Use Of Fk506 For The Treatment Of Pulmonary Arterial Hypertension

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AU 2012250974 B2
(12) STANDARD PATENT (11) Application No. AU 2012250974 B2 
(54) Title 
Use of FK506 for the treatment of pulmonary arterial hypertension 
(51) International Patent Classification(s) 
A61K 31/40 (2006.01) 
(21) Application No: 2012250974 (22) Date of Filing: 2012.04.30 
(87) WIPO No: W012/151153 
(30) Priority Data 
(31) Number (32) Date (33) Country 
61/481,317 2011.05.02 US 
(43) Publication Date: 2012.11.08 
(44) Accepted Journal Date: 2017.05.04 
(71) Applicant(s) 
The Board of Trustees of The Leland Stanford Junior University 
(72) Inventor(s) 
Spiekerkoetter, Edda;Rabinovitch, Marlene;Beachy, Philip A.;Solow-Cordero, David 
(74) Agent / Attorney 
FB Rice, L 14 90 Collins St, Melbourne, VIC, 3000, AU 
(56) Related Art 
FUJIWARA, M. et al. Circulation Journal (2008) Vol.72 pages 127 to 133 
MACHADO, R.D. et al. "Genetics and Genomics of Pulmonary Arterial 
Hypertension" Journal of the American College of Cardiology (2009) Vol. 54 No. 1 
Supply S, pages S32 to S42 
RABINOVITCH, M. "Pathobiology of Pulmonary Hypertension" Annual Review of 
Pathology: Mechanisms of Disease (2007) Vol.2 No.1 pages 369 to 399 
ALBINANA, V. et al. MOLECULAR PHARMACOLOGY (2011) Vol.79 No.5 pages 
833 to 843 
US 2005/0119330 Al 
US 6489335 B2 
WO 2010/031968 Al

(19) World Intellectual Property 
International Bureau 
(10) International Publication Number 
(43) International Publication Date W O 2012/151153 A1 
8 November 2012 (08.11.2012) W I PO I P CT 
(51) International Patent Classification: (74) Agent: KEDDIE, James S.; Bozicevic, Field & Francis 
A61K31/40 (2006.01) LLP, 1900 University Avenue, Suite 200, East Palo Alto, 
(21) International Application Number: CA 94303 (US).  
PCT/US2012/035793 (81) Designated States (unless otherwise indicated, for every 
) .a kind of national protection available): AE, AG, AL, AM, (22) International Filng Date: AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, 
April 2012 (30.04.20 12) CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, 
(25) Filing Language: English DZ, EC, EE, EG, ES, Fl, GB, GD, GE, GH, GM, GT, HN, 
HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, KR, (26) Publication Language: English KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, 
(30) Priority Data: MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, 
61/481,317 2 May 2011 (02.05.2011) US OM, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SC, SD, 
SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, 
(71) Applicant (for all designated States except US): THE TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.  
FORD JUNIOR UNIVERSITY [US/US]; 1705 El Cam- (84) Designated States (unless otherwise indicated, for every 
mo Real, Palo Alto, CA 94306-1106 (US). kind of regional protection available): ARIPO (BW, GH, 
GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, 
(72) Inventors; and UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, 
(75) Inventors/Applicants (for US only): SPIEKERKOET- TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, 
TER, Edda [DE/US]; 300 Pasteur Drive, Grant S102J, EE, ES, Fl, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, 
Stanford, CA 94305-5110 (US). RABINOVITCH, Mar- MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, 
lene [CA/US]; 269 Campus Drive, CCSR 1215, Stanford, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, 
CA 94305-5162 (US). BEACHY, Philip, A. [US/US]; 265 ML, MR, NE, SN, TD, TG).  
Campus Drive, Room G3120A, Stanford, CA 94305-5463 Published (US). SOLOW-CORDERO, David [US/US]; 269 Cam
pus Drive, CCSR Room 0133A, Stanford, CA 94305-5174 - with international search report (Art. 21(3)) 
(57) Abstract: A method of reducing pulmonary arterial hypertension in a mammal that employs FK506 is provided. In certain em
bodiments, the method comprises administering FK506 to a mammal having pulmonary arterial hypertension associated with defect
ive MBPR2 signaling at a dosage sufficient to reduce blood pressure in the pulmonary artery of the mammal.

This invention was made with Government support under contract HL089989 
awarded by the National Institutes of Health. The Government has certain rights in this 
Pulmonary artererial hypertension is abnormally high blood pressure in the arteries of 
the lungs. Because symptoms may develop very gradually, patients may delay seeing a 
physician for years. Common symptoms are shortness of breath, fatigue, non
productive cough, angina pectoris, fainting or syncope, peripheral edema (swelling around 
the ankles and feet), and rarely hemoptysis(coughing up blood).  
A method of reducing pulmonary arterial hypertension in a mammal that employs 
FK506 is provided. In certain embodiments, the method comprises administering FK506 to 
a mammal having pulmonary arterial hypertension associated with defective BMPR2 
signaling at a dosage sufficient to reduce blood pressure in the pulmonary artery of the 
mammal. In some cases, the mammal is a mouse, rat or a human. In particular cases, the 
mammal has hereditary pulmonary arterial hypertension caused by, for example, a mutation 
in BMPR2, ALKI or endoglin. In particular cases the FK506 may be administered at a dose 
that provides an FK506 blood concentration of 0.05 to 1 ng/ml, e.g. 0.1-0.2 ng/ml.  
In a further embodiment of the invention there is also provided a method of reducing 
pulmonary hypertension in a mammal, comprising: administering FK506 to a mammal 
having a pulmonary arterial hypertension wherein FK506 is administered at a dosage that 
provides a blood concentration of below 5ng.5mL.  
Throughout this specification the word "comprise", or variations such as "comprises" 
or "comprising", will be understood to imply the inclusion of a stated element, integer or 
step, or group of elements, integers or steps, but not the exclusion of any other element, 
integer or step, or group of elements, integers or steps.  
Any discussion of documents, acts, materials, devices, articles or the like which has 
been included in the present specification is not to be taken as an admission that any or all of 
these matters form part of the prior art base or were common general knowledge in 
the field relevant to the present disclosure as it existed before the priority date of each claim 
of this application.  
The method described herein is for treating pulmonary arterial hypertension in 
patients that have defect in BMPR2 (bone morphogenetic protein receptor 2) signaling.  
Pulmonary arterial hypertension (PAH) is a progressive lung disorder which, untreated, often 
leads to death on average within a few years after being diagnosed. An increasing 
constriction of the pulmonary circulation leads to increased stress on the right heart, which 
can develop into right heart failure. By definition, the mean pulmonary arterial pressure 
(mPAP) in a case of chronic pulmonary hypertension is >25 mmHg at rest or >30 mmHg 
during exertion, where the normal values are <20 mmHg. The pathophysiology of 

WO 2012/151153 PCT/US2012/035793 
pulmonary arterial hypertension is characterized by vasoconstriction and remodeling of the 
pulmonary vessels. In chronic PAH there is neomuscularization of initially unmuscularized 
pulmonary vessels, and the vascular muscles of the already muscularized vessels increase in 
circumference. This increasing obliteration of the pulmonary circulation results in 
progressive stress on the right heart, which leads to a reduced output from the right heart and 
eventually ends in right heart failure (Humbert et al., J. Am. Coll. Cardiol. 2004, 43, 13S
Defects in BMPR2 signaling may, for example, be caused by a mutation in BMPR2 
(see accession no. 600799 in NCBI's OMIM database), a mutation in ALKi (the activin A 
receptor; see accession no. 601284 in NCBI's OMIM database) or a mutation in endoglin 
(see accession no. 131195 in NCBJ's OMIM database). A defect in BMPR2 signaling can be 
detected, for example, by measuring the expression of Idl (inhibitor of differentiation 1) 
mRNA or protein, which is a well-known downstream read-out for BMPR2 signaling.  
Decreased BMPR2 signaling decreases the amount of Idlin pulmonary artery smooth muscle 
is cells. In particular cases, the pulmonary arterial hypertension may be hereditary pulmonary 
arterial hypertension. In particular embodiments, a subject may be pre-screened to identify 
whether they have a mutation that effects BMPR2 signaling, or they may be assayed to 
determine if they have abnormal expression of Idl.  
FK-506 (also known Tacrolimus or Fujimycin) is an immunosuppressive drug that is 
mainly used after allogeneic organ transplant to reduce the activity of the patient's immune 
system and so lower the risk of organ rejection. It is also used for the treatment of severe 
atopic dermatitis (eczema), severe refractory uveitis after bone marrow transplants, and the 
skin condition vitiligo. FK-506 is a 23-membered macrolide lactone discovered in 1984.  
In T-cells, activation of the T-cell receptor normally increases intracellular calcium, 
which acts via calmodulin to activate calcineurin. Calcineurin then dephosphorylates the 
transcription factor NF-AT (nuclear factor of activated T-cells), which moves to the nucleus 
of the T-cell and increases the activity of genes coding for IL-2 and related cytokines. FK
506 prevents the dephosphorylation of NF-AT. Specifically, FK-506 reduces peptidyl-prolyl 
isomerase activity by binding to the immunophilin FKBP12 (FK506 binding protein) 
creating a new complex. This FKBP12-FK506 complex interacts with and inhibits 
calcineurin thus inhibiting both T-lymphocyte signal transduction and IL-2 transcription.  
In some embodiments, the FK506 is administered at a dose and regimen that provides an 
FK506 serum concentration that is much lower than the FK506 serum concentration 
commonly used in immunosuppressive applications (which is typically 5-15 ng/ml). For 
WO 2012/151153 PCT/US2012/035793 
example, in certain embodiments of the instant method, the FK506 is administered at a dose 
and regimen that provides an FK506 serum concentration of as 0.05 ng/ml to 1 ng/ml, e.g., 
0.1 ng/ml to 0.5 ng/ml, 0.15 ng/ml to 0.3 ng/ml or e.g. 0.1-0.2 ng/ml. In part because FK
506 is metabolized by the cytochrome P450 system, the exact dosing may vary between 
patients. The FK506 may be administered once a day or more, e.g., twice per day. In 
immunosuppressive applications, FK506 is normally given twice daily with the goal to reach 
FK-506 serum levels of 5 - 15 ng/ml. The treatment is started at 0.5 mg twice daily and then 
up-titrated according to the measured FK506 serum level. In some cases a dosing of 0.075 
mg/kg/day is recommended to reach a serum levels of 5-10 ng/ml. In some embodiments of 
the instant method, the goal is to reach a serum level of about 0.2 ng/ml, which is about 1/20 
of the immunosuppressive serum level. In this case, an initial dose of 0.001 mg/kg day to 
0.01 mg/ kg day (e.g., .002 mg kg / day to 0.05 mg/ kg/ day may be sufficient, and the does 
can be up-titrated according to the measured FK506 serum level. The subject may be any 
mammal, e.g., a human, rat, or mouse, for example. In particular cases, the FK506 may reach 
is a serum concentration as low as 0.1-0.2 ng/ml (e.g., 0.10 to 0.12, 0.12 to 0.14, 0.14 to 0.16, 
0.16 to 0.18 or 0.18 to 0.20, however serum a concentration in the range of 0.2 to 2 ng/ml, 
e.g., 0.2, 0.5, 1 and 2 ng/ml may be acceptable. In particular cases, the FK506 may reach a 
serum concentration of < 1.0, 1.5-2.5, or 3-5ng/ml.  
The FK506 may be administered alone or in combination with other active 
compounds that treat or prevent PAH. The other active compound may be administered at a 
different time or at the same time as the FK506 and in certain embodiments the FK506 and 
the other active compound may be present in the same formulation, or as separate 
formulations in the same kit. Exemplary other active compounds that treat PAH include, 
e.g., prostacyclin analogues, endothelin receptor antagonists, phosphodiesterase-5 inhibitors, 
high-dose calcium channel blockers, anticoagulants, diuretics or antiproliferative agents. In 
particular cases, the other active compound may be, for example, Isordil (isosorbide 
dinitrate), Revatio (sildenafil), Tracleer (bosentan), Letairis (ambrisentan), Flolan 
(epoprostenol), Adcirca (tadalafil), Remodulin (treprostinil) Ventavis (iloprost), Tyvaso 
(treprostinil), Dilatrate-SR (isosorbide dinitrate), Isordil Titradose (isosorbide dinitrate), 
IsoDitrate (isosorbide dinitrate) or Isochron (isosorbide dinitrate).  
Administration of FK506 to a subject may decrease pulmonary arterial pressure by 
about at least 1 mm Hg, e.g., at least 2 mm Hg, at least 3 mm Hg, at least 4 mm Hg, at least 5 
mm Hg or at least 10 mm Hg or more, thereby returning the pulmonary arterial pressure to a 
level that may be considered normal for the subject.  
WO 2012/151153 PCT/US2012/035793 
In general terms, the FK506 may be administered to the subject in the instant method 
in a similar way to how FK506 is administered in immunosuppressive applications. For 
example, the FK506 may be present in a pharmaceutically acceptable excipient, and it may 
be administered intravenously. Alternatively, it may be administered orally.  
Because the FK506 is being administered at a lower dose, its usual side effects may 
be decreased. Typical side effects include infection, cardiac damage, hypertension, blurred 
vision, liver and kidney problems (tacrolimus nephrotoxicity), hyperkalemia, 
hypomagnesemia, hyperglycemia, diabetes mellitus, itching, lung damage (sirolimus also 
causes lung damage), and various neuropsychiatric problems such as loss of appetite, 
insomnia, Posterior reversible encephalopathy syndrome, confusion, weakness, depression, 
cramps, neuropathy, seizures, tremors, and catatonia.  
Pharmaceutical Compositions 
A pharmaceutical composition comprising a subject compound may be administered 
to a patient alone, or in combination with other supplementary active agents. The 
pharmaceutical compositions may be manufactured using any of a variety of processes, 
including, without limitation, conventional mixing, dissolving, granulating, dragee-making, 
levigating, emulsifying, encapsulating, entrapping, and lyophilizing. The pharmaceutical 
composition can take any of a variety of forms including, without limitation, a sterile 
solution, suspension, emulsion, lyophilisate, tablet, pill, pellet, capsule, powder, syrup, elixir 
or any other dosage form suitable for administration.  
A subject compound may be administered to the host using any convenient means 
capable of resulting in the desired reduction in disease condition or symptom. Thus, a 
subject compound can be incorporated into a variety of formulations for therapeutic 
administration. More particularly, a subject compound can be formulated into 
pharmaceutical compositions by combination with appropriate pharmaceutically acceptable 
carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or 
gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, 
suppositories, injections, inhalants and aerosols.  
Formulations for pharmaceutical compositions are well known in the art. For 
example, Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., 
Easton, Pa., 19th Edition, 1995, describes exemplary formulations (and components thereof) 
suitable for pharmaceutical delivery of disclosed compounds. Pharmaceutical compositions 
comprising at least one of the subject compounds can be formulated for use in human or 
veterinary medicine. Particular formulations of a disclosed pharmaceutical composition may 
WO 2012/151153 PCT/US2012/035793 
depend, for example, on the mode of administration and/or on the location of the infection to 
be treated. In some embodiments, formulations include a pharmaceutically acceptable 
carrier in addition to at least one active ingredient, such as a subject compound. In other 
embodiments, other medicinal or pharmaceutical agents, for example, with similar, related or 
complementary effects on the affliction being treated can also be included as active 
ingredients in a pharmaceutical composition.  
Pharmaceutically acceptable carriers useful for the disclosed methods and 
compositions are conventional in the art. The nature of a pharmaceutical carrier will depend 
on the particular mode of administration being employed. For example, parenteral 
formulations usually comprise injectable fluids that include pharmaceutically and 
physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, 
aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, 
pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, 
pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to 
biologically neutral carriers, pharmaceutical compositions to be administered can optionally 
contain minor amounts of non-toxic auxiliary substances (e.g., excipients), such as wetting 
or emulsifying agents, preservatives, and pH buffering agents and the like; for example, 
sodium acetate or sorbitan monolaurate. Other non-limiting excipients include, nonionic 
solubilizers, such as cremophor, or proteins, such as human serum albumin or plasma 
Some examples of materials which can serve as pharmaceutically-acceptable carriers 
include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch 
and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, 
ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; 
(8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, 
cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, 
such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene 
glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, 
such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free 
water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered 
solutions; (21) polyesters, polycarbonates and/or polyanhydrides; and (22) other non-toxic 
compatible substances employed in pharmaceutical formulations.  
The disclosed pharmaceutical compositions may be formulated as a pharmaceutically 
acceptable salt of a disclosed compound. Pharmaceutically acceptable salts are non-toxic 
WO 2012/151153 PCT/US2012/035793 
salts of a free base form of a compound that possesses the desired pharmacological activity 
of the free base. These salts may be derived from inorganic or organic acids. Non-limiting 
examples of suitable inorganic acids are hydrochloric acid, nitric acid, hydrobromic acid, 
sulfuric acid, hydroiodic acid, and phosphoric acid. Non-limiting examples of suitable 
organic acids are acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, malonic 
acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic 
acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p
toluenesulfonic acid, methyl sulfonic acid, salicylic acid, formic acid, trichloroacetic acid, 
trifluoroacetic acid, gluconic acid, asparagic acid, aspartic acid, benzenesulfonic acid, p
toluenesulfonic acid, naphthalenesulfonic acid, and the like. Lists of other suitable 
pharmaceutically acceptable salts are found in Remington's Pharmaceutical Sciences, 17th 
Edition, Mack Publishing Company, Easton, Pa., 1985. A pharmaceutically acceptable salt 
may also serve to adjust the osmotic pressure of the composition.  
A subject compound can be used alone or in combination with appropriate additives 
is to make tablets, powders, granules or capsules, for example, with conventional additives, 
such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline 
cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as 
corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or 
magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, 
preservatives and flavoring agents. Such preparations can be used for oral administration.  
A subject compound can be formulated into preparations for injection by dissolving, 
suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or 
other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or 
propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic 
agents, suspending agents, emulsifying agents, stabilizers and preservatives. The 
preparation may also be emulsified or the active ingredient encapsulated in liposome 
vehicles. Formulations suitable for injection can be administered by an intravitreal, 
intraocular, intramuscular, subcutaneous, sublingual, or other route of administration, e.g., 
injection into the gum tissue or other oral tissue. Such formulations are also suitable for 
topical administration.  
In some embodiments, a subject compound can be delivered by a continuous delivery 
system. The term "continuous delivery system" is used interchangeably herein with 
"controlled delivery system" and encompasses continuous (e.g., controlled) delivery devices 
WO 2012/151153 PCT/US2012/035793 
(e.g., pumps) in combination with catheters, injection devices, and the like, a wide variety of 
which are known in the art.  
A subject compound can be utilized in aerosol formulation to be administered via 
inhalation. A subject compound can be formulated into pressurized acceptable propellants 
s such as dichlorodifluoromethane, propane, nitrogen and the like.  
Furthermore, a subject compound can be made into suppositories by mixing with a 
variety of bases such as emulsifying bases or water-soluble bases. A subject compound can 
be administered rectally via a suppository. The suppository can include vehicles such as 
cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are 
solidified at room temperature.  
The term "unit dosage form," as used herein, refers to physically discrete units 
suitable as unitary dosages for human and animal subjects, each unit containing a 
predetermined quantity of a subject compound calculated in an amount sufficient to produce 
the desired effect in association with a pharmaceutically acceptable diluent, carrier or 
vehicle. The specifications for a subject compound depend on the particular compound 
employed and the effect to be achieved, and the pharmacodynamics associated with each 
compound in the host.  
The dosage form of a disclosed pharmaceutical composition will be determined by 
the mode of administration chosen. For example, in addition to injectable fluids, topical or 
oral dosage forms may be employed. Topical preparations may include eye drops, 
ointments, sprays and the like. Oral formulations may be liquid (e.g., syrups, solutions or 
suspensions), or solid (e.g., powders, pills, tablets, or capsules). Methods of preparing such 
dosage forms are known, or will be apparent, to those skilled in the art.  
Certain embodiments of the pharmaceutical compositions comprising a subject 
compound may be formulated in unit dosage form suitable for individual administration of 
precise dosages. The amount of active ingredient administered will depend on the subject 
being treated, the severity of the affliction, and the manner of administration, and is known 
to those skilled in the art. Within these bounds, the formulation to be administered will 
contain a quantity of the extracts or compounds disclosed herein in an amount effective to 
achieve the desired effect in the subject being treated.  
Each therapeutic compound can independently be in any dosage form, such as those 
described herein, and can also be administered in various ways, as described herein. For 
example, the compounds may be formulated together, in a single dosage unit (that is, 
combined together in one form such as capsule, tablet, powder, or liquid, etc.) as a 
WO 2012/151153 PCT/US2012/035793 
combination product. Alternatively, when not formulated together in a single dosage unit, 
an individual subject compound may be administered at the same time as another therapeutic 
compound or sequentially, in any order thereof.  
Methods of Administration 
The route of administration may be selected according to a variety of factors 
including, but not necessarily limited to, the condition to be treated, the formulation and/or 
device used, the patient to be treated, and the like. Routes of administration useful in the 
disclosed methods include but are not limited to oral and parenteral routes, such as 
intravenous (iv), intraperitoneal (ip), rectal, topical, ophthalmic, nasal, and transdermal.  
Formulations for these dosage forms are described herein.  
An effective amount of a subject compound will depend, at least, on the particular 
method of use, the subject being treated, the severity of the affliction, and the manner of 
administration of the therapeutic composition. A "therapeutically effective amount" of a 
composition is a quantity of a specified compound sufficient to achieve a desired effect in a 
is subject (host) being treated.  
Therapeutically effective doses (or growth inhibitory amounts) of a subject 
compound or pharmaceutical composition can be determined by one of skill in the art, with a 
goal of achieving local (e.g., tissue) concentrations that are at least as high as the IC50 of an 
applicable compound disclosed herein.  
The specific dose level and frequency of dosage for any particular subject may be 
varied and will depend upon a variety of factors, including the activity of the subject 
compound, the metabolic stability and length of action of that compound, the age, body 
weight, general health, sex and diet of the subject, mode and time of administration, rate of 
excretion, drug combination, and severity of the condition of the host undergoing therapy.  
In order to further illustrate the present invention, the following specific examples are 
given with the understanding that they are being offered to illustrate the present invention 
and should not be construed in any way as limiting its scope.  
Mouse study 
A loss of function mutation in bone morphogenetic protein (BMP) receptor II 
(BMPRII) is present in >80% of familial and -20% of sporadic idiopathic (I) PAH 
WO 2012/151153 PCT/US2012/035793 
(Machado et al. Hum Mutat 2006, 27:121-32). Even patients with IPAH without a BMPRII 
mutation or with other causes of PAH have reduced expression of BMPRII, reinforcing the 
importance of BMPRII signaling in the pathogenesis of PAH (Humbert M et al. Eur Respir J 
2002, 20:518-23). Furthermore BMPR2 receptor gene therapy attenuates experimental 
s hypoxic pulmonary hypertension in rats (Reynolds et al. Am J Physiol Lung Cell Mol 
Physiol 2007). Therefore increasing BMPRII signaling in patients with pulmonary arterial 
hypertension might prevent or reverse disease.  
3600 FDA approved drugs and bioactive compounds were screened for their ability 
to activate BMP signaling, using a C2C12 mouse myoblast cell line stably transfected with a 
reporter plasmid expressing a BMP response element (BRE) from the Idl promoter fused to 
the luciferase-gene (BRE-luc). Whether the best qHTS-BMPRII activator can induce Smad 
phosphorylation (phospho), Idl expression and promote PAEC survival and tube formation 
was determined using BMP4 as a positive control. Whether the qHTS-BMPRII-activator 
is would prevent PAH in mice with a conditional deletion in BMPRII in ECs (BMPRII-SCL
CreERT) that develop exaggerated PAH after 3 weeks of hypoxia (10% 02) was determined.  
In order to assess whether the BMPRII activator could also reverse PAH, we used 2 models 
of severe experimental PH in rats: 1. Monocrotaline induced pulmonary hypertension with 
development of severe medial hypertrophy of the pulmonary arteries 3 weeks after injection.  
2. SUGEN (VEGF-Receptor blocker) and 3-week chronic hypoxia induced pulmonary 
hypertension with development of neointima formation in pulmonary arteries 8 weeks after 
initiation of the stimulus. Both groups were treated with FK-506 for 3 weeks via sc osmotic 
pump (0.05mg/kg/d) after PAH and remodeling of the pulmonary arteries was established.  
The serum level of FK-506 in mice and rats was aimed to be 0.2ng/ml.  
FK-506, an agent that can induce BMPRIA phosphorylation, was the main activator 
of Idl expression. FK-506, at a dose of 15 ng/ml, the therapeutic serum level used to induce 
immunosuppression, and at a much lower dose of 0.2ng/ml increased Idl protein expression 
1h following stimulation, in a manner comparable to BMP4 (10 ng/ml) (n=3, p<0.06). This 
was preceded by phospho-Smad 1/5/8 at 15 min, similar to BMP4 (n=3, p<0.001). FK-506 
induced p-Smad 1/5/8 and Idl expression in PAECs harvested from six different IPAH 
patients at the time of transplant, including 3/6 patients that did not respond to BMP4. Both 
BMP4 and FK-506 improved survival of PAECs (n=5, p<0.001) and induced tube formation 
in an angiogenesis assay (n=3, p<0.01). A 3-week preventive treatment with FK-506 
WO 2012/151153 PCT/US2012/035793 
(0.05mg/kg/d) (serum levels 0.2ng/ml) in mice with a conditional deletion in BMPRII in 
ECs exposed to 3 weeks of hypoxia prevented the development of PAH and right ventricular 
hypertrophy (RVH); RV systolic pressure: 32±0.9 vs 21±2.3 mmHg, p<0.001; RVH: 
36.2±2.5 vs 26.9±4.5, p<0.01, both n=5. To test whether FK-506 could also reverse PAH, 
we induced PAH in rats with monocrotaline (60mg once s.c) and began treatment with FK
506 3 weeks after injection, a time when PAH was established (RVSP 50.8 ± 2.7mmHg, 
n=7). The survival after a 3-week treatment with FK-506 did not differ in the FK-506 (57%) 
compared to the vehicle group (66%), yet of those that that survived the PAH was 
significantly reduced after treatment with FK-506 compared to vehicle treated animals 
(RVSP 39.5 ± 4.7 vs 68.6 ± 4.2 mmHg, n=14).  
It was determined that the combined stimulus of SUGEN (20mg/kg s.c) and 3-weeks 
of chronic hypoxia induced PAH in rats when rats were returned to RA and left for another 5 
weeks (RVSP 55.1±10.7 vs control 25.1±0.5mmHg, RVH 0.24±0.005 vs 0.44±0.07, n=4, 
p<0.05) but that a 3-week sc treatment of FK-506 at the time of established PAH could 
is prevent progression and induce regression of PAH in FK-506 treated vs vehicle treated 
animals (RVSP 66.5±4.lmmHg vs 39.5±0.6 mmHg, RVH 0.49±0.07 vs 0.34±0.02, n=4, 
p<0.05). Neointima formation in small pulmonary arteries (alveolar wall and alveolar duct 
vessels) per total vessel number decreased from 61.2±6.1% to 16.2±5.8% (n=4, p<0.01). At 
the low dose of FK-506 of 0.2 ng/ml no effect on total or differential WBC count was 
observed, nor was an immunosuppressive effect of decreased nuclear NFATc2 measured.  
FK-506 (Tacrolimus) was identified in a quantitative high throughput screen (qHTS) 
of FDA approved drugs and bioactive compounds as a drug that activates BMPRII signaling, 
restores normal function of pulmonary artery endothelial cells (PAECs), prevents and 
reverses experimental PAH in mice and rats.  
Clinical Study 
Patients are invited to participate in this study because they have pulmonary 
hypertension (PH) and are currently treated with one or multiple drugs for PH such as PDE-5 
inhibitors (sildenafil, tadalafil), prostacyclins (Flolan, Remodulin, Iloprost) and/or the 
endothelin antagonist Ambrisentan. While all these drugs are effective as vasodilators, new 
medications are sought that could reverse the pathological remodeling of the pulmonary 
arteries. Whether subjects have a familial form of pulmonary hypertension or not, it is 
WO 2012/151153 PCT/US2012/035793 
known that a certain pathway (BMPR2) is impaired in PH. Studies have shown that the 
immunosuppressive drug FK-506 (Tacrolimus) activates the BMPR2 pathway and prevents 
and reverses pulmonary hypertension in experimental pulmonary hypertension.  
This study is open to male or female subjects, 18 - 70 years of age, with PH. If a 
patient agrees to participate in this study, the patient will be one of 40 subjects participating 
in the study.  
If a patient agrees to participate and the patent qualifies, the patient will be allocated 
to the study drug through a process called randomization. Randomization means that the 
study drug that the patient will receive is selected by chance (like the flip of a coin). The 
study drug options for this study are placebo, and 3 different doses of FK-506 (blood level < 
1.0, 1.5-2.5, and 3-5ng/ml; as a reference: the immunosuppressive dose is 5-15 ng/ml). The 
study drug will be added to the patient's baseline PH therapy. The randomization for this 
study is 1:3 which means patients have a chance of 75% of receiving treatment with FK-506.  
The purpose of this study is to confirm that adding FK-506 to a PH treatment at a 
is dose below the normal dose that is used for immunosuppression is safe and whether it will 
improve pulmonary hypertension. Heart function will be assessed by echo, 6-min walk and 
the biomarker NT-proBNP.  
Study medication 
FK-506 (Tacrolimus) is an FDA approved immunosuppressive drug used in organ 
transplantation as well as in autoimmune diseases. As the metabolism of FK-506 differs in 
patients quite widely, therapy is directed by measuring drug levels in whole blood. The 
blood will be drawn shipped to a testing lab to measure FK-506 levels. The goal 
immunosuppressive doses are 5-15 ng/ml. In this study we aim for much lower doses (see 
above). Patients will receive the study drug for the duration of study. The drug will be 
delivered in a prepared bottle, which allows monitoring of drug intake. This device is called 
a Medication Event Monitoring System (MEMS) and for it to monitor drug intake properly.  
Patients should always take out one tablet at a time from the bottle.  
Participation in the study lasts for approximately 16 weeks. During this time, patients 
will be required to visit the clinic approximately 4-5 times.  
Study procedures 
If a patient agrees to take part in this study, they will first sign this consent form.  
After the patients have signed, dated and received a copy of this consent form, they will have 
the study screening visit to ensure the patient is eligible to take part in this study. Previous 
WO 2012/151153 PCT/US2012/035793 
test results (echocardiogram, physical examination, pulmonary function tests, Right Heart 
Catheterization (RHC) may also be used to determine patient eligibility.  

WO 2012/151153 PCT/US2012/035793 
What is claimed is: 
1. A method of reducing pulmonary arterial hypertension in a mammal, comprising: 
administering FK506 to a mammal having pulmonary arterial hypertension 
associated with defective BMPR2 signaling, wherein said FK506 is administered at a dosage 
that is below 5ng/ml and is sufficient to reduce blood pressure in the pulmonary artery of 
said mammal 
2. The method of claim 1, wherein said mammal is human.  
3. The method of claim 1, wherein said mammal has reduced expression of BMPR2 
ALKi or endoglin.  
4. The method of claim 1, wherein said mammal has hereditary pulmonary arterial 
hypertension caused by a mutation in BMPR2.  
5. The method of claim 1, wherein said mammal has hereditary pulmonary arterial 
hypertension caused by a mutation in ALKi.  
6. The method of claim 1, wherein said mammal has hereditary pulmonary arterial 
hypertension caused by a mutation in endoglin.  
7. The method of claim 1, wherein said dosage provides an FK506 serum concentration 
of 0.1 - 0.2 ng/ml.  
8. The method of claim 1, wherein said administering is oral.  

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