`Pullman et al.
`
`111111
`
`1111111111111111111111111111111111111111111111111111111111111
`US006943166Bl
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 6,943,166 Bl
`Sep.13,2005
`
`(54) COMPOSITIONS COMPRISING
`PHOSPHODIESTERASE INHABITORS FOR
`THE TREATMENT OF SEXUAL
`DISFUNCTION
`
`(75)
`
`Inventors: William Ernest Pullman, Far Hills, NJ
`(US); John Steven Whitaker,
`Woodinville, WA (US)
`
`(73) Assignee: Lilly ICOS LLC., Wilmington, DE
`(US)
`
`( *) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`
`(21) Appl. No.:
`
`10/031,556
`
`(22) PCT Filed:
`
`Apr. 26, 2000
`
`(86) PCTNo.:
`
`PCT/USOO/l1129
`
`§ 371 (c)(l),
`(2), ( 4) Date: Oct. 19, 2001
`
`(87) PCT Pub. No.: W000/66099
`
`PCT Pub. Date: Nov. 9, 2000
`
`Related U.S. Application Data
`( 60) Provisional application No. 60/132,036, filed on Apr. 30,
`1999.
`
`Int. Cl? ....................... A61K 31/495; A61K 31/50
`(51)
`(52) U.S. Cl. ....................................................... 514/250
`(58) Field of Search .......................................... 514/250
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`6,451,807 B1
`
`9/2002 Emmick et a!.
`
`FOREIGN PATENT DOCUMENTS
`wo 95/19978
`wo 97/03675
`wo 99 59584
`wo 99/59584
`wo 00 53148
`wo 00 66114
`wo 01 80860
`
`......... C07D/471/14
`......... A61K/31!495
`
`......... A61K/31!415
`
`wo
`wo
`wo
`wo
`wo
`wo
`wo
`
`7/1995
`2/1997
`11/1999
`11/1999
`9/2000
`11/2000
`11/2001
`
`OTHER PUBLICATIONS
`
`Israel, The Pharmaceutical Journal, 261, pp. 164-165
`(1998).
`Goldenberg, Clinical Therapeutics, 20, No. 6, pp.
`1033-1048 (1998).
`WPIOS AN 2000-339026, Furitsu et al, JP 19990276134,
`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
`
`(57)
`
`ABSTRACT
`
`The present invention relates to highly selective phosphodi(cid:173)
`eterase (PDE) enzyme inhibitors and to their use in phar(cid:173)
`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(cid:173)
`uct at about 1 to about 20 mg unit dosage are useful for the
`treatment of sexual dysfunction.
`
`1!1999 Daugan ...................... 514/249
`5,859,006 A
`6,140,329 A * 10/2000 Daugan ...................... 514/250
`
`12 Claims, No Drawings
`
`INTELGENX 1001, pg 1
`
`
`
`US 6,943,166 Bl
`
`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(cid:173)
`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
`(PDES) that when incorporated into a pharmaceutical prod(cid:173)
`uct is useful for the treatment of sexual dysfunction. The unit
`dosage form described herein is characterized by selective
`PDES inhibition, and accordingly, provides a benefit in
`therapeutic areas where inhibition of PDES is desired, with
`minimization or elimination of adverse side effects resulting
`from inhibition of other phosphodiesterase enzymes.
`
`BACKGROUND OF THE INVENTION
`
`25
`
`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,
`s erythrityl 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
`10 remains a need to identify improved pharmaceutical prod(cid:173)
`ucts that are useful in treating sexual dysfunction.
`Daugan U.S. Pat. No. 5,859,006 discloses certain tetra(cid:173)
`cyclic derivatives that are potent inhibitors of cGMP(cid:173)
`specific PDE, or PDES. The IC50 of the compounds dis-
`15 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
`20 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-me thy lenedioxypheny 1)-pyrazino[ 2' ,1': 6,1 ]pyrido[ 3,
`4-b ]indole-1,4-dione, alternatively named ( 6R-trans)-6-(1,
`3-benzodioxol-5 -yl)-2,3,6, 7, 12, 12a-hexahydro-2-
`methy lpyrazino-[1' ,2': 1,6 ]pyrido[3,4-b ]indole-1 ,4-dione,
`and referred to herein as Compound (I), can be administered
`in a unit dose that provides an effective treatment without the
`30 side effects associated with the presently marketed PDES
`inhibitor, sildenafil. Prior to the present invention such side
`effects were considered inherent to the inhibition of PDES.
`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(cid:173)
`bined effects of a PDES inhibitor and an organic nitrate.
`40 Thus, the contraindication once believed necessary for a
`product containing a PDES 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-
`45 viduals who previously were untreatable or suffered from
`unacceptable side effects, including individuals having car(cid:173)
`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,
`50 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-
`55 tration comprising about 1 to about 20 mg Compound (I).
`
`The biochemical, physiological, and clinical effects of
`cyclic guanosine 3',5'-monophosphate specific phosphodi(cid:173)
`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 cGMP-specific phosphodi(cid:173)
`esterase (PDES) is the major CGMP hydrolyzing lyzing 35
`enzyme in vascular smooth muscle, and its expression in
`penile corpus cavernosum has been reported (Taher et al.,J.
`Ural., 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 PDES
`inhibitor, is currently available and marketed under the
`trademark VIAGRA®. The active ingredient in VIAGRA®
`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 PDES than other known phos(cid:173)
`phodiesterases (greater than 80 fold for PDEl inhibition,
`greater than 1,000 fold for PDE2, PDE3, and PDE4
`inhibition). The IC50 for sildenafil against PDES 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 PDES versus PDE3, and only a
`10-fold selectivity for PDES 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). 60
`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
`
`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-methy lenedioxypheny l)pyrazino[2' ,1': 6,1]
`pyrido[3,4-b ]indole-1,4-dione in a unit dosage form suitable
`for oral administration.
`The present invention further provides a method of treat-
`65 ing conditions where inhibition of PDES 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
`
`INTELGENX 1001, pg 2
`
`
`
`US 6,943,166 Bl
`
`said unit dosage form suitable for oral administration, and
`method of treating sexual dysfunction using the pharmaceu(cid:173)
`tical unit dose composition.
`
`DETAILED DESCRIPTION
`
`3
`PDES 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 PDES inhibitor at a dosage of
`about 1 to about 20 mg for the treatment of sexual dysfunc(cid:173)
`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(cid:173)
`maceutical unit dosage composition comprising about 1 to
`about 20 mg of a compound having the structural formula:
`
`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(cid:173)
`aged as an article of manufacture for human pharmaceutical
`5 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,
`10 pharmacist, and patient to make an informed decision
`regarding the use of the product. The package insert gener(cid:173)
`ally is regarded as the label of the pharmaceutical product.
`The package insert incorporated into the article of manu(cid:173)
`facture indicates that Compound (I) is useful in the treatment
`15 of conditions wherein inhibition of PDES 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
`20 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(cid:173)
`tion (including male erectile dysfunction; and female sexual
`dysfunction, and more preferably female arousal disorder
`25 (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
`30 a retinal disease, for example, diabetic retinopathy or retini(cid:173)
`tis pigmentosa, or in patients who are using organic nitrates.
`Thus, the package insert preferably is free of contraindica(cid:173)
`tions associated with these conditions, and particularly the
`administration of the dosage form with an organic nitrate.
`For purposes of the present invention as disclosed and
`More preferably, the package insert also is free of any
`described herein, the following terms and abbreviations are 35
`cautions or warnings both associated with retinal diseases,
`defined as follows.
`particularly retinitis pigmentosa, and associated with indi(cid:173)
`The term "container" means any receptacle and closure
`viduals prone to vision abnormalties. Preferably, the pack(cid:173)
`therefor suitable for storing, shipping, dispensing, and/or
`age insert also reports incidences of flushing below 2%,
`handling a pharmaceutical product.
`The term "IC50" is the measure of potency of a compound 40 preferably below 1%, and most preferably below 0.5%, of
`the patients administered the dosage form. The incidence
`to inhibit a particular PDE enzyme (e.g., PDE1c, PDES, or
`rate of flushing demonstrates marked improvement over
`PDE6). The IC50 is the concentration of a compound that
`prior pharmaceutical products containing a PDES inhibitor.
`results in 50% enzyme inhibition in a single dose-response
`The container used in the article of manufacture is con(cid:173)
`experiment. Determining the IC50 value for a compound is
`ventional in the pharmaceutical Iarts. Generally, the con(cid:173)
`readily carried out by a known in vitro methodology gen- 45
`tainer is a blister pack, foil packet, glass or plastic bottle and
`erally described in Y. Cheng et al., Biochem. Pharmacal., 22,
`accompanying cap or closure, or other such article suitable
`pp. 3099-3108 (1973).
`for use by the patient or pharmacist. Preferably, the container
`The term "package insert" means information accompa(cid:173)
`is sized to accommodate 1-1000 solid dosage forms, pref-
`nying the product that provides a description of how to
`50 erably 1 to 500 solid dosage forms, and most preferably, 5
`administer the product, along with the safety and efficacy
`to 30 solid dosage forms.
`data required to allow the physician, pharmacist, and patient
`Oral dosage forms are recognized by those skilled in the
`to make an informed decision regarding use of the product.
`art to include, for example, such forms as liquid
`The package insert generally is regarded as the "label" for a
`formulations, tablets, capsules, and gelcaps. Preferably the
`pharmaceutical product.
`55 dosage forms are solid dosage forms, particularly, tablets
`The term "oral dosage form" is used in a general sense to
`comprising about 1 to about 20 mg of Compound (I). Any
`reference pharmaceutical products administered orally. Oral
`pharmaceutically acceptable excipients for oral use are
`dosage forms are recognized by those skilled in the art to
`suitable for preparation of such dosage forms. Suitable
`include such forms as liquid formulations, tablets, capsules,
`pharmaceutical dosage forms include coprecipitate forms
`and gelcaps.
`60 described, for example, in Butler U.S. Pat. No. 5,985,326,
`The term "vision abnormalities" means abnormal vision
`incorporated herein by reference. In preferred embodiments,
`characterized by blue-green vision believed to be caused by
`the unit dosage form of the present invention is a solid free
`of a coprecipitate form of Compound (I), but rather contains
`PDE6 inhibition.
`solid Compound (I) as a free drug.
`The term "flushing" means an episodic redness of the face
`and neck attributed to vasodilation caused by ingestion of a 65
`Preferably, the tablets comprise pharmaceutical excipi(cid:173)
`ents generally recognized as safe such as lactose, microc(cid:173)
`drug, usually accompanied by a feeling of warmth over the
`rystalline cellulose, starch, calcium carbonate, magnesium
`face and neck and sometimes accompanied by perspiration.
`
`INTELGENX 1001, pg 3
`
`
`
`US 6,943,166 Bl
`
`10
`
`5
`
`5
`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(cid:173)
`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 PDES inhi(cid:173)
`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(cid:173)
`mizes undesirable side effects previously believed unavoid(cid:173)
`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 PDES
`inhibitor to patients suffering from a retinal disease, like
`diabetic retinopathy or retinitis pigmentosa.
`Compound (I) has the following structural formula:
`
`6
`PREPARATIONS
`Human PDES Preparation
`Recombinant production of human PDES 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 ADHl 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
`15 cells were grown in 2xSC-leu medium, pH 6.2, with trace
`metals, and vitamins. After 24 hours, YEP medium contain(cid:173)
`ing glycerol was added to a final concentration of 2xYEP/
`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 MgC12 ,
`0.25 mM dithiothreitol, 1 mM benzamidine, and 10 ,uM
`ZnS0 4 ). Cells were lysed in a microfiuidizer with N2 at
`20,000 psi. The lysate was centrifuged and filtered through
`25 0.45 ,urn 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 MgC12 , 0.25 mM
`dithiothreitol, 10 ,uM ZnS0 4) and eluted with a step gradient
`30 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 MgC12 , 0.25 mM
`35 dithiothreitol, 10 ,uM ZnS0 4 , 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(cid:173)
`cipitated with 60% ammonium-sulfate, and resuspended in
`40 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
`45 were about 85% pure by SDS-PAGE.
`Assay for PDE Activity
`Activity of PDES 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
`50 separation technique were performed essentially as
`described in Loughney et al., (1996), The Journal of Bio(cid:173)
`logical Chemistry, 271:796-806. In this assay, PDES activ(cid:173)
`ity converts [32p ]cGMP to [32p ]S'GMP in proportion to the
`amount of PDES activity present. The [32P]5'GMP then is
`55 quantitatively converted to free [32P] phosphate and unla(cid:173)
`beled adenosine by the action of snake venom
`5'-nucleotidase. Hence, the amount of [32P] phosphate lib(cid:173)
`erated is proportional to enzyme activity. The assay is
`performed at 30 C in a 100 ,uL reaction mixture containing
`60 (final concentrations) 40 mM Tris-Cl (pH 8.0), 1 ,uM ZnS0 4 ,
`5 mM MgCl, and 0.1 mg/mL bovine serium albumin. PDES
`is present in quantities that yield <30% total hydrolysis of
`substrate (linear assay conditions). The assay is initiated by
`addition of substrate (1 nM [32P]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
`
`(I)
`
`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(cid:173)
`fold greater decrease in systolic blood pressure over a
`placebo, which leads to the contraindications in the VIA(cid:173)
`GRA insert, and in warnings to certain patients.
`The following illustrates the PDES and PDE6 IC50 values
`for the compound of structural formula (I) determined by the
`procedures described herein.
`
`Compound
`
`PDE5 IC50 (nM)
`
`PDE6 IC50 (nM)
`
`PDE6/PDE5
`
`2.5
`
`3400
`
`1360
`
`The compound of structural formula (I) additionally dem- 65
`onstrates an IC50 against PDElc of 10,000, and a ratio of
`PDElc/PDES of 4,000.
`
`INTELGENX 1001, pg 4
`
`
`
`US 6,943,166 Bl
`
`10
`
`7
`stopped by addition of 200 mL of activated charcoal (25
`mg/mL suspension in 0.1 M NaH2 P0 4 , pH 4). After cen(cid:173)
`trifugation (750 xg for 3 minutes) to sediment the charcoal,
`a sample of the supernatant is taken for radioactivity deter(cid:173)
`mination in a scintillation counter and the PDE5 activity is 5
`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., FEES 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(cid:173)
`cally followed procedures described by Schichi et al., J.
`Bioi. Chern., 224:529 (1969). In a typical experiment, 35
`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
`25,000xg for 20 minutes. The pellet was homogenized in 7.5
`mL 0.006 M phosphate buffer (40% in sucrose), and care(cid:173)
`fully layered under 7.5 mL of phosphate buffer (containing
`no sucrose). Centrifugation was conducted in a swing-out
`rotor at 45,000xg for 20 minutes, and produced a pellet
`which is black at the bottom, and also a red band at the 30
`interface 0.066 M. phosphate-40% sucrose/0.066 M phos(cid:173)
`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
`25,000xg 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 lOO,OOOxg for
`30 minutes.
`The preparation was reported to have a specific activity,of
`about 35 nmoles cGMP hydrolyzed per minute per milli- 45
`gram protein.
`PDElc 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 ,uM ZnS0 4 , 0.1 mM
`CaC12 , 1 mM DTT, 2mM benzamidine HCl, 5 ,ug!ml each of
`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 TI45 rotor. The supernatant was discarded and the
`resultant pellet was resuspended with 40 ml of Solubiliza(cid:173)
`tion Buffer (Lysis Buffer containing 1M NaCl, O.lM MgC12 ,
`1 mM CaC12 , 20 ,ug/ml calmodulin, and 1% Sulfobetaine
`SB12 (Z3-12) by sonicating using a VibraCell tuner with a
`microtip for 3x30 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(cid:173)
`lizing membrane bound proteins. This mixture was centri(cid:173)
`fuged in a Beckman ultracentrifuge using a type TI45 rotor 65
`at 36,000 rpm for 45 minutes. The supernatant was diluted
`with Lysis Buffer containing 10 ,ug!ml cal pain 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 ZnS0 4 , 5mM MgC12 , 0.1 mM CaC12 , 1 mM
`DTT, 2 mM benzamidine HCl).
`The solubilized sample was applied to the column at a
`15 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(cid:173)
`umes of Column Buffer B (Column Buffer A containing 20
`20 mM 5'-AMP), and followed by 5 column volumes of Col(cid:173)
`umn Buffer C (50 mM MOPS pH 7.4, 10 ,uM ZnS0 4 , 0.1
`mM CaC12 , 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
`25 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 1M NaCl. The final
`pool of enzyme consisted of a 5 bed volume wash with
`Column Buffer C containing 1M NaCl and 20 mM cAMP.
`The active pools of enzyme were collected and the cyclic
`nucleotide removed via conventional gel filtration chroma(cid:173)
`tography or chromatography on hydroxy-apatite resins. Fol(cid:173)
`lowing removal of cyclic nucleotides, the enzyme pools
`were dialyzed against Dialysis Buffer containing 25 mM
`35 MOPS pH 7.4, 10 ,uM ZnS0 4 , 500 mM NaCl, 1 mM CaC12 ,
`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.
`IC50 Determinations
`The parameter of interest in evaluating the potency of a
`competitive enzyme inhibitor of PDE5 and/or PDElc 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
`50 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
`55 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
`60 ,uM.
`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 (!(,). Under these conditions, the IC50
`
`INTELGENX 1001, pg 5
`
`
`
`US 6,943,166 Bl
`
`9
`will closely approximate the K;. This is because of the
`these two parameters:
`Cheng-Prusoff equation relating
`approximately 1 at low
`IC50=K;(l+SJK,), with (1+S/K,)
`values of SJK,.
`The IC50 value is estimated from the data points by fitting 5
`the data to.a suitable model of the enzyme inhibitor inter(cid:173)
`action. When this interaction is known to involve simple
`competition of the inhibitor with the substrate, a two(cid:173)
`parameter model can be used:
`
`Y~A!(1+x!B)
`
`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
`10 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
`15 compound.
`
`20
`
`25
`
`35
`
`Component
`
`Selective PDE5 Inhibitor')
`Hydroxypropyl Methylcellulose
`Phthalate
`Microcrystalline Cellulose
`Croscarmellose Sodium
`Sodium Laury! Sulfate
`Povidone K30
`Purified Water, USP (water for
`irrigation)
`Croscarmellose Sodium
`Sodium Laury! Sulfate
`Colloidal Anhydrous Silica
`Magnesium Stearate
`
`Formulations
`(mg per tablet)
`
`5
`5
`
`213.87
`5.00
`2.50
`9.38
`q.s.
`
`5.00
`2.50
`0.50
`1.25
`
`221.87
`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)
`
`250.00
`about 8 mg
`
`250.00
`about 8 mg
`
`')Compound (I).
`
`EXAMPLE 2
`
`The following formula is used in preparing the finished
`40 dosage form containing 10 mg of Compound (I).
`
`where the y is the enzyme activity measured at an inhibitor
`concentration of x, A is the activity in the absence of
`inhibitor and B is the IC50 . See Y. Cheng et al., Biochem.
`Pharmacal., 22:3099-3108 (1973).
`Effects of inhibitors of the present invention on enzymatic
`activity of PDE5 and PDE6 preparations as described above
`were assessed in either of two assays which differed