and mass spectrometer. It differs from GC/MS in that the
`
`mobile phase is liquid, usually a combination of water and organic solvents, instead of gas. Most
`
`commonly, an electrospray ionization source is used in LC/MS.
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`25
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`[0034] Tandem mass spectrometry (MS/MS) involves multiple steps of mass selection or
`
`analysis, usually separated by some form of fragmentation. A tandem mass spectrometer is one
`
`capable of multiple rounds of mass spectrometry. For example, one mass analyzer can isolate
`
`one peptide from many entering a mass spectrometer. A second mass analyzer then stabilizes the
`
`peptide ions while they collide with a gas, causing them to fragment by collision-induced
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`dissociation (CID). A third mass analyzer then catalogs the fragments produced from the
`
`peptides. Tandem MS can also be done in a single mass analyzer over time as in a quadrupole
`
`ion trap. There are various methods for fragmenting molecules for tandem MS, including
`
`collision-induced dissociation (CID), electron capture dissociation (ECD), electron transfer
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`5
`
`dissociation (ETD), infrared multiphoton dissociation (IRMPD) and blackbody infrared radiative
`
`dissociation (BIRD). One of skill in the art will appreciate that other assays for testing
`
`mifepristone levels are known to one of skill in the art.
`
`[0035]
`
`In some embodiments, the assay can be performed as follows. Blood is collected from
`
`a patient in a vacutainer containing sodium heparin. The blood is centrifuged and the resulting
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`10
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`plasma frozen at an appropriate temperature until assay. In some embodiments, the temperature
`
`is about -70 °C. In other embodiments, other blood components can be collected and stored.
`
`Prior to analysis, the plasma is thawed and a fraction of the plasma is mixed with an internal
`
`standard in a solvent such as acetonitrile, to obtain a fixed concentration of the standard. In
`
`some embodiments, the internal standard can be mifepristone-d4 . The concentration of the
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`15
`
`internal standard is selected in order to be greater than the expected concentration of
`
`mifepristone in the plasma. For example, the internal standard can have a concentration of 2000
`
`ng/mL. One of skill in the art will appreciate that other internal standards, and other
`
`concentrations, are useful in the present invention.
`
`[0036] Base is then added to the sample solution. The base can be any amine or ammonium
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`20
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`base, such as ammonium hydroxide. One of skill in the art will appreciate that other bases are
`
`useful in the present invention.
`
`[0037] Solvent is then added to the solution and the mifepristone (along with the internal
`
`standard) are extracted from the plasma. Solvents useful for the extraction of mifepristone
`
`include, but are not limited to, hexanes, pentanes, ethers (such as diethylether, tetrahydrofuran
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`25
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`and methyl-t-butyl ether (MTBE)), ethyl acetate, chloroform and methylene chloride. One of
`
`skill in the art will appreciate that other solvents are useful in the present invention.
`
`[0038] Following separation and concentration of the organic layer, the sample is reconstituted
`
`in a solvent mixture comprising water, acetonitrile and formic acid. The ratio of the solvent
`
`components can vary. In some embodiments, the solvent mixture is water:acetonitrile:formic
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`acid (75:25:0.1, v/v/v). One of skill in the art will appreciate that other solvent mixtures are
`
`useful in the present invention.
`
`[0039] The sample can then be analyzed by reverse-phase high pressure liquid
`
`chromatography (HPLC). In some embodiments, the reverse-phase HPLC is performed using a
`
`5 water:acetonitrile:formic acid (60:40:0.1) mobile phase (isocratic) at a flow rate of 0.3 mL/min.
`
`One of skill in the art will appreciate that other mobile phases and flow rates are useful in the
`
`present invention.
`
`[0040] The reverse-phase HPLC column can be a phenyl column maintained at 50°C.
`
`Mifepristone elutes at 4.2 minutes. Following elution, the mobile phase can be nebulized using
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`10
`
`heated nitrogen in a Z-spray source/interface and the ionized compounds detected using a
`
`tandem quadrupole mass spectrometer. Mifepristone (molecular weight of 430 g/mol) can be
`
`detected at m/z 372.30. The internal standard mifepristone-d4 can be detected at m/z 376.30.
`
`The ratio of the mifepristone peak height to the peak height for the internal standard can then be
`
`calculated.
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`15
`
`[0041] The plasma concentration of mifepristone is then calculated by comparing the
`
`experimental ratio to a standard curve of mifepristone:mifepristone-d4 peak height ratio v.
`
`mifepristone concentration. The standard curve is generated by first measuring the
`
`mifepristone:mifepristone-d4 peak height ratios for mifepristone samples at 10, 20, 50, 100, 200,
`
`500, 1000 and 2000 ng/mL where the mifepristone-d4 internal standard has a concentration of
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`20
`
`2000 ng/mL. The mifepristone:mifepristone-d4 peak height ratios of these known solutions are
`
`then fit to a power equation (Mass Lynx by Micromass, Beverly, MA), against which future
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`samples with unknown concentrations of mifepristone are compared.
`
`[0042] The plasma levels of mifepristone derivatives such as RU42633, RU42698 and
`
`RU42848, among others, can also be determined using the assay described above.
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`25
`
`E.
`
`Kits for Treating Cushing's syndrome with Mifepristone
`
`[0043] The present invention provides kits. The kits of the present invention comprise seven
`
`daily doses and a plasma sampling collection device. The kits of the present invention can also
`
`comprise any other component necessary for a kit, such as a container.
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`12
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`[0044] Patient plasma can be collected by any known plasma collection device. Some plasma
`
`collection devices useful in the present invention include, but are not limited to, vacutainers. The
`
`plasma collection devices of the present invention can optionally comprise additives in the
`
`device, such as anticoagulants (EDTA, sodium citrate, heparin, oxalate), a gel with intermediate
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`5
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`density between blood cells and blood plasma, particles causing the blood to clot, a gel to
`
`separate blood cells from serum, thrombin and fluoride, among others.
`
`[0045] The kits can also contain additional vessels used for further analysis of the plasma. For
`
`example, when the plasma is centrifuged, the centrifuged plasma can be transferred to a vessel,
`
`such as a cryostat tube. One of skill in the art will appreciate that other vessels and containers
`
`10
`
`are useful in the present invention.
`
`IV.
`
`EXAMPLES
`
`Example 1: Determination of Mifepristone Plasma Level
`
`[0046] This example provides a procedure for determining the plasma level of mifepristone in
`
`a patient.
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`15
`
`[0047] Three (3) mL of blood was collected from a patient in a vacutainer containing sodium
`
`heparin. The blood was centrifuged and the resulting plasma frozen at -70 to -80°C until assay.
`
`For analysis, the plasma samples were warmed and prepared as follows:
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`20
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`25
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`1.
`
`2.
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`3.
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`4.
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`5.
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`6.
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`7.
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`8.
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`9.
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`Using a pipette, 50.0 !lL of the sample was aliquoted into a 16 x 100-mm glass
`test tube. When a partial volume aliquot was needed, the aliquot was added to the
`tube and diluted to full volume with blank human plasma.
`
`20.0 !lL of the internal standard, mifepristone-d4 (5.00 11g/mL in acetonitrile), was
`added to the tube, resulting in 2000.0 ng/mL mifepristone-d4 in plasma.
`
`The tube was vortexed for approximately 1 minute.
`
`50.0 !lL of 6% ammonium hydroxide was added to the tube.
`
`The tube was vortexed for approximately 1 minute.
`
`2.00 mL ofMTBE was added to the tube.
`
`2.00 mL of hexane was added to the tube.
`
`The tube was vortexed for at least 15 minutes.
`
`The tube was centrifuged for at least 10 minutes at 2500 RPM (575 x g).
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`30
`
`10.
`
`The aqueous layer was frozen in a freezer set to maintain -70°C.
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`13
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`14
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`11.
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`12.
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`13.
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`14.
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`15.
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`16.
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`17.
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`18.
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`The upper organic layer was pou