(12) United States Patent
`Pullman et al.
`
`US0069.43166B1
`US 6,943,166 B1
`Sep. 13, 2005
`
`(10) Patent No.:
`(45) Date of Patent:
`
`(54)
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`(75)
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`(73)
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`(*)
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`(21)
`(22)
`(86)
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`
`COMPOSITIONS COMPRISING
`PHOSPHODIESTERASE INHABITORS FOR
`THE TREATMENT OF SEXUAL
`DISFUNCTION
`
`Notice:
`
`Inventors: William Ernest Pullman, Far Hills, NJ
`(US); John Steven Whitaker,
`Woodinville, WA (US)
`Assignee: Lilly ICOS LLC., Wilmington, DE
`(US)
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`Appl. No.:
`10/031,556
`PCT Filed:
`Apr. 26, 2000
`PCT No.:
`PCT/US00/11129
`§ 371 (c)(1),
`Oct. 19, 2001
`(2), (4) Date:
`PCT Pub. No.: WOOO/66099
`PCT Pub. Date: Nov. 9, 2000
`Related U.S. Application Data
`Provisional application No. 60/132,036, filed on Apr. 30,
`1999.
`Int. Cl." ....................... A61K 31/495; A61K 31/50
`U.S. Cl. ....................................................... 514/250
`Field of Search .......................................... 514/250
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`5,859,006 A
`1/1999 Daugan ...................... 514/249
`6,140,329 A * 10/2000 Daugan ...................... 514/250
`
`
`
`6,451,807 B1
`FOREIGN PATENT DOCUMENTS
`
`9/2002 Emmick et al.
`
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`
`WO 95/19978
`WO 97/03675
`WO 99 59584
`WO 99/59584
`WO 00 53148
`WO ()() 661.14
`WO 01 80860
`
`......... CO7D/471/14
`7/1995
`2/1997 ......... A61K/31/495
`11/1999
`11/1999
`9/2000
`11/2000
`11/2001
`
`......... A61K/31/415
`
`20, No. 6, pp.
`
`OTHER PUBLICATIONS
`Israel, The Pharmaceutical Journal, 261, pp. 164–165
`(1998).
`Goldenberg, Clinical Therapeutics,
`1033–1048 (1998).
`WPIOS AN 2000–339026, Furitsu et al., JP 1999.0276134,
`Sep. 1999, abstract.
`NDA 20–895 (New Drug Application) Sildenafil for Male
`Impotence, pp. 99–103 and 183–187, Jan. 22, 1998, author
`unknown.
`* cited by examiner
`Primary Examiner—Rebecca Cook
`(74) Attorney, Agent, or Firm—Marshall, Gerstein & Borun
`LLP
`ABSTRACT
`(57)
`The present invention relates to highly selective phosphodi
`eterase (PDE) enzyme inhibitors and to their use in phar
`maceutical articles of manufacture. In particular, the present
`invention relates to potent inhibitors of cyclic guanosine
`3',5'-monophosphate specific phosphodiesterase type 5
`(PDE5) that when incorporated into a pharmaceutical prod
`uct at about 1 to about 20 mg unit dosage are useful for the
`treatment of sexual dysfunction.
`
`12 Claims, No Drawings
`
`MYLAN - EXHIBIT 1001
`
`

`
`1
`COMPOSITIONS COMPRISING
`PHOSPHODIESTERASE INHABITORS FOR
`THE TREATMENT OF SEXUAL
`DISFUNCTION
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`This is the U.S. national phase application of International
`Application No. PCT/US00/11129, filed on Apr. 26, 2000,
`which claims the benefit of provisional patent application
`Ser. No. 60/132,036, filed Apr. 30, 1999.
`FIELD OF THE INVENTION
`The present invention relates to a highly selective phos
`phodiesterase (PDE) enzyme inhibitor and to its use in a
`pharmaceutical unit dosage form. In particular, the present
`invention relates to a potent inhibitor of cyclic guanosine
`3',5'-monophosphate specific phosphodiesterase type 5
`(PDE5) that when incorporated into a pharmaceutical prod
`uct is useful for the treatment of sexual dysfunction. The unit
`dosage form described herein is characterized by selective
`PDE5 inhibition, and accordingly, provides a benefit in
`therapeutic areas where inhibition of PDE5 is desired, with
`minimization or elimination of adverse side effects resulting
`from inhibition of other phosphodiesterase enzymes.
`BACKGROUND OF THE INVENTION
`The biochemical, physiological, and clinical effects of
`cyclic guanosine 3',5'-monophosphate specific phosphodi
`esterase (cGMP-specific PDE) inhibitors suggest their utility
`in a variety of disease states in which modulation of smooth
`muscle, renal, hemostatic, inflammatory, and/or endocrine
`function is desired. Type 5 c6MP-specific phosphodi
`esterase (PDE5) is the major CGMP hydrolyzing lyzing
`enzyme in vascular smooth muscle, and its expression in
`penile corpus cavernosum has been reported (Taher et al., J.
`Urol., 149, p. 285A (1993)). Thus, PDES is an attractive
`target in the treatment of sexual dysfunction (Murray,
`DN&P 6(3), pp. 150–56 (1993)).
`A pharmaceutical product, which provides a PDE5
`inhibitor, is currently available and marketed under the
`trademark VIAGRAB). The active ingredient in VIAGRAB)
`is sildenafil. The product is sold as an article of manufacture
`including 25, 50, and 100 mg tablets of sildenafil and a
`package insert. The package insert provides that sildenafil is
`a more potent inhibitor of PDE5 than other known phos
`phodiesterases (greater than 80 fold for PDE1 inhibition,
`greater than 1,000 fold for PDE2, PDE3, and PDE4
`inhibition). The ICso for sildenafil against PDE5 has been
`reported as 3 rM (Drugs of the Future, 22(2), pp. 138–143
`(1997)) and as 3.9 nM (Boolel et al., Int. J. of Impotence, 8,
`pp. 47–52 (1996)). Sildenafil is described as having a
`4,000-fold selectivity for PDE5 versus PDE3, and only a
`10-fold selectivity for PDE5 versus PDE6. Its relative lack
`of selectivity for PDE6 is theorized to be the basis for
`abnormalities related to color vision.
`While sildenafil has obtained significant commercial
`success, it has fallen short due to its significant adverse side
`effects, including facial flushing (10% incidence rate).
`Adverse side effects limit the use of sildenafil in patients
`suffering from vison abnormalities, hypertension, and, most
`significantly, by individuals who use organic nitrates (Welds
`et al., Amer. J. of Cardiology, 83(5A), pp. 21(C)–28(C)
`(1999)).
`The use of sildenafil in patients taking organic nitrates
`causes a clinically significant drop in blood pressure which
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`US 6,943,166 B1
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`2
`could place the patient in danger. Accordingly, the package
`label for sildenafil provides strict contraindications against
`its use in combination with organic nitrates (e.g.,
`nitroglycerin, isosorbide mononitrate, isosorbide nitrate,
`erythrity1 tetranitrate) and other nitric oxide donors in any
`form, either regularly or intermittently, because sildenafil
`potentiates the hypotensive effects of nitrates. See C. R.
`Conti et al., Amer. J. of Cardiology, 83(5A), pp. 29C–34C
`(1999). Thus, even with the availability of sildenafil, there
`remains a need to identify improved pharmaceutical prod
`ucts that are useful in treating sexual dysfunction.
`Daugan U.S. Pat. No. 5,859,006 discloses certain tetra
`cyclic derivatives that are potent inhibitors of c(3MP
`specific PDE, or PDES. The ICso of the compounds dis
`closed in U.S. Pat. No. 5,859,006 is reported in the range of
`1 nM to 10 um. The oral dosage for such compounds is 0.58
`mg daily for an average adult patient (70 kg). Thus, unit
`dosage forms (tablets or capsules) are reported as 0.2 to 400
`mg of active compound. Significant adverse side effects
`attributed to compounds disclosed in U.S. Pat. No. 5,859,
`006 are not disclosed.
`Applicants have discovered that one such tetracyclic
`derivative, (6R,12aR)-2,3,6,7,12,12a-hexahydro-2-methyl
`6-(3,4-methylenedioxyphenyl)-pyrazino? 2,1':6,1]pyrido[3,
`4-blindole-1,4-dione, alternatively named (6R-trans)-6-(1,
`3-benzodioxol-5-yl)-2,3,6,7,12,12a-hexahydro-2
`methylpyrazino-[1,2':1,6]pyrido[3,4-blindole-1,4-dione,
`and referred to herein as Compound (I), can be administered
`in a unit dose that provides an effective treatment without the
`side effects associated with the presently marketed PDE5
`inhibitor, sildenafil. Prior to the present invention such side
`effects were considered inherent to the inhibition of PDE5.
`Significantly, applicants’ clinical studies also reveal that
`an effective product having a reduced tendency to cause
`flushing in susceptible individuals can be provided. Most
`unexpectedly, the in product also can be administered with
`clinically insignificant side effects associated with the com
`bined effects of a PDE5 inhibitor and an organic nitrate.
`Thus, the contraindication once believed necessary for a
`product containing a PDE5 inhibitor is unnecessary when
`Compound (I) is administered as a unit dose of about 1 to
`about 20 mg, as disclosed herein. Thus, the present invention
`provides an effective therapy for-sexual dysfunction in indi
`viduals who previously were untreatable or suffered from
`unacceptable side effects, including individuals having car
`diovascular disease, such as in individuals requiring nitrate
`therapy, having suffered a myocardial infarction more than
`three months before the onset of sexual dysfunction therapy,
`and suffering from class 1 congestive heart failure, or
`individuals suffering from vision abnormalties.
`The present invention provides Compound (I) in a unit
`dosage form. That is, the present invention provides a
`pharmaceutical unit dosage form suitable for oral adminis
`tration comprising about 1 to about 20 mg Compound (I).
`SUMMARY OF THE INVENTION
`The present invention provides a pharmaceutical dosage
`form for human pharmaceutical use, comprising about 1 to
`about 20 mg of (6R,12aR)-2,3,6,7,12,12a-hexahydro-2
`methyl-6-(3,4-methylenedioxyphenyl)pyrazino? 2,1':6,1]
`pyrido[3,4-blindole-1,4-dione in a unit dosage form suitable
`for oral administration.
`The present invention further provides a method of treat
`ing conditions where inhibition of PDE5 is desired, which
`comprises administering to a patient in need thereof an oral
`dosage form containing about 1 to about 20 mg of a selective
`
`

`
`3
`PDE5 inhibitor, as needed, up to a total dose of 20 mg per
`day. The invention further provides the use of an oral dosage
`form comprising a selective PDE5 inhibitor at a dosage of
`about 1 to about 20 mg for the treatment of sexual dysfunc
`tion.
`Specific conditions that can be treated by the present
`invention, include, but are not limited to, male erectile
`dysfunction and female sexual dysfunction, particularly
`female arousal disorder, also known as female sexual
`arousal disorder.
`In particular, the present invention is directed to a phar
`maceutical unit dosage composition comprising about 1 to
`about 20 mg of a compound having the structural formula:
`
`
`
`O
`
`sº N 2. CH3
`
`said unit dosage form suitable for oral administration, and
`method of treating sexual dysfunction using the pharmaceu
`tical unit dose composition.
`DETAILED DESCRIPTION
`For purposes of the present invention as disclosed and
`described herein, the following terms and abbreviations are
`defined as follows.
`The term “container” means any receptacle and closure
`therefor suitable for storing, shipping, dispensing, and/or
`handling a pharmaceutical product.
`The term “ICso” is the measure of potency of a compound
`to inhibit a particular PDE enzyme (e.g., PDE1c, PDE5, or
`PDE6). The ICso is the concentration of a compound that
`results in 50% enzyme inhibition in a single dose-response
`experiment. Determining the ICso value for a compound is
`readily carried out by a known in vitro methodology gen
`erally described in Y. Cheng et al., Biochem. Pharmacol., 22,
`pp. 3099–3108 (1973).
`The term “package insert” means information accompa
`nying the product that provides a description of how to
`administer the product, along with the safety and efficacy
`data required to allow the physician, pharmacist, and patient
`to make an informed decision regarding use of the product.
`The package insert generally is regarded as the “label” for a
`pharmaceutical product.
`The term “oral dosage form” is used in a general sense to
`reference pharmaceutical products administered orally. Oral
`dosage forms are recognized by those skilled in the art to
`include such forms as liquid formulations, tablets, capsules,
`and gelcaps.
`The term “vision abnormalities” means abnormal vision
`characterized by blue-green vision believed to be caused by
`PDE6 inhibition.
`The term “flushing” means an episodic redness of the face
`and neck attributed to vasodilation caused by ingestion of a
`drug, usually accompanied by a feeling of warmth over the
`face and neck and sometimes accompanied by perspiration.
`
`US 6,943,166 B1
`
`4
`The term “free drug” means solid particles of drug not
`intimately embedded in a polymeric coprecipitate.
`The presently claimed dosage form preferably is pack
`aged as an article of manufacture for human pharmaceutical
`use, comprising a package insert, a container, and a dosage
`form comprising about 1 to about 20 mg of Compound (I)
`The package insert provides a description of how to
`administer a pharmaceutical product, along with the safety
`and efficacy data required to allow the physician,
`pharmacist, and patient to make an informed decision
`regarding the use of the product. The package insert gener
`ally is regarded as the label of the pharmaceutical product.
`The package insert incorporated into the article of manu
`facture indicates that Compound (I) is useful in the treatment
`of conditions wherein inhibition of PDE5 is desired. The
`package insert also provides instructions to administer one
`or more about 1 to about 20 mg unit dosage forms as needed,
`up to a maximum total dose of 20 mg per day. Preferably, the
`dose administered is about 5 to about 20 mg/day, more
`preferably about 5 to about 15 mg/day. Most preferably, a 10
`mg dosage form is administered once per day.
`Preferred conditions to be treated include sexual dysfunc
`tion (including male erectile dysfunction; and female sexual
`dysfunction, and more preferably female arousal disorder
`(FAD)). The preferred condition to be treated is male erectile
`dysfunction.
`Significantly, the package insert supports the use of the
`product to treat sexual dysfunction in patients suffering from
`a retinal disease, for example, diabetic retinopathy or retini
`tis pigmentosa, or in patients who are using organic nitrates.
`Thus, the package insert preferably is free of contraindica
`tions associated with these conditions, and particularly the
`administration of the dosage form with an organic nitrate.
`More preferably, the package insert also is free of any
`cautions or warnings both associated with retinal diseases,
`particularly retinitis pigmentosa, and associated with indi
`viduals prone to vision abnormalties. Preferably, the pack
`age insert also reports incidences of flushing below 2%,
`preferably below 1%, and most preferably below 0.5%, of
`the patients administered the dosage form. The incidence
`rate of flushing demonstrates marked improvement over
`prior pharmaceutical products containing a PDE5 inhibitor.
`The container used in the article of manufacture is con
`ventional in the pharmaceutical Iarts. Generally, the con
`tainer is a blister pack, foil packet, glass or plastic bottle and
`accompanying cap or closure, or other such article suitable
`for use by the patient or pharmacist. Preferably, the container
`is sized to accommodate 1–1000 solid dosage forms, pref
`erably 1 to 500 solid dosage forms, and most preferably, 5
`to 30 solid dosage forms.
`Oral dosage forms are recognized by those skilled in the
`art to include, for example, such forms as liquid
`formulations, tablets, capsules, and gelcaps. Preferably the
`dosage forms are solid dosage forms, particularly, tablets
`comprising about 1 to about 20 mg of Compound (I). Any
`pharmaceutically acceptable excipients for oral use are
`suitable for preparation of such dosage forms. Suitable
`pharmaceutical dosage forms include coprecipitate forms
`described, for example, in Butler U.S. Pat. No. 5,985,326,
`incorporated herein by reference. In preferred embodiments,
`the unit dosage form of the present invention is a solid free
`of a coprecipitate form of Compound (I), but rather contains
`solid Compound (I) as a free drug.
`Preferably, the tablets comprise pharmaceutical excipi
`ents generally recognized as safe such as lactose, microc
`rystalline cellulose, starch, calcium carbonate, magnesium
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`

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`US 6,943,166 B1
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`stearate, stearic acid, talc, and colloidal silicon dioxide, and
`are prepared by standard pharmaceutical manufacturing
`techniques as described in Remington’s Pharmaceutical
`Sciences, 18th Ed., Mack Publishing Co., Easton, Pa.
`(1990). Such techniques include, for example, wet granula
`tion followed by drying, milling, and compression into
`tablets with or without film coating; dry granulation fol
`lowed by milling, compression into tablets with or without
`film coating; dry blending followed by compression into
`tablets, with or without film coating; molded tablets; wet
`granulation, dried and filled into gelatin capsules; dry blend
`filled into gelatin capsules; or suspension and solution filled
`into gelatin capsules. Generally, the solid dosage forms have
`identifying marks which—are debossed or imprinted on the
`surface.
`The present invention is based on detailed experiments
`and clinical trials, and the unexpected observations that side
`effects previously believed to be indicative of PDE5 inhi
`bition can be reduced to clinically insignificant levels by the
`selection of a compound and unit dose. This unexpected
`observation enabled the development of a unit dosage form
`20
`that incorporates Compound (I) in about 1 to about 20 mg
`per unit dosage forms that, when orally administered, mini
`mizes undesirable side effects previously believed unavoid
`able. These side effects include facial flushing, vision
`abnormalities, and a significant decrease in blood pressure,
`when Compound (I) is administered alone or in combination
`with an organic nitrate. The minimal effect of Compound (I),
`administered in about 1 to about 20 mg unit dosage forms,
`on PDE6 also allows the administration of a selective PDE5
`inhibitor to patients suffering from a retinal disease, like
`diabetic retinopathy or retinitis pigmentosa.
`Compound (I) has the following structural formula:
`
`6
`PREPARATIONS
`Human PDE5 Preparation
`Recombinant production of human PDE5 was carried out
`essentially as described in Example 7 of U.S. Pat. No.
`5,702,936, incorporated herein by reference, except that the
`yeast transformation vector employed, which is derived
`from the basic ADH2 plasmid described in V. Price et al.,
`Methods in Enzymology, 1985, pages 308–318 (1990),
`incorporated yeast ADH2 promoter and terminator
`sequences rather than ADH1 promoter and terminator
`sequences and the Saccharomyces cerevisiase host was the
`protease-deficient strain BJ2-54 deposited on Aug. 31, 1998
`with the American Type Culture Collection, Manassas, Va.,
`under accession number ATCC 74465. Transformed host
`cells were grown in 2×SC-leu medium, pH 6.2, with trace
`metals, and vitamins. After 24 hours, YEP medium contain
`ing glycerol was added to a final concentration of 2×YEP/
`3% glycerol. Approximately 24 hours later, cells were
`harvested, washed, and stored at –700C.
`Cell pellets (29 g) were thawed on ice with an equal
`volume of lysis buffer (25 mM Tris-Cl, pH 8, 5 mM MgCl2,
`0.25 mM dithiothreitol, 1 mM benzamidine, and 10 um
`ZnSO4). Cells were lysed in a microfluidizer with N2 at
`20,000 psi. The lysate was centrifuged and filtered through
`0.45 um disposable filters. The filtrate was applied to a 150
`mL column of Q Sepharose Fast Flow (Pharmacia). The
`column was washed with 1.5 volumes of Buffer A (20 mM
`Bis-Tris Propane, pH 6.8, 1 mM MgCl2, 0.25 mM
`dithiothreitol, 10 uM ZnSO) and eluted with a step gradient
`of 125 AM NaCl in Buffer A followed by a linear gradient
`of 125–1000 mM. NaCl in Buffer A.
`Active fractions from the linear gradient were applied to
`a 180 mL ceramic hydroxyapatite column in Buffer B (20
`mM Bis-Tris Propane (pH 6.8), 1 MM MgCl2, 0.25 mM
`dithiothreitol, 10 um ZnSO, and 250 mM KCl). After
`loading, the column was washed with 2 volumes of Buffer
`B and eluted with a linear gradient of 0–125 mM potassium
`phosphate in Buffer B. Active fractions were pooled, pre
`cipitated with 60% ammonium-sulfate, and resuspended in
`Buffer C (20 mM Bis-Tris Propane, pH 6.8, 125 mM NaCl,
`0.5 mM dithiothreitol, and 10 um ZnSO,). The pool was
`applied to a 140 mL column of Sephacryl S-300 HR and
`eluted with Buffer C. Active fractions were diluted to 50%
`glycerol and stored at -20° C. The resultant preparations
`were about 85% pure by SDS-PAGE.
`Assay for PDE Activity
`Activity of PDE5 can be measured by standard assays in
`the art. For example, specific activity of any PDE can be
`determined as follows. PDE assays utilizing a charcoal
`separation technique were performed essentially as
`described in Loughney et al., (1996), The Journal of Bio
`logical Chemistry, 271:796–806. In this assay, PDE5 activ
`ity converts [**p]cGMP to [32p]5'GMP in proportion to the
`amount of PDE5 activity present. The [*P]5'GMP then is
`quantitatively converted to free [*P] phosphate and unla
`beled adenosine by the action of snake venom
`5'-nucleotidase. Hence, the amount of [*P] phosphate lib
`erated is proportional to enzyme activity. The assay is
`performed at 30 C in a 100 uD reaction mixture containing
`(final concentrations) 40 mM Tris-Cl (pH 8.0), 1 um ZnSO,
`5 mM MgCl, and 0.1 mg/mL bovine serium albumin. PDE5
`is present in quantities that yield ~30% total hydrolysis of
`substrate (linear assay conditions). The assay is initiated by
`addition of substrate (1 nM [*P]cGMP), and the mixture is
`incubated for 12 minutes. Seventy-five (75) ug of Crotalus
`atrox venom then is added, and the incubation is continued
`for 3 more minutes (15 minutes total). The reaction is
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`(I)
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`wº N 2. CH3
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`The compound of structural formula (I) was demonstrated in
`human clinical studies to exert a minimal impact on systolic
`blood pressure when administered in conjunction with
`organic nitrates. By contrast, sildenafil demonstrates a four
`fold greater decrease in systolic blood pressure over a
`placebo, which leads to the contraindications in the VIA
`GRA insert, and in warnings to certain patients.
`55
`The following illustrates the PDE5 and PDE6 ICso values
`for the compound of structural formula (I) determined by the
`procedures described herein.
`
`50
`
`Compound
`I
`
`PDE5 ICso (nM)
`2.5
`
`PDE6 ICso (nM)
`3400
`
`PDE6/PDE5
`1360
`
`60
`
`The compound of structural formula (I) additionally dem
`65
`onstrates an ICso against PDE1c of 10,000, and a ratio of
`PDE10/PDE5 of 4,000.
`
`

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`US 6,943,166 B1
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`stopped by addition of 200 mL of activated charcoal (25
`mg/mL suspension in 0.1 M NaH2PO4, pH 4). After cen
`trifugation (750 ×g for 3 minutes) to sediment the charcoal,
`a sample of the supernatant is taken for radioactivity deter
`mination in a scintillation counter and the PDE5 activity is
`calculated. The preparations had specific activities of about
`3 umoles cGMP hydrolyzed per minute per milligram pro
`tein.
`Bovine PDE6 Preparation
`Bovine PDE6 was supplied by Dr. N. Virmaux, INSERM
`U338, Strasbourg. Bovine retinas were prepared as
`described by Virmaux et al., FEBS Letters, 12(6), pp.
`325–328 (1971) and see also, A. Sitaramayya et al., Exp. Eye
`Res., 25, pp. 163–169 (1977). Briefly, unless stated
`otherwise, all operations were done in the cold and in dim
`red light. Eyes were kept in the cold and in the dark for up
`to four hours after slaughtering.
`Preparation of bovine retinal outer segment (ROS) basi
`cally followed procedures described by Schichi et al., J.
`Biol. Chem., 224:529 (1969). In a typical experiment, 35
`20
`bovine retinas were ground in a mortar with 35 mL 0.066 M
`phosphate buffer, pH 7.0, made up to 40% with sucrose,
`followed by homogenization in a Potter homogenizer (20 up
`and down strokes). The suspension was centrifuged at
`25,000×g for 20 minutes. The pellet was homogenized in 7.5
`mL 0.006 M phosphate buffer (40% in sucrose), and care
`fully layered under 7.5 mL of phosphate buffer (containing
`no sucrose). Centrifugation was conducted in a swing-out
`rotor at 45,000×g for 20 minutes, and produced a pellet
`which is black at the bottom, and also a red band at the
`interface 0.066 M. phosphate—40% sucrose/0.066 M phos
`phate (crude ROS). The red material at the interface was
`removed, diluted with phosphate buffer, spun down to a
`pellet, and redistributed in buffered 40% sucrose as
`described above. This procedure was repeated 2 or 3 times
`until no pellet was formed. The purified ROS was washed in
`phosphate buffer and finally spun down to a pellet at
`25,000×g for 20 minutes. All materials were then kept
`frozen until used.
`Hypotonic extracts were prepared by suspending isolated
`40
`ROS in 10 mM Tris-Cl pH 7.5, 1 mM EDTA, and 1 mM
`dithioerythritol, followed by centrifugation at 100,000×g for
`30 minutes.
`The preparation was reported to have a specific activity,of
`about 35 nmoles cGMP hydrolyzed per minute per milli
`gram protein.
`PDE1c Preparation from Spodoptera fugiperda Cells (Sf9)
`Cell pellets (5g) were thawed on ice with 20 ml of Lysis
`Buffer (50 mM MOPS pH 7.4, 10 ulo? ZnSO, 0.1 mM
`CaCl2, 1 mM DTT, 2m M benzamidine HCl, 5 Aug/ml each of
`50
`pepstatin, leupeptin, and aprotenin). Cells were lysed by
`passage through a French pressure cell (SLM-Aminco)
`while temperatures were maintained below 10° C. The
`resultant cell homogenate was centrifuged at 36,000 rpm at
`4° C. for 45 minutes in a Beckman ultracentrifuge using a
`Type TIA5 rotor. The supernatant was discarded and the
`resultant pellet was resuspended with 40 ml of Solubiliza
`tion Buffer (Lysis Buffer containing 1M NaCl, 0.1M MgCl2,
`1 mM CaCl2, 20 ug/ml calmodulin, and 1% Sulfobetaine
`SB12 (Z3-12) by sonicating using a VibraCell tuner with a
`microtip for 3×30 seconds. This was performed in a crushed
`ice/salt mix for cooling. Following sonication, the mixture
`was slowly mixed for 30 minutes at 4° C. to finish solubi
`lizing membrane bound proteins. This mixture was centri
`fuged in a Beckman ultracentrifuge using a type TI45 rotor
`at 36,000 rpm for 45 minutes. The supernatant was diluted
`with Lysis Buffer containing 10 ug/ml calpain inhibitor I and
`
`8
`II. The precipitated protein was centrifuged for 20 minutes
`at 9,000 rpm in a Beckman JA-10 rotor. The recovered
`supernatant then was subjected to Mimetic Blue AP Agarose
`Chromatography.
`In order to run the Mimetic Blue AP Agarose Column, the
`resin initially was shielded by the application of 10 bed
`volumes of 1% polyvinylpurrolidine (i.e., MW of 40,000) to
`block nonspecific binding sites. The loosely bound PVP-40
`was removed by washing with 10 bed volumes of 2M NaCl,
`and 10 mM sodium citrate pH 3.4. Just prior to addition of
`the solubilized PDE1c3 sample, the column was equilibrated
`with 5 bed volumes of Column Buffer A (50 mM MOPS pH
`7.4, 10 um ZnSO1, 5m M MgCl2, 0.1 mM CaCl2, 1 mM
`DTT, 2 mM benzamidine HCl).
`The solubilized sample was applied to the column at a
`flow rate of 2 ml/min with recycling such that the total
`sample was applied 4 to 5 times in 12 hours. After loading
`was completed, the column was washed with 10 column
`volumes of Column Buffer A, followed by 5 column vol
`umes of Column Buffer B (Column Buffer A containing 20
`mM 5'-AMP), and followed by 5 column volumes of Col
`umn Buffer C (50 mM MOPS pH 7.4, 10 ulo? ZnSO, 0.1
`mM CaCl2, 1 mM dithiothreitol, and 2 mM benzamidine
`HCl). The enzyme was eluted into three successive pools.
`The first pool consisted of enzyme from a 5 bed volume
`wash with Column Buffer C containing 1 mM camp. The
`second pool consisted of enzyme from a 10 bed volume
`wash with Column Buffer C containing 1 M NaCl. The final
`pool of enzyme consisted of a 5 bed volume wash with
`Column Buffer C containing 1 M NaCl and 20 mM cAMP
`The active pools of enzyme were collected and the cyclic
`nucleotide removed via conventional gel filtration chroma
`tography or chromatography on hydroxy-apatite resins. Fol
`lowing removal of cyclic nucleotides, the enzyme pools
`were dialyzed against Dialysis Buffer containing 25 mM
`MOPS pH 7.4, 10 ulo? ZnSO,500 mM NaCl, 1 mM CaCl,
`1 mM dithiothreitol, 1 mM benzamidine HCl, followed by
`dialysis against Dialysis buffer containing 50% glycerol.
`The enzyme was quick frozen with the aid of dry ice and
`stored at –70° C.
`The resultant preparations were about >90% pure by
`SDS-PAGE. These preparations had specific activities of
`about 0.1 to 1.0 umol cAMP hydrolyzed per minute per
`milligram protein.
`ICso Determinations
`The parameter of interest in evaluating the potency of a
`competitive enzyme inhibitor of PDE5 and/or PDE1c and
`PDE6 is the inhibition constant, i.e., K. This parameter can
`be approximated by determining the ICS, which is the
`inhibitor concentration that results in 50% enzyme
`inhibition, in a single dose-response experiment under the
`following conditions.
`The concentration of inhibitor is always much greater
`than the concentration of enzyme, so that free inhibitor
`concentration (which is unknown) is approximated by total
`inhibitor concentration (which is known).
`A suitable range of inhibitor concentrations is chosen (i.e.,
`inhibitor concentrations at least several fold greater and
`several fold less than the K, are present in the experiment).
`Typically, inhibitor concentrations ranged from 10 nM to 10
`AuM.
`The concentrations of enzyme and substrate are chosen
`such that less than 20% of the substrate is consumed in the
`absence of inhibitor (providing, e.g., maximum substrate
`hydrolysis of from 10 to 15%), so that enzyme activity is
`approximately constant throughout the assay.
`The concentration of substrate is less than one-tenth the
`Michaelis constant (Ka). Under these conditions, the ICso
`
`30
`
`35
`
`45
`
`55
`
`60
`
`65
`
`

`
`US 6,943,166 B1
`
`9
`will closely approximate the K. This is because of the
`Cheng-Prusoff equation relating these two parameters:
`ICso-K;(1+S/Ka,), with (1+S/Ka,) approximately 1 at low
`values of S/K,.
`The ICso value is estimated from the data points by fitting
`the data to...a suitable model of the enzyme inhibitor inter
`action. When this interaction is known to involve simple
`competition of the inhibitor with the substrate, a two
`parameter model can be used:
`
`10
`2 minutes. The blend was compressed to a target
`compression/weight of 250 mg using 9 mm round normal
`concave tooling.
`The core tablets were coated with an aqueous suspension
`of Opadry OY-S-7322 using an Accelacota (or similar
`coating pan) using inlet air at 50° C. to 70° C. until the tablet
`weight was increased by approximately 8 mg. Opadry
`OY-S-7322 contains methylhydroxypropylcellulose Ph.
`Eur, titanium dioxide Ph. Eur, Triacetin USP. Opadry
`increases the weight of each tablet to about 258 mg. The
`amount of film coat applied per tablet may be less than that
`stated depending on the process efficiency.
`The tablets are filled into blister packs and accompanied
`by package insert describing the safety and efficacy of the
`compound.
`
`Component
`Selective PDE5 Inhibitor”
`Hydroxypropyl Methylcellulose
`Phthalate
`Microcrystalline Cellulose
`Croscarmellose Sodium
`Sodium Lauryl Sulfate
`Povidone K30
`Purified Water, USP (water for
`irrigation)
`Croscarmellose Sodium
`Sodium Lauryl Sulfate
`Colloidal Anhydrous Silica
`Magnesium Stearate
`
`Formulations
`(mg per tablet)
`5
`5
`
`1
`1
`
`221.87
`5.00
`2.50
`9.38
`Q.S.
`
`5.00
`2.50
`0.50
`1.25
`
`213.87
`5.00
`2.50
`9.38
`Q.S.
`
`5.00
`2.50
`0.50
`1.25
`
`Total core subtotal
`(Film coat Opadry OY-S-7322)
`°Compound (I).
`
`250.00
`about 8 mg
`
`250.00
`about 8 mg
`
`EXAMPLE 2
`The following formula is used in preparing the finished
`dosage form containing 10 mg of Compound (I).
`
`Ingredient
`
`Granulation
`
`Selective PDE5 Inhibitor")
`Lactose Monohydrate
`Lactose Monohydrate (spray dried)
`Hydroxypropylcellulose
`Croscarmellose Sodium
`Hydroxypropylcellulose (EF)
`Sodium Lauryl Sulfate
`
`Outside Powders
`
`Microcrystalline Cellulose (granular-102)
`Croscarmellose Sodium
`Magnesium Stearate (vegetable)
`
`Total
`Film coat (approximately)
`
`Quantity (mg)
`
`10.00
`153.80
`25.00
`4.00
`9.00
`1.75
`0.70
`35.00
`
`37.50
`7.00
`1.25
`
`250 mg
`11.25
`
`Purified Water, USP is used in the manufacture of the
`tablets. The water is removed during processing and mini
`mal levels remain in the finished product.
`Tablets are manufactured using a wet granulation process.
`A step-by-step description of the process is as follows. The
`drug and excipients to be granulated are security sieved. The
`selective PDE5 inhibitor is dry blended with lactose mono
`
`10
`
`15
`
`20
`
`25
`
`where the y is the enzyme activity measured at an inhibitor
`concentration of x, A is the activity