CA2971071C - Use of milrinone and derivatives thereof in the treatment of heart failure with preserved ejection fraction (hfpef) - Google Patents

Abstract

The present invention relates to the use of a sustained-delivery formulation of l,2- dihydro-3-cyano-6-methyl-5-(4-pyridinyl)-2(1H)-pyridinone (milrinone) or a pharmaceutically acceptable salt thereof in treating a patient having heart failure with preserved ejection fraction (HFpEF).

Description

TITLE OF THE INVENTION "USE OF MILRINONE AND DERIVATIVES THEREOF IN THE TREATMENT OF HEART FAILURE WITH PRESERVED EJ.ECTION FRACT.ION (HFpE.Fr [00011 This application claims the benefit of Australian Provisional Patent 5 AppHeation No. 2014905194, filed on 22.

December 2014.

FIELD OF THE INVENTION

[0002] The pres.ent. .invention relates to .metho:ds of treating subjects having heart failure with preserved ejection fraction (HFpEF) with a sustai.ned~delivery formulation of 10 .cardioto,nic 5-(pyridinyl)..:2(1H)-pyridinone compoi;Jnds, BACKGROUND OF THE INVENTION

[0003] BibliQgraphic details of publications referred to in this specification are collected alphabetically at the end offhe description. [0.004] The re£erenoe in this :specification to fill;\' ·prior pqblic~tfon. (or information 15 derived from. it), or to any matter which is known, is not, and .should not be taken as an ack:now]oogment or admission or any form of suggestion that that prior publication (or information. derived .from it) or known. matterfonns part of the common ge:n.eral knowledge in the field of endeavor to which this specification relates,

[0005] Heart faUure (HF) is a. complex: disease state broadiy defined by an .in.ability 20 •O•f the heart to pump sufficiently to cope with its venous return and/or to deliver sufficient output to meet the m.etabolic demand& of the body.

Scvcr.e heart failure is an i:ncrcasfo.gl y commo.n:1 life-:threatoo.ing oardiovas.cular diimrder~ characterized by marked disability, frequent ho.spitalization and high mortality, HF is incre!;tsingly prevalent in o1derindivi.duaJs ( up to l 0% of the population) and it has become the most common cause for hospitalization in 25 peopl,e >65 :Yrs. HF is :a leading cause or c.ontributot to hospi:taHzation and therefore i.s emerging as a substantial contributor to healthcare Sp.ending.

The parti.cular cHnical manifestations of HF are determined by the underlying cause of the heart failure. (00061 The term. heart failure (HF) refers broadly to a pathopby1sioll)gi'c disorder h1 which cardiac performance is incapable of delivering sufficient blood to meet metabolic 30 demand (e.g. duri,t\S p:h:y.s:ioal activity or in severe .oases at rest)~. or to accommodate venous Date Rer;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 return. A range of further sub-classifications can then be applied, .however in the commonest clinical paradigm HF is considered according to symptoms of reduced cardiac output leading to easy fatigue and organ dysfunction (e.g. terrnJ), ancl to syrnptoms .related to congestion .either in the .lungs (causing b.reathlessness) or peripherally (le!iding to swelling of the lower 5 limbs and abdomert). HF is the most common chronic cardiovascular d1sorder.

In the US approximately 5,000,000 patients have heart failure and of these up to 15-20% have the most advanced forms.

It is particularly prevalenHn older individuals .(up to 10% of the general population >70 yrs) }!.Ild it has become the most common cause for hospitalization in pee>ple >65yn~.

Recurrent hospitalization is frequent? with 25% of patients re-:admitted within one 10 month .of an admissi.on and >50% will be. re-admitted within 6 months.

The average US cost of an HF admission is >$20.~000, with an average length of stay of fourto five days. [0007J Many patients suffering from HF have impai.red le.ft ventri.cu]ar (LV) myocardial function.

However, HF may be associated with a wide variety of L V dysfunctkms ..

These range from patients with normal LV size and preserved ejectionfraction 15 to. those wi.th severe dilation of LV and/or markedly reduced ejection fraoti.on (Yan.cy et al).

[0008] Ejection fraction (EF) is considered an important classification in heart failure patients because of patient demographics, comorbid conditions; prognosis and response to therapies and the patients for clinical trials are often selected on the basis of EF (Yanqy et.al}

[0009] HF with reduced EF. (HFrEF) has an EF or s 40%, Randomised controlled therapel.ltic trials mainly enroll patients with HFrEF and it is only these patients that have . . efficacious therapies to date (Yancy etctl),

[0010] HF with preserved EF (HFpEF) refers to patients having an EF of> 40%, with those having an EF from 40to 49%being considered borderline HFpEF.

Several criteria 25 have been proposed to define Ol' diagnose HFpEF including: L clinical signs and symptoms ofHF; ii. evidetme of preserved or normal LVEF; and iii. evidence of L V diastolic dysfunction that can be determined by Doppler echocardiography or cardiac :c:atheteriz.ation.

[0011] At present, in contrast to HFrEF there are ilo efficacious therapies for HFpEF (Yancy et al, Loffredo et al).

Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820

[0012] For patients with advanced HFrEF that .requir:e. hospitalization, the use of positive inotropes such as intravenous dobutamine and milrinone, to stimulate cardiac contraction is. CQmmon.

Recently an oral controlled-release formulation for treating such patfonts has been developed (WO 20.13/023250).

Furthermore, the use of long-term inotropii:: 5 support for ''rto option" patients, that is, those patients not suitable fot heart transplantation or artificial heart transplant, has recently been advocated by the American Heart Association Guidelines for treatment ofHF:rEF,

[0013] A number of therapies for HFpEF have been propo®d (Kamajda and Lam, Sharm.a. and Kass) includin~ ~-blockers and ¢aJch.un channel blocker$, ACE inhi):,itqrs and 10 angiotensin receptor blockers and digoxin, each withlittle or no conclusive benefit. A recent study with spironolactone (Edelmann et al), an aldosterone receptor blocker improved left veniricular diastolic function but did not affect maximal exercise capacity, patient symptoms or quality of life in HFpEF patients.

Another recent study with the phosphosiesterase-5 inhibitor sildepafil (RELAX study) did not result in improvement i11 exercise .. capacit)t' or 15 clinical status in HFpEF patients (Redfield et al). A.clinical trial with Ranolazine, a s.elective inhibitor of late sodium current, also did not result in a change in echocardiographic parameters ,or exercise performance in HFpEF patients (Komajda and Lam).

Some new approaches have had come promising effects in preclinical or early clinical studies, including neprilysin inhibitors, sc:,luble gμanylate cyclase stimulators and advanced glycation end 20 products, b1,1t have not been yet fully investigated:

[0014] Inotropes have not been investigated in HFpEF patients because contractile function.is generally thought to be normal or only mildly reduced.

Hence those treating heart failure patients would not recommend .the use of drugs such as milrinpne to treat HFpEF patients bas.ed on present literature.

[0015] There is a need for therapies that improve one or more of the clinical symptoms ofHFpEF, Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 SUMMARY OF THE INVENTION

[0016] The present invention is predicated, at least in part, by the discovery that controlled-telease Milrinone, is effective in improvin:g the clinical symptoms of patients with HFpEF. 5

[0017] In a first aspect of the present invention, there is provided a method of treating a patient having heart failure with preserved ejection fraction (HFpEF) comprising administering to the patient a sustained-'(jcelivery formulation of a 5-(pyridiny1)~2(1H)pyridinone compound of formula, (I): (I) 10 wherein R1 is hydrogen, _;C 1-C6a1kyl or -C1-C6alkyl-OH; R2 is -G1-C6a]kyl; R3 is hydrogen, -Nlf2, -CN, -C(O)NHz, haJ'o, ".NH(C1.C6alkyl), -N(C1.C<lalkyl)2, -NH(COC1-C6aJkyl), -CO2H or -CO2C1-C6alkyl; and PY is 4~, 3- or 2-pyridinyl optionally substituted with one or two. C1-C6allwl 15 groups; or a pharmaceutically acceptable salt thereof; wherein the formulation permits delivery of the compound of formula rn in .an amount to achiev~ steady state plasma levels effective to allevjate the $ymptoms of fJFpEF; wherejn delivery of the compound offornmla (I) is in the range of between 0.1 μglkg body weight per 20 minute to 20 μg/kg body weight per minute,

[0018] In another aspect, the present invention further provides a sustained-delivery formulation ofa.5-(pyricjiny1}2(1H)-pyridinone compound of formula (I): R3 (I) Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 wherein R1 is hydrogen, -C1-C6alkyl or -C1-C6alkyl-OH; R2 is -C1-C6alkyl; PCTiAU2015/050820 Ra is hydrpgen, -NH2, -CN, -C(O)NH2, halo, -NJ-I(C1.C6alkyl), -N(C1 .. C6alkyl)2, -NH(COC1-Ce;alkyl)1 -CO2:H or -CO2C1-Ce;alkyl; and PY is 4-, 3- ot 2-pyridinyl optionally substituted with one ot two Ci-C6alkyl groups; or ·a pharmaceutically acceptable salt thereof; wherein the formulation p.ermi ts deli very of the compound of formula (I) in an amount to ach.ieve steady state. plasma levels effective to. al1eviate the ,symptoms <1r HFpEF; 10 wherein .delivery of the compound of fontrnla (I} is in the range ofbetween OJ μg/kg body weight per minute to 20 μg/kg body weight per minute for use in: the treatment of heart failure with preserved ejection fraction (HFpEF),

[0019] In a further aspect, the presen:t invention also provides use of a sustaineddelivery form.ulation.ofa 5-(pyridinyl)-2(lH)-pyridinone compound offormuJa (I): (I) wherein R1 is hydrogen, -C1-C6a1kyl .or -C1-C6a1ky1-OH; R2 is -C1-C!'ialkyl; R:, is hydrogen, .:Nff2, -CN, -C(O)NH2, halo, -NH(C1.C6alkyl), .:N(C1.C6alkyl}t, -NH(COC1-Cr;alkyl), -CO2Hor -CO2C1-C5alkyl; and PY is 4-, 3- or 2-:Pyrldinyl optionally substituted with one or two C1-C6alkyl .groups; or a pharmaceutically acceptable salt thereof; wherein the formulation permits deliv.ery ofthe compound of formula (I) in an amount to achieve steady state plasma levels effective to .alleviate the symptoms of HFpEF; 25 wherein delivery of th.e compound of fon-uula (I) is in the range of between Q, 1 μg/kg body weight per minute to 20 μg,lkg body weight per minute in the manufacture of a medicament for use in the treatment of heart failure with preserved ejection fraction (HFpEF), Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820

[0020] In a yet further aspect the invention further provides the use of of a sustained-delivery fonnulation of a 5-(pyridinyl)-Z(IH)-pyridinone compound of fonnula (I): py, R3 (I) wherein R1 is hydrogen, -C1-C6alkyl or -C1-C6alkyl-OH; 5 R2 is -C1-C6alkyl; R3 is hydrogen, -NH2, -CN, -C(O)NH2, halo, -NH(C1.C6alkyl), -N(C\~C6alkyl)2, ~NH(COC1-C6alkyl), -CO2Hor -CO2C1-Csalkyl; and PY is 4-, 3- or 2-pyridinyl optionally substituted with one or two C1-C6alkyl groups; 1 o ot a pharmaceutically acceptable salt thereof~ wherein the formulation permits deliv.ecy of the compound offormula(I) ili an amount to achieve steady state plasma levels effective to alleviate the symptoms of HFpEF; wherein delivery of the compound of formula (I) is in the range of between OJ μg/kg body weight per minute fo 20 μg/kg body weight per minute, 15 in the treatment of heart faihire with preserved ejection fraction (HFpEF),

[0021] In yet another aspect the invention further provides a method of preparing a .sustained-delivery formulation of a :S-(pyridinyl)-2(lH)-pyridinone compound of formula (I): R3 (I) 20 Wherein R1 is hydrogen, -C1-C6alkyl or -C1-C6alkyl-OH; R2 is ...:Ci.-C6alkyl; R3 is hydrogen, -NH2, -CN, -C(O)NH2, halo, . .,Nff(Ci.Ctialky1), -N(C1.C6a.lkyl):i, -NH(COC1-C5alkyl); ~C02H or -C02C1-C6alkyl; and Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 groups; PY is 4 ... ~ 3- or 2-pyridinyl optionally substituted with one or two C1-C6alkyl or.a pharmaceutic.ally acceptable salt thereof; wherein theJortnulation permits deli.very of the compound offommla (I) in an amoJJnt to achieve steady state plasma levels effective to alleviate the symptoms of HFpEF; wherein delivery of the .compound of formula (I) is in the range of between 0.1 μg/kg body weight per minute to 20 μg/kg body weight per minute, for the treatment of heart failure with preserved ejection fraction (HFpEF) comprising formulating a S-(pyri.dinyl)-:2(1EI)-pyridinone compound of 10 formula (I) as hereinbefote defined with one ot mote po1ymers to provide an extended release matrix formulation; and testing to .confirm that the formulation provides the desired release profile forthe compound of formula (I). (0022] In some embodiments, the sustained:--d:elivery formulation is .a formulation 15 suitable for intravenous administration.

In other embodiments, the sustained delivery formulation is an oraLcontrolled-release formulation.

[0023] In some embodiments~the patienthas an ejection fraction of> 50%.

[0024] In one embodiment of the invention the compound of formula (I) is l,2- dihydro-3-cyano-6-methyl-5-( 4-pytidinyl )-2(1H)-pyridinone (Milrinone).

DETAILED DESCRIPTION OF THE INVENTION J. l)ef!i,niti:ons

[0025] Unless defined otherwise, all technical. and sci.enti:l:ic terms used hereillhave the same meanin$ as. conunonly u.nderstood by those of' ordirtary skill in the art to wbii;;h the 25 invention belongs.

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present. invention, preferred methods and materials .are described., For the purposes of the present invention; th(;l following terms are defined below. (0026] The articles "a" and ''an" are used herein to refer to. one or to more than one 30 (i.e. to at least one) of the grammatical object ofthe article.

By way of example, "an element?' 1ueans one element or more than one element Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820

[0027] Throughout this specification, unless the context requires otherwise1 the word "comprise", or variations such as "comprises'' or "'comprising", will be understood to impfy the inclusion of a stated element or integer or group of elements or integers but not the .exdu.sion ofa:tty other element orintege:t' orgroup of element$ or integers. 5

[0028] The term «sustainedc-delivery formulation" as used herein refers to a formulation that is capable of delivering the compound of formula m to a patient over a. sustained length of time.

The formulation may be a formulatfon for intravenous delivery in which tlu; compound of formula (I) is delivered over a period of hours, days or weeks, The sustai11ed-delivery fornrulation may be a "controlled-rel.ease fo.rniulation" formulation, 10

[0029] The term "controlled-release fomiulation:" refers to a formulation in which the compound of formula (I) is administered as a bolus dosage but the formulation releases the drug in a controlled manner.

The .objective of a. controlled-release formulation is tp provide zero order kinetics of drug delivery (i.e. a linear delivery with respect to time).

Controlled reiea:se ofdrug from the dosage fpnn relies upon two processes: dissolution. and 15 release,

[0030] As used herein, the term "heart failure with pres.erved ejection fraction" (HFpEF) Jiefers t() heart. failut.e fo which the ejection :fraction (EF) is ;;-:: 40%, with .those having an EF from 40to 49% being considered borderline HFpEF.

[0031] As used herein, the term "heart failure with reduced ejection fraction" 20 (HFrEF) refers to heart failure in which the ejection fraction (EF) is :S 40%.

[0032] As used herein, the term ''alkyl!' refers to a straight c:bltfo or .branche.d .saturated hydrocatbo.n group having J to 6 carbon. atoms.

Where appropriate, the alkyl group may have a specified number of carbon atoms, for example, C1-6alkyl which includes alkyl groups having 1, 2; 1, 4; 5 or 6 carbon atoms fo a linear or branched arrangement.

Examples 25 of suitable alkyl groups .include, but are not limited to, methyl, ethyl, n-propyl, /.:propy1, nbutyl, i-butyl, t-butyl, n~pentyl, 2.;methylbutyl, 3-methyl.butyl, 4-methylbuty], n-hexyl, 2- methylpentyl, 3--methylpentyl, 4-:methylpenty1, 5-:methylpert~y.1, 2-ethylbutyl and.3-ethylbutyl ..

[0033] As used herein, the term ''halogen11 or "halo" refers to fluorine (fluoro), chlorine (chloro), brQmine (bromo) and iodine (iodo} 30

[0034] As used herein, the term "pyridine" or "pyridinyl" refers to a 6:-membered aromatic cyclic group having one nitrogen atom in the ring having the formula: Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820

[0035] The pyridine ring may be attached to the structure of fonnula (I) where indicated with the PY at any of the carbon atom~ at the 2-, 3- or 4- position.

[0036] The compounds of formula (I) may be in the form of pharmaceutically S acceptable salts.

Suitable phartn.a:ceutically acceptable s.alts include, but are not limited to, salts of phartnaceutically acceptable in:organic acids such as hydrochloric, su1phuric, phm~phoric, nitric, carbonic,. boric, sulfamic and hydrobmmic acids, or salts of pharmaceutically acceptable organic acicls such a.s acetic; propionic, butyric, ta~rlc, maleic, hydroxymaleic, fuma:ric, citric, lactic, mucic, gluconic, benzoic, succinic; oxalic, 10 phenylacetic, methanesulphonic, toluenesulphonic, benzenesulphonic, salicylic:; sulphanHic, aspartic; glutamic, edetic, stearic, palmitic, oleic, !auric, pantothertic, tannic; ascorbic and val.eric acids.

[0037] Base salts include, but are not limited to, those formed with pharmaceutically acceptable cations, such as sodium, potassium, lithium, calcium, 15 magnesium, anini.onium.and alkylammonium.

[0038] Basic nitrogen-containing. g.r oups may be .quaternized with such a.g ents as lower alkyl halide, such as methyl, ethyl; propyl and butyl chlorides, bromides and iodides, dialkyl sulfates like dimethyl and diethyJsulfate, and others.

[0039] The teml "subject" generally means a human.

However, the present 20 invention extends to the treatmer1t of animal model systems including non-human primates as well as pigs, sheep, dogs and horses..

Non.:.humah commercial applications include. the treatment of race. animals such as horses, dogs and camels as well as work animal.s such as horses 11nd dogs..

By "human'1 mean.s a person of any age from infant, child1 adolescent1 teenager, young adult, adult, middle age .and aging indiyidual.

Age ranges from 1 day old to 25 120 years old ate contemplated herein.

In extreme emergencies, in utero treatments of unborn babies may be contemplated and is ehc.ompas.sed by the present h1vention.

Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 2.

Methods of the invention

[0040] The present invention relates to methods of treating patients with HFpEF with a comp.01,111d of formula (I), Ip one aspect the invention rela,tes to a metllod of treating patients with HFpEF with a sustai:ned~delivery formulation of a compound of formula (I) 5 wherein the formulation permits delivery of the compound of formula (I) in an amount to achieve steady state plasma levels effective to alleviate the symptoms of IIBpEF:; wherein delivery of the com pound of formula (I): is in the range of between O .1 μglkg body weight per minute to 20 μg/kg b9dy weight P'er minute. [0041) In some embodiments, the patients with ffl\'pEF ate patients. having 10 borderline HFpEF (an ejection fraction between> 40 and 49%). 1h other embodiments, the patients with HFpEF are patients having an ejection fraction of 2"' 50%.

[0042] The.admiuistra,tionis generally μnder conditions suftfoient to achieve. levels of the compound of formula (I) which are not overly toxic and which is effective to al.leviate the symptoms of HFpEF.

Conveniently, the compound offonnula (I) is formulated to enable 15 compound delivery into the blood stream at a rate of between above 0.1 μg/kg body weight/minute to about 20μglkg body weig}lt/minute..

This range includes 0.1, 0.2, 0.3, 04, 0 .. 5, 0.6, 0.7, 0.8, 0.9, l, 2, 3, 4, $, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,. 18, 19 and ZOμg/kg; body weight/minute as well as fractions in between.

In a particular embodiment, the compound of fonnula (I) is delivered at a rate of from about 0,3 to l μg/kg body 20 weight/minute including from about 0.37 to 0.75μg/kgbody weight/minute.

[0043] The amm.mt of compound of formula (I)that i;, admi.nistere<l will depend on the subject being treated1 their physical condition, their weight and the fonnulation being used.

Suitable otal dosages are in the range of 5 mg to 75 mg, especially 10 to 50 mg or 10 to 40 mg.

Suitable fatravenous dosing includes administration in the range of 0.1 to 0.75 25 μg/kglmin as a continuous infusion.

[0044] When in the form of an oral controlled-release dosage form, the dosage may .b.e provided in ti single dos.e. per day, for example, one dose of 30 to 40. mg, or may be provided in divided dosages for example, two, three or four times a day.

Amounts 15 to 30 mg or 15 to 20 mg every 12 hours is a useful therapeutic amount in accordance with the 30 present invention and allows for 12 hourly or twice daily dosing.

Amounts of 10 to 15 mg every 8 hours allows for dosing three times per day and amounts of 7.5 to 10 mg every 6 Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 hours allows for dosing four times a day.

In particular .embodiments~ administration is twice daily.

[0045] Th~ optimal plasma level ofa compound off o.rmula (I) such as milrinone, is in the range of JOO ngltnL to 400 nglmL, especially 100 nglmL to 300 nglmL Plasma 5 clearance of the compound of formula (I) is affected by the presence of either renal or cardiovascular disease.

The optimal dose of sustained-delivery compound of formula (I) may need to be determined for an individual patient by a stepwise upward titration of the dose accompanied by regular monitoring of the patiellt's plasma lev©ls of compound of formula (I) until the required steady state level is achievecl. 1n some embodiments, the method further 10 comprises the step of monitoring plasma concentrations of the compound of formula (I) and if necessary, adjusting the dosage to achieve a plasma concentration in the range of 100 to 400 ng/mL. f0046] In some embodiments, in the compound of fonnula (I), at least one of the foUowin:g applies:. 15 R1 is selected from hydrogen, ~C1-C3alky1 or ~C1-C3alkylOH, especially hydrogen, -CH3 or -CH20H, more especially hydrogen; R2 is selected from ~C1-C3alkyl, especially methyl or ethyl, more esp.ecia1ly methyl; R.3 is selected from -CN (cyano), -NH2, halo, --NH(C1-C3a1ky1), -N(C1-C3alkyl)2, -C02H or -C02C1.3alkyl, especially -CN, -NH2~ -C02H and -C02CH:;, more especially -CN.~ and 20 PY is unsubstituted 4-, 3- or2-pyridinyl, especially unsubstituted 4--pyridinyl [00471 In a particular embodiment, the .compoμnd of formula (I) is l,2-dihydm-3- cyano-6-methyl--5-(4--pyridinyl)-2(lH)-pyridinone.

This compound is also known as :2- rnethyl-6-oxcHlihydro-3,4' -bipyridine-S-carbonitrile and milrinone.

[0048] Methods of making compounds •Of formula (I) including milrinone are 25 known in the art and can be found, for example, in GB Patent No. 2065642 and US Patent No. 4,313,95[ [0049} In some embodiments; the intravenous administration may be a continuous intravenous infusion administered over a period of l to 48 hours, but is not limited to this period.

Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820

[0050] In other embodiments, the sustained-delivery formulation is an oral controlled.release fonnuli:ition.

In some embodiments, the oral contrdlled•release formuhition comprises: i) a core comprising the compound .offormula (J) and one or mo.re polymers 5 and one otmore excipients; and ii) a sustaine<i-release coating.

[0051] The epmpound of formula (I) may be blended with one or more polymers, to provide.a matrix that is either formed into a.partic;le (small orlatge), or is coated on an inert particle to fonn the core of the formulation.

The polymers of the core are selected from 10 hydrophilic~ hydrophobic or plastic, Hydrophilic polymers are water soluble and hydrate in ,contact with water to form a hydrogel as they dissolve and swell; hydrophobic polymers do not dissdlve but may be subject to erosion as the matrix releases soluble constituents; plastic polymers form insoluble or skeletal matrices. but do not erode.

Upon exposure t.o the fluid in the stomach, small intestine and colon~ hydrophilic polymers hydrate and form a hycltogel that 15 acts as a diffusion barrier to drug release; hydrophobic polymers release drug through diffusion through pores and through erosion.

Drug release from plastic matrices is controlled by the rate .. ofliquid pe11etration and is accelerated by the presence of .channel fonning agents: soluble components that are added in addition to drug.

[0052] The, behaviour of some polymer.s is dependent upon pH This is. particul.arl y 20 true where the polymer contains acidic. or basic moieties. as pH will .affect the ionization state.

Ionization can transform a polymer from hydrophobic to hydrophilic., with an accompanying transformation in release properties.

[0053] The release of the dissolved compound of formula (I) into, for example, the gastrointestinal (Gl) tract may also.be controlled by the coating on the particle.

This,coating is 25 typ1ca1ly a polymer .or btend of polymers that is. relatively stab1e towards the conditions. encountered in the gut.

In many cases; the coating includes at least one hydrophilic polyrirer that will swell on contact with fluid in the gut to form a hydrogel barrier that is homogenous and. stable to changes that may take place to the underlying matrix.

The hydrogel also assists with slow release. of dissolved compound .. offotmula (I}, The properties of the surface coating 30 can be pH dependent depending upon the presence of acidic or basic moieties in .the polymer. constituents.

Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820

[0054] A particular disadvantage of some controlled-release formulations is the potential for a burst release of drug to occur immediately following contact of the dosage form with the dissolution fluid, The use of a hydrophilic polymer in the film coating o.r in the matrix:, wherein the hydrophilic polymer forms a hydi;:ogel rapidly after hydration, can 5 significantly reduce the incidence of the burst release phenomenon.

[0055] Controlled~release ora:l formulations include a monolithic tablet dosage form in which one or more drug~polymer matrices provide the core and or particulate or bead dosage forms in which an inert particle coated with drug provides the core.

These types of formulations may include. an optional surface film co.ating to provide additfort~ controi over 10 drug release.

Particulate dosage forms may be formed into a tablet or filled irtto a capsule, This differs from immediate release (TR) formulations which are designed to disintegrate, 'dissolve promptly and release a bolus dose of drug,

[0056] The core matrix containing the compound of fonnula (I) may be fonned by g:ranulatipn or directcomptession and .may be.heterogeneous to provide porosity, 15

[0057] In particular, a core matrix may comprise either ot both hydrophilic polymers and hydrophobic polymers in order to achieve the appropriate release profile.

Further, one or more of the polymers may .swell upon hydration it1, a manner that may additionally be dependent upon pR. to form a hydrogel that is viscous and gelatinous and thus provides a b.atrier to drug: release.

The composition of hydrogel determinces its properties,. 20 which can thus be manipulated in order to achieve appropriate drug release kinetics.

[0058] The optional surface film coating provides a diffusion release mechanism where the permeability is often directly related to hydration leading to polymer swelling and the installation of hydrogel dynamics.

[0059] At least one combination of matrix and optional surface film coating 25 provided in the descri.ptidn below can be us.ed in the fonnu]ation of the invention to achieve the desired release profile across the different environments encountered during transit through the GI tract

[0060] Sustained release formulations of compounds of formula (I), and in particular sustained relea:se. fonnulations 01 milrin.one, which achieve the desired telease 30 profile across the .different environments encountered during transit through the GI tract ate described in PCT application PCT/AU2012/000967; published as WO 2013/023250 AL The release profile pf a sustained release formulation of a Qompound of formtila (I) can be Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 determined in accordance with the dissolution study methods .described. in WO 2013/023250 Al and as described in the Examples below, A sustained-release fonnμlation of a compound of formula (I) preferably provides zero order kinetics of drug delivery (i.e. a linear delivery with respect to tirrH~). 5

[0061] The invention further provides a method of preparing a sustained-delivery formulation of a 5-;(pyridinyl)-2(1H)~pyridirrone compound of formula (I) as hereinbefore defineq, or a pharmace4tically acceptable salt thereof; wherein the formulation permits delivery of the e-0mpound of formula (I) in an amount to r;ichieve steady state plasma levels effective to alleviate the symptoms of HFpEF; wherein delivery of the compound of formula 10 (I) is fo the range of between 0 .1 μg/kg body weight per minute to 20 μg/kg body weight per minute, for the treatment of heart failure with preserved ejection fraction (HFpEF) comprising the steps of: formulating a 5-(pyridinyl )-2(ll-I)-pyridinone compound of formula (I) as hereinbefore defined with one or more polymers to provide an extend.ed release matrix. formulation; and testing to confirm that the formulation provides the desired release profile for the compound of formula (I).

[0062] In some embodiments the sustained-delivery formulation is for oral administration.

In some embodiments the compound of formula (1) is fqrrnulated with one or 20 more pharmaceutical excipients.

In s.ome embodiments the compound of formula (I) is formulated as a core comprising the compound of formula (I) and one or more polymers and one or more pharmaceutically acceptable ex.cipients and a sustained. release coating.

In some embodiments the formulati<;m is provided with one or more seal coatings, In some embodiments the formulation is provided with one or more enteric coatings.

In some 25 embodiments the i;ompound offormula(I).isformulated 'as a.unit dose form, for example as a tninitablet or as beads.

In Some embodiments the sustained~delivery fonnulatkm is a composition as herein defined.

In some embodiments, the sustained delivery formulation comprises: iii) iv) a core comprising the compound of formula (I) and one or more polymers and one or more excipients; and. a sustained~release coating.

[0063] In a particular embodiment the sustained deliveiy formulation .comprises a compound of formula (I) in a polymeric matrix, the polymeric matrix and: compound of Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 fonnula (I) mixture having a seal coating, The seal..-coated polymeric matrix .compound of formula (I) has a sustained-:release coating and the formulation further comprises an enteric,. relea.se coating.

Optionally. there is a buffer-coating between the sustained-release coating and the entetip--release coating. 5

[0064] Methods of testing to confirm that the formulation provides the desired release profile of the compound of fommla (I) are known in the art and may include dissolution orreleas.e studies such as those described herein.

Preferably the sustained delivery formulation provides zero order kinetics of drug delivery (i.e. a linear delivery with respect to time). 10

[0065] Polymers that are of use in the.formation of core drug-polymer matrices are as follows; • Acrylic ahd :methactylic polymersincluding hydroxypropyl methacrylates (HPMA) and hydroxyethyl methacrylates (HEMA), as well as N-isopropyl acrylamides; • Polyethylene oxides (PEQ) also known as polyethylene glyqols (PEG) and 15 polypropylene oxides(PPO), as well as block copolymers of PEQ andPPO (also known a.s PluronicS> (Registered Trade Mark); .IP Cellulose ethers including hydroxypropyl methylcellulose (HPMC), hydroxypropylcellulose (HPC), hydroxyethyl celluJose (HEC), methylcellalose (MC), ethyl cellulose (EC). a,id caiboxymethylcellulose (CMC); 20 • Polylactides{PLA), polyglucolides (PGA), copolymers ofpolylactide and polyglucolide in varioμs proportions (PLGA); • Poly(suct(lse acrylates); • Polylysine; polyvinylamine, polyethylimine (PEI), polyglutarnic acid, polyvinyl alcohol (PY A); copolyrnen11 of ethylene and vinyl acetclte (pEV A); 25 • Polyethylen:eglycol terephthalate, polybutylene terephthalate and copolymers thereof (also known as Locteron [Registered Trade Mark]); Copolymers of PEG and PLGA also known as Re ..

Qel (Registered Trade Mark); Polyorthoesters also known as Chronomer (Registered Trade Mark); polyarihydrides; copolymers of acrylic acids and esters, or methacrylic aci.ds atid esters of various molecular weight and proportion also 30 known as Budragit (Registered Trade Mark) in particular RL30D, RLPO. RLIO0, RSJ0D, RSPO, RS100, NE30D, NM30D, NB40D, Ll00; copolymers of phthalic acid cellulose and phthalic ester cellulose also known as CAP (Registered Trade Mark); Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 • Polyvinylpyrro)idone also known as Kollidon (Registered Trade Marl<) and copolynters thereof with polyvinyl acetate also known as Kolli don SR {Registered Trade Mark)1 • Polymers of natural origin .including non--ionic, amino, carboxylated and sulfated polysaccharidei:;, optionally chemically modified through partial hydrolysis and/or 5 conjugation of modifiers such as carboxylates or long chain fatty acids (C8-Cl6), in.clud.e:.

Guar ~rum; ac.acia gum, tragacanth gum, xanthan gUm, carra:geenans (both io.ta .and lambda); Linn gum, alginates, scleroglucans, dextrans, chitins and chitosans, pectins, galactomannans including locust bean gum. 10 f0066] In addition, it isfreqvently (ound that polymer blends are particularly useful for providing the appropriate release profiles for controlled . ..:release formulations, for example mixins polymers with hydrophilic and hydrophobic properties, and such polymer blends would include: • Methyl methacrylates polymers with starch or cellulose polym.ers;. 15 • Polyacrylic acid-Pluronic-polyacrylic acid block copolymers; • Multilayer polyelectrolytes using cationic polymers selected from chitosan, polylysine, polyallylatnine ot potyviri,ylamine with anionic polymers selected from Carbopols including 971NF, catrageenan, xanthan gum~ alginate, hyaluronic acids, Eudragit® including LlOO and carbox:ymethylcellulose; 20 • Hydrophobic cellulose polymers such as ethylcellulose or Compritol 888 ATO are often mixed with hydrophilic polymers such as HPMC.

NaCMC, sodium alginate, xanthan gum or Methocel (Registered Trade Mark); • Hydrophilic swelling polymer such as HPMC is mixed with a pH dependantpolymer such as Eudragit{Registe:red Trade Mark) Ll 00-55; 25 • Polymer blends may be crosslinked either by covalent bonds or, particularly for polymers of natural origin, through the addition of polyvalent cations including borate, calcium, magnesium and zinc; • Natural gums ate often used in p.olymer blends, in particular carrageena:ns with cellulose ethers, xantban gum with locust bean gum. :rn [00671 Whilst ternary blends are less. common, one example js a blend of non-ionic water solu.ble polymt!r Polyox with a swellable high molecular weight crosslinked acrylic polymer Catbopol and lactose.

Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820

[0068] Film coatings are contemplated for use with multi-'unit dosage forms other than m()nolithic tablets.

Coatings are selected which include polymer, solvent and a. plasticiser, particularly triethyl citrate, dibutyl sebacate., diethyl phthalate or propylene glycol.

Plasticisers may not be ne¢essarywhen poly(dimethylsiloxane) or other silane,elastomers are 5 used. [006.9] Particular examples .of surface coatings which can provide a hydrogel b.arrier upon hydration include the cellulose polymers, Eudragit (Registered Trade Mark) polymers and graft copolymers of polyvinyl acetaw, polyvinyl alcohol and PEG, also known as Kallie.oat (R(;'lgistered Trade.

Mark), for example Kollicoat (Registered Trade.

Mark) SR m)d 10 Kollicoat (Registered Trade Mark) IR, used with propyleneglycol as plasticiser.

The properties ofthis.coatin,g are independent of pH.

[0070] Polyelectrolyte multilayers. (PEM) are one particular example of a film coating which can provide an appropriate ra,te of drug release through a combination of vatiablesincluding: 15 • The selection of positive and negatively charged polyelectrolytes; • The number Ciflayers that are deposited; .IP The molecular weight of the polyelectrolytes used to form the film.

[0071] The permeability of PEMs can be responsive to stimuli whereby a change in pH, ionic s.trength or wroperature has tM potential to change the permeability to. particular 20 solutes.

[0072] Multilayer tablet formulations are particularly useful for highly soluble drugs, Such dosage forms include a hydrophilic matrix core with one or two semipermeable coatings, which may be implemented as a film or compressed barrier.

Typical polymers inc.lu<le celluloS"e derivatives particularly HPMC, NaCMC? If.PC, EC or MC, or natural gums 2.5 particularly tragacanth or guar gum.

[0073] In one embodiment, the core comprises a compound of formula (I), hydroxypropylmethylcellulose or hydroxypropylcellulose having a viscosity of 801000 to 120,000 cps; .hydroxypropylmethylcellulose having a viscosity of about 50 cps; 30 and at least one.phatmaceutically acceptable excipient; wherein the hydroxyptopylmethylcelhilose ot hydroxypropylcellulose (80,000 to 120,000 'CPS) and the hydroxypropylmethylcellulose (50 cps) are in a ratio of 2:•l to 1:2, and the ratio Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 of compound of formula (I) to total hydroxY,propylmethylcellulose or hydroxypropybnethyleellulose and hydroxypropylcellulose is 1 ;2 to 1 :6.

[0074] Hydroxypropylmethylcellulos¢, also known as. hypromellose or HPMC, is available i11 different viscosities.

In the present invention,. the hydrox.ypropylmethylceilu1ose 5 is present in two viscosities, 80;000 to 120,000 cps and about 50 cps. A suitable HPMC having a viscosity of 80,000 to 120,000 is hypromellose .2208 USP which comprises 1 9-24% methoxy ether.substitution and 7-12% hydroxypropyloxy ether.substitutfon on glqco.se C2~ C3 and C6 hydroxyl moieties and has a viscosity of about 100,000 cps.

The viscosity is mea.sured at .2% conoentration in water at 1Q°C. A suitable Hl1MC (80,000 to 120,000) is 10 HPMC Kl OOM. A suitable HPMC having a viscosity of about 50 cps isHPMC E50 L V.

[0075] In some embodiments~ the HPMC (80,000 to 120,000) may be substituted by hydroxyprqpylc.ellulose (HPC) having a, vtscosity of 80,000 to 120,000 cps,

[0076] In some embodiments, tbe HPMC or HPC (80,000 to 120,000) is HPMC (80,000 to 1201000), especially HPMC KlOOM. 15

[0077] In some embodiments, the HPMC(about 50 cps) is HPMC E50 LV.

[0078] In some embodiments the ra:tio of HPMC or HPC (80;000 to 120,000) to HPMC (c1:bout 50 qps)is in the range ofl.5:1 to 1:L5, especially aboutl:.L

[0079] In some embodiments~ the ratio of compound of formula (I) to total HPMC ot HPMC or HPC (80>000 to 120,000) and HPMC (about 50 cps)1 is l:2 to 1 :.6, especially 20 about 1:3 to L5, more especially about1~3.

[0080] In some e,mbodiQ1.ents the compound .of fonnula (I) is present .in an amount of 10 to 300/4 w/w of the core, especially 15 to 25% w/w of the core, more especially about 20% w/w of the core.

[0081] In some embodiments the. HPMC or .HPC (80;000 to. 120,000) is present in 25 an amount of 20 to 40% w/w of the ccore, especially 25 to 35% w/w of the core, more especially about 30% w/w of the core.

[0082] In some embodiments, the HPMC .(about50 cps) is present in an amount of 10 to 40% w/w of the core, especial! y 20 to 35% w/w or 25 to 35% w/w of the core, more especially about 30% w/w of the core. 30

[0083] In some embodiments the core also comprises pharmaceutically acceptable excipients. s.uch as. binders .a,nd/or .lubricants.

Suitable binders include di saccharides such as Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 sucrose and lactose, polysaccharides such as starches and cellulose derivatives, for exampleJ microcrystalline cellulose, cellulose ethers and hydrQxypropylcellulose (HPC), sugar alcohols such .as xylitol, sorbitol or maltitol, protein.s such as gelatine.!Uld. synthetic polymers such as polyvinylpyrrolidon.e (PVP) and polyethylene glycol (PEG).

In a particuhtr em.bodiment, the 5 binder is tnicrocrystalline cellulose.

[0084] In some embodiments, the binderis presentin an amount .of 10 to 30% w/w of the core, especially about 15 to 25% w/w of the core., more especially about 18% w/w of the core; In some embodiments, the pompound of formula (I) such as milrinone and the binder .such as microorysta11inti cellulos<:i w-e together present in the c.ore. in about 30 to 50%, 10 especially about 40% w/w of the core.

Ih some embodiments, the. ratio of compound of formula (I) to binder is 1 :2 to 2: 1, especially about 1 :1.

[0085] Suitable lubricants inclu.de fats such as. magnesiμm stearate, vegetable stearin and stea:ric acid, talc or silica.

In particularembodiments, the lubricant is magnesium .stearate. 15

[0086] In some embodiments, the lubricant is present in an amount of 0.5 to 5% w/w of the core, especially about 1 to 3% w/w of the core, especially about 2% w/w of the core.

[0087] In another embodiment the core comprises a compound of formula {I)? a hydrophilic matrix comprising at least two natural gums, and :at least one pharmaceutically 20 acceptable excipient; wherein the two natural gums are in a ratio. of 2: 1 to .t2; and the ratio ofthe compound of formula (I)to the hydrophili.c matrix is 1: I to 1 :2.5. (0088] Suitable natural gums include guar gum, acacia gum, tragacartth gum, xanthan gum, carrageenans (both iota and lambda), Linn gum, alginates~ scleroglucans, 25 dextrans., chi tans and chitosans, pectins, and galactomannans including locust bean gum.

In some embodiments the hydrophilic matrix includes x.anthan gum or locust bean gum.

In a particular embodiment the hydrophilic matrix includesxanthan gum and locust bean gum.

[0089] In some embodiments, the ratio ofxanthan gum to locust bean gum is.about 1.:5:1 to l :J.5, especially about 1 :L 30

[0090] In some embodiments, the ratio of compound of formula (I) to hydrophilic rnatri:t!: hi 1: 1 to 1 :;2, .especially about l: l.5,.

Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820

[0091] In some embodiments, the compound of formula (I) is present in an amount of 15 to 25% w/w of the core, especially 1.8 to 22% w/w of the core, more especially about 20% w/w of the core. [Q09l:) In some embodim:e.nts, the hydrophilic matrix is present irt an amount of20 5 to 40% w/w of the cote, especially 25 to 35% w/w of the core. more especially about 30% w/w of the core.

For a ratio of 1 :1 xanthan gum to locust bean gum, the amount of each gum will be aboufl 5% w/w of the core.

[0093] In, sQme embodiments, the exdpients are selected from binders, fillers, .'glidants, h1bricants and mixtures there!)( 10

[0094] Suitable binders include disaccharides such as sucrose and lactose, polysaccharides s:uch as starches and ceJlulose derivatives such as microcrystalline celhilose, cellulose ethers and hydroxypropytcellulose (HPC), sugar alcoh,01$ such as xylitol, sorbitol or maltitol, proteins such as gelatine and synthetic polymers such as polyvinylpyrrolidone (PVP) and polyethylene glycol (PEG).

In particular embodiment, the binder is microctystalline 15 cellulose; polyvinylpyrrolidone (PVP) otmixtures ofmicrocrystalline cellulose andPVP.

[0095] In some embodiments the hinder is .present in an amount of 17 to 30% w/w of the core, more especially about 23.5%w/w of the core.

In ~ome embodime:nts the binder comprises about20% w/w of microcrystalline cellulose and about 3. 5% w/w PVP. (0096] Suitable fillers or bulking ag~nts include lactose, sucrose, glucose, mantiitol, 20 sorbitol, calcium carbonate and dibasic calcium phosphate.

In a particular embodiment, the fllleris lactose. (0097] In $On1e embodiments, the filler is present in the cpre in an amount of 20% w/w of the core, especially about25% w/w of the core. [0.098] Suitable giiclants include fumed silica,, talc and magnesium carbonate.

In a 25 particular embodiment, the glidant is fumed silica.

[0099] In some embodiments the gliqantis present in an amount of about 0,5 to J. .5 % w/w of the core, especially about 1 % w/w •ofthe core.

[00100] Suitable lubricants include fats such as magnesium stearate, vegetable stearin and stearic acid, talc or ,silica.

In particular ,.embodiments, the lubricant is magnesium 30 stearate.

Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820

[00101] In some .embodiments the lubricant is present in an amount.of 0.25 to 1 % w/w of the core; especially about 0.5% w/w of the core. [00102;] In yetanothet embodiment the core comprises (i) a coating composition contprising a compound of formula (I), one or more 5 polymers, and orte or more exci pients, and (ii) inert spht:irical particles; wherein the coating composition is on coated on the surface of the spherical particles; wherein the ratio of c.ompound of formula (() to the spherical parti.cles is about 1 :5 to l:25; and 10 whereinthe c00;ted particles further comprise a seal coa,ing. (00103] The inert spherical particles may be any inert spherical particles commonly used in microparticulate systems.

Typically, the inert spherical particles have a diameter of 0.06 to 2 mm.

Suitable inert spherlcal particles are sugar and/ot starch spherical particles.

Such particles are suitable for fonnulation into a capsule or tablet Microparticle dosage 15 systems can provide the following benefits for extended release formμlations;. • Less dependent on gastric emptying, re:sultingin less intra/inter individual variability in gastric transit time (sizes less than 2 mm .are able to continuously leave stomach even when pylorus is ~losed); • Particles are better distributed, avoiding possibility oflocalised irritation; 20 • Drug safety is improved for modified release formulations, as less susceptible to performance failure if damaged; • Multiparticulate formulations are popular for selective delivery to the colon when that is the only absorption window1 they .can also be used for continuous GI absorption.

Furthermore it is possible to mix particles with different release profiles to optimise 25 exposure in different regions of gut [00t04] In some embodiments, the compound of formula (I) is prepared in a coating composition comprising a coating polymer and excipients such as binders.

The coating composition is then coated onto.the spherical particles.

[00105] Suitable coating compositions comprise, in addition to compound of 30 formula (l), a polymer, plasticiser and binder.

If required~ the coating qomposition may b.e dissolved or suspended in a suitable solvent such as watel;', for application.

Suitable polymers Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 include polyvinyl alcohol (PVA) or cellulose polymers such as HPMC, hydroxypropykeJlulose (HPC), hydroxyeth.yfoellulose (HBC), methylcellulose (MC), ethylcdlulose. (EC) and carboxy111ethylcellulose (CMC), Suitable plasticisers incl4d.e propyl~n~ glycol 1 polyethylene glycol (PEG), dibutyJ sebacate, glycerine, trietbyl chrate and 5 diethyl phthalate.

In one particular embodiment, the polymer is HPMC and the plasticiser is PEG, for example, the coating composition sold under the trade mark OPADRY CLEAR (Registered Trade Mark): In another particular embodiment, the polymer is PVA and the plasticiser is PEG and/or glycerine, for example, the coating composition sold under the trade mark OJ>ADRY II (Registered Trade Mark). 10 (00106] The coating composition may also comprise a binder.

Suitable binders include disaccharides such as sucrose and lactose, polysaccharides such as starches and 'cellulose oerivatives such as microcrystalline cellulose, cellulose ethers and. hydroxypropylcellulose (HPC), sugar alcohols such as xylitol, sorbitol or maltitol, proteins .such as gelati11e and synthetic polymers such a$ polyvinylpyrrolidone (J>VP) and polyethylene 15 glycol (PEG).

In a particular embodiment, the binder is. PVP.

[00107] In some embodiments, the ratio of compound of formula (I) to the polymer/plasticiser blend is about 1.5: 1 to 2:1,. especially about 1.6;1 to L8:L

[00108] In some embodiments, the ratio of compound of formula (I) to binder is in the range of8:l to 12:1, especially 1-'tbout 11:L 20

[00109] In some embodiments, the ratio of compound of formula (I) to spherical particles is about 1:10 to 1:25, especially about 1:15 to 1:2U.

Seal Coating/Buffer Coating

[00110] In some embodiments, the formulations of the invention may comprise a seal coating.

The seal coating may be applied over the core,. fot example over the dtug 25 coating of the spherical particles or may be used as a coating on a tablet formed. by compression of the core, also for example between layers of the formulation., such as between the core and .the sustcJ,ined-releas.e coating (seal coat) or b~ween the sustained-release coating andthe enteric-release coating{buffer coat).

The seal coating orbuffer coating may comprise a polymer and a plasticis.er.

Suitable polymers include PVA and cellulose polymers such as 30 HPMC, hydroxypropykellulose (HPC), hydtoxyethylcellulose (HEC), methylcellulose (MC), ethylcellulose {EC) and carboxymethylcellulose (CMC).

Suitable plasticisers include propylene .glycol, polyethylene glycol (PEG), dibutyl sebacate, glycerine, triethyl citrate .11nd Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 diethyl phthalate.

In a particular embodimen1; the polymer is HPMC and the plasticiser is PEG~ for example, the coating composition sold under the trade mark OPADRY CLEAR (Registered Trade Mark).

In another particular embodiment, the polymer is PVA and the plasticis.et fa. PEG and/or glycerine,· tor example1 the coating composhicm sold under the trade 5 mark OPADRY II (Registered Trade Mark).

The seal coating or buffer coating may also include a pigment to give a desired.colour.,for example, titanium dioxide to give white.

The seal coating or buffer coating may be present in an amount of 3 to 15% w/w of the formulation, especially 5 to 12% w/w, more especially 5 to 10% w/w.

Sustained-Rele3se Coating 10

[00111] The formulations above include a sustained-release coating.

Suitable sustained-release coatings include cellulose derivative coatings such as HPMC, HPC, HEC, EC,. MC and CMC or co-polymers of acrylic acids and. their esters or methacrylic acids or their esters, such as those sold under the trade mark Eμdragit® including llL30D, RLPO, RLlOO, R830D, RSPO, RSIOO, NE30D, NB4()D and LWO.

Irt particular embodhnents, the. 1 S sustained-release coating may comprise ethyfoellulose (EC), which is insoluble in water, in which case, the sustained-release coating may optionally include a low content of water soluble polymer such as a low viscosity HPMC (11,1g: 6cps), for example Opadry Clear™., In other embodiments, the sustained-release coa;ting may comprise an acrylic acid, actyHc ester, methacrylic acid or methacryli<: ester optionally including a low content of a methacrylic acid 20 .ester with quatetnaty ammonium groups (trimethylammonioethyl methacrylate ,chloride) copolymer.

This sustained-release coat may be<comprised of one or more copolymers of ethyl acrylate (A), methyl methacrylates (B) and a low content of a methacrylie acid ester with quaternary ammonium groups (trimqthylammonioethyl methacrylate chloride) {C).

For the polymeric materials of'this embodiment, the molar ratio of the monomers A:B are in the range 25 1:1-1:3 preferably 1:2;themolarratio of the monomers A:C arein the rangel:0.01: to l:0.5,. preferably irt the range 0,05-0.25.

When one or more of the layers comprises a blend of two copolymers, The molar ratio of the monomers A:B:C in the first of the copolymers is approximately 1:2:0,2 and the molar ratio of the monomers A:B:C in the second of the copolymers is 1:.2:0.1, and the ratio of the first and the second copolym~r is in the range l:5 to 30 1: 15, especially .about 1 :9.

[00112] The sustained-release coatings may also comprise lubricants.

The sustained~ release coatings may also comprise plasticisers.

The sustained-release coatings may also comprise anti-tacking agents.

Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820

[00113] In a particular embodiment, the sustained-release coating comprises ethyl cellulose as Aquacoat ECD 30 and HPMC 6cps as Opadry Clear wherein the ratio of EC and HPMC is in the range 19: 1 to 4: I especially about 9: I.

[00114] In a particular embodiment, the sustained~release coating comprises ethyl 5 cellulose as Aqua.coat ECD 30 and HPMC 6cps as Opadry Clear, and a plasticiser~ wherein the ratio ofEC and HPMC is in the range 19:1 to4:1 especially about 9:1 and the ratio of EC to plasticiser isin the range 9:1 to 2:l especially about 3:1.

[00115] In, a particular embodiment, the sustained-release coati,ng comprises ethyl c.ellulose as Aquacoat ECO 30 and HPMC 6cps as Opadry Clear~ and further comprises talc 10 and a plasticiser, wherein the ratio of EC and RPMC is in the range 19: 1 to 4: 1 especially about 9:l; the ratio of EC to talc is in the range 19:1 to 4: 1 especially about 9: 1, and the ratio of EC to plasticiser is in the range 9: 1 to 2: 1 especially about 3: 1.

[00116] In a particular embodiment, the sustained release coating compnses Eudragit RS30D, Eudragit RL3QD or mixtures thereof wherein: the ratio of the first and 15 second copolymer is.in the range of 1:5 to 1:15, especially about 1:9.

[00117] The i;.ustained release cofl.ting may be applied to the formulation in tablet form or to the drug~coated sphedcal partich:is. [00118} In some embodiments, the formulation may comprise more th.an one sustained,..telease coating.

In some embodiments, a .first sustained release coating may be 20 present followed by a second sustained-release coating.

The first and second sustained release coatings may be the same or different FQr example, the first coating may be an ethylcellulose coati:ng and the second coating a Eudragit coating such as a combination of Eudragit RS30D and Eudragit RL30D or the first coating may be a combination ofEudragit RS30D and Budragit RI.'30D and the second toating'may be Eudragit RS30D. 25

[00119] Typically, the sustained-release coatings will be present in an amount ofl to 40% w/w of the sustained-release coated formulation, especially 3 to 30%, more especially 5 to 25%,. 'In one embodiment, an ethylcellulose coating may be present in an amount of 3 to 15% w/w of th¢ sustained.,.release coatedJormqlation, especially 5 to 10%, for example, about 7Ji% .or may be present in an amount of about 5% w/w of the sustained-release coated 30 formulation.

In another embodiment, an ethylcellulose coating.may be present in an amount of about 10% w/w of the sustained.,.release coated formulation. fu yet another embodiment, a sustained.,.relea$e coating of Eudr:11git RL30D and Eudragit R$30D may be. present in an Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 amount of about 25% w/w of the sustained..,release. coated formulation and may further comprise a sustained-release coating of Eudragit RS30D which may be present in an amount of about 15% w/w of the sustained-release coated formulation, Enteric~Release Coat 5

[00120] Optionally, any of the formulations above may include an enteric-release coating.

St,titable enteric-release. coatings include cellulose coatings such as cellulose acetate phthalate polymers or hydroxypropyl methylcellulose phthalate polymers or co-polymers of acrylic acids and their esters or methacrylic acids or their esters, such as thm;e sold under the trade mark Eudragit® including Ll00, Ll00-55 and.

SlO0, In particular embodfments, the 10 enteric-release coating may comprise poly(methacrylic acid-co-ethyl acrylate) 1:1 (Eudragit Ll00-55); poly(methacrylic .acid..,co-ethyl acrylate) 1:1 (Eudragit Ll00) and methacrylic acid - methyl methacrylate copolymer (I:2) (Eudmgit S100) ..

In a preferred embodiment, the enteric release coating is poly(methacrylic acid-co-ethyl acrylate) 1 • 1 (Eudragit LI00-55) or an aqueous dispersion thereof (Eudra;git L)0 D-55); 15

[00121] The enteric-release coatings may also comprise lubricants.

The entericreleas. e coatings may also comprise plasticisers.

The enteric-release coatings may also comprise anti .. tacking agents, [0012.2] In a particular embodiment, the enteric-release coating comprises Eudragit LI00-55. 20

[00123] In a particular embodiment, the enteric-release coating comprises Eudragit Ll00-55 and a plasticiser wherein the ratio of polymer and plasticiser is in the range 19; 1 to 4: I especially aboμt 9:1.

[00124] In a particular embodiment, the enteric-telease coating comprises Eudragit Ll00-55, plasticiser and a:n .anti-tacking agent, wherein the ratio of polymer and plasticiser is 25 in the range 19:1 to 4:1 especially about 9: 'L and the ratio of polymer to anti-tacking agent is in the range 4:1 to L:4, preferably 3:1 to 1:31 more preferably 3:2 to 2:3, for example3:2 or 1:1.

Typically, the enteric-release coatings will be present in an amount of 20-60% w/w of the enteric-i::elease coated formulation, forexample . 20 to 50% w/w, especially 25 to 40:% wlw, for 30 example about 40% w/w .or 30% w/w of the enteric .. release coated formulation.

Irt one embodiment., a coating of poly(methacylic acid~co~ethyf acrylate) LI {Eudragit Ll00-55). m;3y be present.in an amount of aboμt 30% w/w of the enteric,relc.;3se coated formulation.

Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 Formulations [00125) In some embodiments, the formulations of the invention may include further excipien:ts sμch as dispersants, solvents:, preservatives, flavours, microbial retardants and the like.

Examples. of dispersing .agents include vegetable oils~ aliphatic o.r aromatic 5 hydrocarbons (e.g. n-decane, n-hexane etc.), aliphatic or aromatic esters (e.g. octanoate) and ketones.

Solvents. that are poorly miscible with water, such as dichloromethane, chloroform and fluorinated hydrocarbons are also examples of dispersing agents.

The dispersing agents may be removed from the formulation in the process of forming the matrix and/or after preparation <,:;f the inven:tio11 but prior to administration.

Suitable preservatives .and 10 antimicrobial agents include for .example, EDTA, benzyl alcohol, bisulphites, monoglyceryl ester of lauric acid (Monolaurin), capric acid and/or .its soluble alkaltne salts or its. m.onoglyceryl ester (Monocaprin), edetate and capric acid and/or its soluble alkaline .salts or its monoglyceryl ester (Monocaprin) and edentate.

[00126] The pharmaceutical compositions used in the methods of the ptes.ent 1 S inventfon may be formulated and administered using methods· known in the art, Techniques for formulation and administration may be found in, for example, Remington: .The Science and Practice of Pharmacy, Loyd V.

Allen; Jr (Ed), The Pharmaceutical Press; London, 22nd Edition, September 2012 ; Martindale: The Complete Drug Reference, Alison Brayfield (Ed), Phannaceutical pr~ss, London, 38th Edition, 2014; and.

Handbook of Pharmac.euticctl W Excipient.s, Raymond C.

Rowe et a/(Eds), PharmacenticaJ Press, London,.

Seventh Edition, 2012 for formulation methods and reagents.

[00127] The pharmaceutical forms sμitable for intravenous use include sterile injectable solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions. 'fhe:y should be stable under the conditions of manufacture and 25 storage and may be preserved asainst reduction or oxidation an:d the contaminating action of microorganisms such as bacteria or fungi.

[00128] The solvent or dispersion medium for the intravenous solution or dispersion may contain any of the conventional solvent or carrier systems for the compound, and may contain, for example, water, ethanpl, polyol (for example, glycerol, propylen.e glycol and 3.0 liquid polyethylene .glycol, and the like), suitable mixtures theteof, and vegetable. oils.

The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

The preventi~.m of the action of microprganisms can be br.oμght about where Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 necessary by the inclusion of various antibacterial and antifungal agents, for example1 parabens, chlorobutanol, phenol, sorbic acid, thimerosal andthe like.

In many cases, it will be preferable to inclμde agents to adju,st osmolarity, for .example, sugars or sodium chloride.

Preferably, the fonn:ulation for injection will be isotonic with blood.

Prolonged iibsorption of 5 the Intravenous compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin,

[00129] Sterile intravenous solutions are prepared by incorporating the active compound in the required amount .in the appropriate solvent with various of the other .ingredie.n:ts such as those mentioned above, l::lS required, followed by ijlteted sterilization. 10 Generally, dispersions are prepared by incorporating the various sterilised active ingredient into a sterile vehicle which contains. the basic dispersion medium and the required other ingredients from those enμmerated above.

In the case of steri.le powders for the preparation of sterile intravenous solt1tions, preferred methods of preparation are vacuum drying or freeze-dryjng of a previously sterile,.filteri;Jd .solution of the active inaredient plus any 15 additional desired ingredients.

[00130] The oral formulations may be any type of solid oral dosage form, for example, tablets:; minitablets qr capsules.

For example, the formulations of the invention may be compressed into tablet form or the coated particles may be filled into a capsule.

Techniqu:es for formulation of solid oral dosage forms are known in .the art. 20 [0013 l] In a particular embodirnertt of the invention there is provided a formulation comprising a compound of formula (1) in a polymeric matrix, the polymeric matrix and compoun<i of formuht (I) mixture having. a .seal coating.

The seal-coated polymeric matrix compound of fonn:ula (I) has a sustained-release coating and the fonn:ulation further comprises .an enteric-r.elease coating..

Optionally, there is a buffer-.coating betwe<.'!11 the 25 sustained.:release coating and the enteric;..telease coating. [00132j In some embodiments, the compound of formula (I) is milrinone.

In sorne embodiments, the polymer matrix of the core is HPMC or HPC (80,000 to 120~000).. and HPMC (50 cp.s) in a ratio of2:1 to 1:21 especially L5:l to 1:1.5, more espe.cially about 1:L In s.ome .embodiments, the seal coating powprises a polymer Selected from BMPC or PVA. and a 3.0 plasticiset selected from PEG and/ot glycerine.

In .s.ome emboditnents, the buffer-coating comprises a polymer selected from HMPC ot PV A and a plasticiser selected from PEG and/or glycerine: In some embodiments, the sustained.,release coating comprises ethylcellulose.

In some embodiments, the enteric-,release cpating comprises cellulose aC1;:tate phthalate Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 pdlymers, hydroxypropyl methylcellulose phthalate polymers or copolymers .of acrylic acids and their esters or methacrylic acid and their esters. [00l3;l,) In some embodiments, the formulation may in(;lude or be administered with, sequ:entiaily and/or separately, Qther medication.s. S:.uch medications include an:giotensin 5 converting enzyme (ACE) inhibitors such as, but not limited to, enalapri.l and ramipril; angiotensin receptor blockers such, as but not limited to, irbesartan and .candesartan; calcium channel blockers such as, but not limited, tb nifedipine and diltiazem; beta blockers such as., but not limited to, metoprolol and carvedilol; diuretics such as, but not limited to, frusemide, .hydrochlorothiazide and. spircmolactone;. arid vasodilatol'i:i such as, hut not limited to; nitrates 10 and hydralazine.

[00134] In .order that the invention may be readily understood and put. into practical effect, particular preferred embodiments will now be de.scribed by way of the f91lowing nonlimiting examples ..

EXAMPLES

[00135] Sustained release formulations of compounds of formula (I), and in particular sustajned release .formulations of milrinone, which achieve the desired release profile across the different environments encountered dμring transit througp the GI tract in accordance with the invention are described in PCT application PCT/AU2012/000967, published.as WO 2013/023250 Al.

Examples off'ormulations that achieve the desired release 20 profile are described below.

The release profile of a sustained release formulation of a compound of formula (I) can be determined in accordance with the dissolution study methods describeq in WO 2013/02325.0 At .Example l: Mhlitablet formulation comprising.hydroxyp.r'.opybnethylcellulo.se matrix 25 Manufacturing Formula: Ingredients mg./Tablet For:700g Milrinone 2.0 140.0 HPMCK 100 3.0 210.,0 HPMCE50 3.0 210.0 Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 Avicel PH 102 l.3 91.0 Extra granular Avicel PH 102 05 35.0 Magnesium Stearate 0.2 14.0 Total 10.0 700.0 Step t: Wei.ghing All ingredients wete weighed sepatate1yinto a double polybag and/or butter paper.

Step 2: Sifting $ 1 .. HPMC .so cps, Miltinone , HPMC l<lOOM ijnQ Avicel PH102 were sifted through ASTM40 mesh.

Step 3: Grantilatiort I.

Above sifted ingredients (intragranular) were added into rapid mixer granulator. 2.

Dry mixing was done for 5 min. at impeller speed of 150rpm. 10 3, 420g Purified wat.er. W!:l.S then added slowly in 2 minutes and wet massing was done for 2 minutes at 150 rpm with Chopper on atJSOOrpm. 4.

Finaliywet granutes were unloaded from the.bowl.

Step 4:.

Drying 1.

Wet mass was drieci in Rapid Dryer at product temperature of 50 °C for 45 min until 15 % w/w moisture reduced to 3 ~ 4% w/w, 2.

Granules were sifted through ASTM 30 mesh Step 5: Milling (granules) 1.

Granules were milled through screen no. 1016 (1 mm) using Co~mil 2.

Step 4 and 5 granules were mixed together 20 Step 6: Sifting I.

Extragran.ular Avicel is sifted through ASTM40 mesh. 2.

Magnesium stearatewas sifted through ASTM 60 m.esh.

Step 7: Blending (Extra~granuhtr) 1, Gi:am.:1les and Extragranular Avicel were mixed together into a double qone blender for 25 $ min at 15 rpm.

Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 2.

Granules and magnesium stearate were mixed together into a double cone blender for 15 mirr~t 15 rpm. 3.

Lubricated granules wer(;) unlo.aded into a .ciouble cone blender anr.t were kept ready for compression. 5 Step 8: Compression .J .

Cadmach CU 20 compression machine was fixed with one "D'' tooling mu:ltitip punch set. a.

Upper pupch: 2mm, r:ound, standard concave (12 tips) b.

Lower punch: 2mm, round, sta:ndatd concave(l2 tips) 10 Step 9: In-process quality control testing of core Minitablets In-process Values/ observations parameters Average Minimum Maximum H&rd11eSf! (N) 20.5 15 25 Thickness (mm) 2.69 2.65 2.78 Weight{mg) 10.10 9..91 1054 Step 10: Coating (seal coat) 1.

Minitablets were sealcoated using Opadry white at 10%w/w weight Bain of film coat. 2, Coating was done μsing bottom spray container (2.4 li~rs) at foHowin~ equipment 15 pa.ram~ters: Coating process Values parameter lnl¢t temperature 45toS5°C Product temperature 38 to42°C E-;:haust temperature 3.$ toA5"C Blower speed 60 to 80% Spray pl.lmp speed ( rpm) 5 tp 15 Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 Atomization (Bars) 0.9 to 1.2 Air flow (cfm) 65 to 94 Step 11: In-process quality control testing o( seal coated Minitablets In-process Values/ observations parameters Average Minimum Maximum Hardness {N) 28.5 25 '57 Thickness (mm) 2,95 2.89 3.02 Weight(mg) lQ.90 11.12 11.35 Step 12: SJJ.s.tainec;t release coating 5 1. 75¾ wlw sustained release coating ofMjnitablets was dot:te using Aquacoat ECO 30 (Ethyl cellulose dispersion)where triethyl citrate was used as a plasticiser.

Ingredients Ratio to EC solids Total Dissolved Solids Quantities Taken (g) (TDS) ($) Aquacoat ECD 24.78 82.76 (as 30% w/w :suspension) Opadry Clear 10% 2.48 2.48 Talc 10% 2.48 2.48 Triethyl Citrate 25% 6.21 6.21 Purified water QS for 15% Solution --- 146.02 Total 36.00 239.95 2.

Coating was done by Wurster coater (bottom spray container 2.4 liters) at following 1 O eq_uipment parameters: Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 Co~.ting proces.s Vab1es parameter Inlet temperature 50 to 60°C Product temperature 38 to 42"C Exhau.st temperature 35 to45°C Blower speed 60to8:4% Spray pump speed (rpm) 5 to 15 Atomization (Bars) 0.9to 1..2 Air flow (cfm) 65 to 95 3.

After coating, euring was done for 2 hours at product temperature around 60°C in Hot air oven. 5 Step 13: In-process quality control testing ofsustained releas.e coated Minitablets In-process Values/ observations parameters Average Minimum Ma:ximum Hardness (N) 37 30 45 Thickness (mm) 3.03 2.98 :us Weight (mg) 11.76 U.65 11.8.8 Step .14: Buffer c(U)ting 1.

Buff er coating was done at 5% w/w weight gain using Opadry white. 2.

Coating was done using bottom spray container {2A liters) at following equipment 10 parameters: Coating process Values parameter Inf et temperature 45 to55°C Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 Product temperature 38 to42°C Exhaust tern perature 35 to 45°C Blower speed 60to.80% Spray pump speed (rpm) 5 to 15 Atomization (Bars) 0.9to 1.2 Air flow (cfm) 65 to 94 Step 15.} Ente.ric co;,1ting I.

Enteric coating of buffer coat.ed .minital?lets was done by using Eydragit LJO D55 polymer at JO% w/w weight gain where talc was used as. an anti~tacking agent and 5 tri:ethyl citrate-was used as a plasticiser.

Ingredients Ratio to Eudragit TDS (g) Quantities Taken (g) .solids Eudragit 130 055 90.00 300.00 Talc 50 45.00 45.00 Triethyl Citrate 10 9.00 9.00 Purified water QS for 20% .,.._;..,, 366.00 Solution Total 144.0 2.

Coating was done μsing bottom spray container (2.4 L) at following equipment parameters, Coating process Values parameter Inlet temperatQ.re 28 to346C Product temperature 25 to 30°C Exhaust tern perature 28 to 32°C Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 Blower speed 50 to98% Spray pump speed (rpm) 5 to 14 Atomization (Bars) 0.8 to 1.3 Air flow (cfm) 60 to 100 3.

After coating curing of Minitablets was done for 2 hour at product temperature 40°G in hot.air oven.

Step 16: In-process quality control testing of enterk coated Minitablets In-:process Values/ observaticms parameters Average Minimum Maximum Hardness (N) 54.5 42 67 Thickness (mm) 3.23 3J8 3.26 Weight(mg) 16.lO 15,80 16.45 Example 2: Minitabletformulation comprising. hydrophilic matrix of natural gums ll1gred,.ients Quantity of matedaJs (g) Milrinone 50.25 Xanthangum 37:50 Locust gum 37.50 Avicel PH102 49.75 Lactose, Anhydrous 62.50 PVPK30 8.75 Aerosil 2.50 Magnesium stearate 1.25 Total 250.0 Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 Step 1: Disp~nsing All the ingredients were weighed separately into double polybags.

Milrinone quantity was weighed based upon following c;alc:ulation: Assay ofMilrinone = 99.70% (as is basis). s Mg/ tablet ofMilrinone = Theoretical quantity of.Milrinone (mg/tablet) x 100 I Assay of Milrlnone = 2.00 x 100/.99,7= 2 .. 01 mg The quantity of API (active pharmaceμtical ingredient) was adjusted with microcrystalline .cellulose, Step 2: Sifting l O l, All the.ingredients ex9ept magnesium stearll,t~ were sifted thrc:iugh ASTM 40 ml:.lsh, 2.

Magnesium stearate was sifl:ed through ASTM 60 mesh.

Step 3: Blending I.

Ingredients 1 to 3 from above.

Table were transferred into a 0.5L Turbula Shaker Mixer container and blending was done for lOmin at 49 rpm. 15 2,.

Ingredients 4 to 7 were then added iind further blending was done for 1 O min at 49 rpm, 3.

Ingredient 8 was then added and h1brication was done for Sm in at 49rpm. 4.

Blend was finally collected into a double polybag.

Step 4: Compression 20 1.

Cadmach CU 20 compression machine was fixed with one 1'D'' tooling multi tip punch set. a.

Upper punch: 2mm, round, standard concave {12 tips) b.

Lower punch: 2mm, round, standard 'cone.ave (12 tips) 2.

Tablets were compressed using Cadmaeh CU 20. compression machine.

Compression 25 was done manually by rotating hand wheel to obtain enough hardness and thickness, Step 5: In-process qnality control testing of core Minitablets Values/ observations In-process parameters Average Minimum Maximum Hardness (N) 28 23 35 Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 Thickness (mm) 2.41 2.42 2.53 Weight(mg) 10 9.0 1-0.0 Frlability Nil Step 5: Seal coatin.g 1.

Seal coating of minitablets was done at 3% w/w weight gain using Opadry white as a film coating agent Opadry film coating system powder was added to water and. mixed for 45 minutes with a propeller $tirt:cr.

The coating suspension can be rnade according to the manufacturer's instructions. 2.

Coating was done by using Gansons coater (GAC-275) at the following parameters: Coating process patameter Values Inlet temperature 60 to 62.3°C Product tern p.erature 38 to40°C Exhaust temperature 39to 40°C Spray pump speeq .(rpm) 2 to 3 Atomization air (kg/cm.2) 0.2 Fan pressure (kglcm:t) 0,2 Step 6: ln-'p.rocess quality control testing.of seal coated Minitablets Values/ observations In-process parameters Average Minimum Maximum Hardness. (N) 32 29 38 Thickness (mm) 252 2.50 2.55 Weight(mg) 10.3 10:1 10.5 Friability Nil Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 Step 7: Sustained release (SR} coating of Minitahlds J.

Mini tablets were 5% wlw SR coated by ethylcellulose dispersion (AquaeoatECD30D) usin~ triethyl citrate as a plasticiser ..

Ingredients Quantities (g) AquacoatECD30D 140.28g Triethyl citrate 8.42g 2.

Coating was done by using Gansons coater (GAC-275) anhe following parameters; Coating pro~ss parameter Values Inlet temperature 56 to 59°c Product temperature 38to40°C Exhaust temperature 39 to40°C Spray pump speed (rpm) 2to2.5 Atomization air (kg/cm1) 0.2 Fan pressure (kg/cm") 0.2 .3.

Curing of minitabletswas clone at 60°C for2 hours in vacuum oven (without vacuum).

Step 8: ln-'p.rocess quality control testing.of SR coated Minitablets ln~process Values/ observations parameters Average Minimum Maximum Hardness (N) 37.4 34 41 Thickness (rnm) 2.65 2.61 2.68 Weight(mg) 10.62 10.2 10.9 Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 Step 9: Buffer'Coatiug 1.

Seal coating of minitablets was done at 5% w/w weight gain using Opadry white as a film coating agent, as des.cribed in ii;tep 5 ofaatch-018. 2 ..

Coating was done by Wurste:r coater 2.4 L .container (GPCG 1.1) at the. following 5 parameters: Coatin~ process parameter Values Inlet temperature 58to 60.3°C Product temperature 39 to 40°C Exhaust temperature 39 to 406C Spray pump speed (rpm) 2 to4 Atomization afr(kg/cmt) 0:2 Fan pressure (kg/cm:·) 0.2 Step 10: In-p.rotess quality co.n.trol testing of Buffer coated Minitablets Values/ observations Tn-process parameters Aerage Minimum ~mum Hardness (N) 41.7 37 46.

Thickness (mm) 2.74 2.70 2.78 Weight(mg) lLl.5 11.03 11.23 Step l1: Ente.ric coating 10 1.

Enteric coating of buffer coated mini tablets was done by Eudragit L30D55 as a enteric polymer along with triethylcitrate asa plasticiser and talc as an anti-tackiqg agent.

Ingredients Quantities (g) EUdr1\git L30D55 333.33g Triethyl citrate 10.00g Talc 50.00g Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 2.

Coating was done by Wurster coater 2.4 L container (GPCG 1.1) at the following patarneters, tCl provide an enteri.~ co.at of 40% w/w.

Coating process p.arameter V:alues Inlet temperature 27 to 32°C Product te:mperatute 26 to 28°C Exhaust temperature 26 to28°C Blower speed{%) .58to 92 Air flow (cfm) 70 to 134 Spray pump SJJeed (rpm) .2to 3 Atomization air (Bars) 1.0 to 1.2 Step 12: In-process quality con.ttol testin3 of enteric coated Minitablets Values/ observations In-process parameters Average Minimμm.

Mipdmum Hardness (N) 89.89 74 107 Thickness (mm) 3.08 3.03 3.14 Weight (mg) 15.61 15.58 15;65 E:J:ample 3: Formulation of Milrinone beads Sr ..

No, Name of equipment/ instrument Manufacturer/ supplier Ol Weighing balance Sartorius 02 Sieves Lab supplies India Pvt.

Ltd. 03 Propeller Mixer Hally Instruments 04 Wurster coater 2A L (GPCG LI) Glatt 05 Homogenizer Silversons Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 1 06 I Vacuum oven I Servewell instruments Ingre<lients Manufacturer % ofSolid:s Quantities (g) Milrinone Chemzam Pharmatech 61 45.00 Kollidon 30 (binder) BASF 6 4.50 Opadry white Colorcon 33 24.50 Puri.fled water FOC In,-house 495.23 Step 1: Drug layering 1.

Procedure for drug dispersion preparation: a.

Milrinone, Kollidon 30 and Opadry white were sifted through ASTM 30 mesh.

All ingredients were collected into a single polybag. b.

Purified water was weighed into a: beaker and was placed under propeller mixer to create Vigorous vortex. c.

Slowly ingredients from step a. were added into water maintaining vortex.

After complete addition, propeller mixer speed was reduced to avoid vortex, Mixingwas done for 30 min. 2. 0mg layering by Wurster coater a. i) ii} iii) b.

Wurster coater was equipped with following accessories,. 2.4 Lbottom spray container Wurster po}ijm:p at 20 mntheight 1.2 mm liquid nozzle insert 350.0g of sugar spheres.(30/35#) [Werner~ Germany] were transferred into the container. c..

St1gar spheres were warmed to reach pro,du.ct temperature of 40°C. d.

Drug dispersion was sprayed on sugar spheres at following parameters recorded overthe period of255 min coating time: Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 CoaJli1g process p.~t'amete.r Values Inlet temperature 45 to 506C Producttemperature 39 to42°C Exhaust temperature 36 to 41°C Blower speed .60 to 77% Spray pump speed (rpm) 2fo6 Atomization ·(Bars) o.s to 1.2 Air flow (cfm) 73 to 92 e.

After coating, peristaltic pump was stopped and product temperature was allowed to reach 440:c and then coating process was stopped. f.

Total yield was 390.43g, S There are two methods of determining% w/w weight gain during spray ,coating of beads.

Method A: Weight gain can be calculated only after complete coating process and then the following formula can be applied to find o.ut weight;gain: % w/w practical weight gain achieved Final weight - Initial weight/ Initial weight :X 100 10 .Method B: Coating dispersion/solution shall be prepared exactly as per described except. 40% w/w for enteric coating with 10% extra solμtion to cov<::r the in-process losses.

Since the solution .quantity equival!;lnt to 40% w/w is sprayed .cornpletely on the beads, ids consid~ted that final weight gain achieved is 40% w/w. 15 Step 2: Seal coating of drug layered beads (10% w/w) L GPCGLI was equipped withfollowing accessories, a. 2.4 L hotto:tn spray container b.

Wurster column.at 20 mm height c. l.2 mm liquid nozzle insert 20 2,.

Preparation of coating solution Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 Jp;gredieots Manufacturer Qua;11tities (g) Opadry white Colorcon 42.9 Purifi.ed water FDC in.,house 493.35 a. A vigorous vortex was created into a weighed quantity of water and slowly Opadry white. was added into it.

After complete addition, speed was reduced to avoid vortex.

Mixing was donefor45 min. b.

Coating solution was sprayed on 390:0 g of drug layered spheres at following parameters recorded over the period of 260 min. of coating time: Coa;ting proc.ess pftra:meter Set Values Actual Values Inlet temperature 47 ±5°C 44 to51°C Product temperature 40±39C 39 to429C Exhaust temperature 40 ±3°C 39 to4l°C Blower speed. 57to70% 57 to70% Spray pump speed (rpm) 2 to 7 2to7 .A tomizati.on (Bars) 0 .. 8 to 1.4 0.8 to.1.4 Air flow (cfm) NA 73 to 92 Note: Before starting coating, beads were wamied to.reach 40°C product temperature. c.

After coating; temperature was allowed to reach 45°C and then coating process was stopped.

Total yield was found to be 412.0g.

Step 3A: First Layer Sustained release (SR) eoating of seal coated beads (using Eudragit R.$30D and Eudrag.~t RL30D at 9:1 ratio) to pre11Jre be.ads with l0%w/:w coating. 15 1, Preparation of coating dispersion: Ingredients .

Manufacturer Ratio to total TDS (g) Quantities .

Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 E.udragh $Olids (g) (%) Eudragit RS30D Evonik 90 22.91 76.37 Eudragit RL30D Evonik 10 2.55 8 .. 50 Talc Luzenac Phanna 50 12.72 12.72 Triethylcitrate Sigma-Aldrich 20 5,09 :5;09 Purified water FDC In~house .272.32 Total 375.00 a.

Eudragit RL30D and Eudtagit RS30D were mixed.together into a beak.er .. b.

Talc and triethylcitrate were homogenized for 10 min. at 4500 rpm in purified water at 4500 rpm of Homogenizer, c.

Polymer dispersion from step a. was then added into b. excipient dispersion ·and mixing was done fo:r 30 min. at380 rpm using propeller mixer. 2. 412:0g of beads were transferred into the 2.4 L bottom spray container of GPCG L 1 and warmed to reach 2S°C. 3.

Coa.ting was done on bead.s (It the. following parameters recordecl over the period of 303 Jilin. of coating process to achieve a first layer SR coating of JO% w/w.

Coating process parameter Values Inlet temperature 27to 31°C Product temperature 25 to 27°C Ex:haust temperature 25 to27°C Blower speed 58 to 71% Spray pump speed (rpm) 2to.3 Atomization (Bars) 0.8 to 1.0 .Air flow (qfm) 7J to 98 Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 4.

Total yie{(i was 451.0 g. 2LOO g of beads were cured at 50°C for 30 minutes in vacuum.oven without vacuum.for analysis (dissolution test).

Step 3B: First Layer Sustained release (SR) coating o.f seal coated. beads (using Eudragit 5 RS30D and EudragitRL30D at 9:1 ratio) to prepare beads with 15o/o w/w coating.

Beads prepared according to Step 2 were coa:t:ed with Sustained Release coating dispersion of Eudragit RS30D and Eudragit RL30D at 9: 1 ratio described in Step 3A accord\ng to the prooedures describl:)dtherein but for sufficient.duration to achieve a Sustained Release cQating .of 15% w/w on b.eads: 10 Step 3C: First Layer Sustained release (SR) coating of seal coated beads (using Eudragit .RS30D and EodragitRL3UD at 9:1 ratio) to prepare be11ds with 200/c.w/w coating.

Beads prepared according to Step 2 were coate4 with Sustained Release coating dispersion of .Eudragit RS.30D and Eudragit RL30D at 9:1 ratio described in Step 3A aco.ording to the procedures described therein.but for sufficient duration to.achieve a .Sustained Release coating 15 of 20% w/w on beads.

Step 31): First Layer Sustained release (SR) cqating of se.al cqated beads (using EudragU RS30D and Eudragit RL30D at 9: 1 ratio) to prepare beads with 25o/o w/w coating.

Beads prepared according to Ste.p 2 wete coated with Sustained Release coating dispersion of Eudragit RS30D and Eudragit RL30D at 9: l ratio described in Step 3A according to the 20 procedures described therein butfor sufficient duration to achieve a .Sustained Release coating of 25% w/w on beads.

Step 3E: Second layer Sustained release (SR) coating of First Layer SR beads using .Eudragit RS30D to prepare beads with total SR 30% w/w coating 1.

Preparation of coating dispersion: 1n.gredi¢nts Manufacturer Quantities .(g) Eudragit RS30D Evonik 84.87 Talc L:uzeμaq PhaflJl.a 1.2.72 Triethylcitrate.

Sigmac.Aldrich 5.09 Purified water FDC Inhouse 169,64 Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 a.

Eudragit RS30D was added to a beaker. b.

Talc and triethylcitrate were homogenised for lOmin. at 4500 rpm in purified water at 4500 rpm or Homogenisef. c, Polymer from step a. was then added .into b. excipient dispersion and mixing was don<;; for 30 min. at 380 rpm using propeller mixer, 2.

Single Layer SR headsfrom Step 3D were transferred into the 2..4 L bottom spray container of GPCGl . 1 .and warmed to tea.ch 28:>C. 3.

Coating was done on beads at the following parameters recorded over a sufficient 1 O period of coating process to achieve a second layer SR .coating of 5% w/w and a total· SR coating of 3 0%.

Coating process parameter Values Inlet tem;perature 21 to 31°c Product temperature 25 to 27°C Exhaust temperature 25 to27PC Blowerspt;ed 58to 71% Spray pump speed (rpm) 2to3 Atomization (Bars) O.Sto LO Air fl.ow (cfm) 71 to98 4.

Total yield of beads. were cured .at 50°C for 30 min. in. vacuum oven without vacuum for analysis (dissolution test). 15 Step 3F: Second layer Sus:tained reteas.e (SR) coating of First Layer SR beads using Eudragit .RS30D to prepare beads with total SR 40% w/w coating Beads prepared accordi11g to.

Step 3D were coated with Sustained.Release coating dispersion of Eud:ragit RS:30D described in Step'3E according:to the procedu:res described therein but for sufficient duration to achieve a second layer Sustained Release coating of 15% wtw and a 20 total SR coating of 40% w/w.

Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 Step 4: Buffer 'Coating of SR coated \leads (at 10% w/w weight gain with Opadry white) 1.

Preparation of coating solution Ingredients Manufacturer Quantities (g).

Qpadiy white Colorcon 43.44 Purified water .FDC in..:house 680.56 PrepcJration procedure was same asJ:hat of step 2 (2) abc;,ve. 2.

Coating was done .on beads using GPCGI .1 bottom spray assembly at the following parameters recorded. over the period. of l 80min. of coating process.

Coating pr.oceu ptramet.er Values Inlet temperature 43 to.52°C Product temperature 39to 43°C Exhaust temperature 35 to 43°C Blower speed 63 to 72% Spray pump speed (rpm) 2to 4 Atomization (Bars) I.Oto 1.2 Air flow ( cfm) 72 to 91 Step 5: Enteric coating with Eudragit L30D55 at 40% w/w enteric weight gain L Coating solution prepar~tion Ingredients Manufattnrer Quantities (g) Eudragit.L30D55 Evonik 395,00 Talc Luzenac Pharma Il.85 Tri ethyl citrate Sigma-Aldrich '59.25 Purified water F'DC Inhouse 797~90 Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 Note: Above solution was.based on 395 .. 00g of pan load for coating and 200/4 extra quantities considering losses. 2.

Talc; and triethylcitrate were ho111cigenised in water :for lO min.

Then this excipient dispersion was poured slowly in.to EudFagit L30l)55 dispersion while stirring slowly at250 rpm.

Finafly speed was reduced to 200 rpm and mixing was done for 30min. 3.

Initially, beads were warmed to reach product temperature of 28°C and then coating was started. whic;b lasted for 765 min and the parameters record.ed .are g;iven below, Coating process parameter Values Inlet tempeni:ture. 28to32°C Product temperature 26 to 28°C EX:hau.st temperature 2p to 29°C Blower speed 63 to 75% Spray pump speed (rpm) 2to7 Atomization (Bars) 1.2td 1.5 Airflow (cfm) 69to 96 3.

Finally; curing. was done for 2 hours between 40 to 43°C in the equipment Total yield. was 543. 00 g at the end of the process.

Exa01ple 4:.

Immedi11~ Rele11se Milrin~ne F9rmulation Sr.110 Composition %w/w Amount/tablet (mg) 01 Milrinone 20 02 02 Avicel J:lH 102. 30 03 03 Lactose anhy:drous 45 4.5 04 KoUidon 30 3,5 0:35 05 Aerosil 01 0.1 Date Rei;ue/Date Received 2022-08-23 s IS WO 2016/101024 PCTiAU2015/050820 06 I Magn~sium .stea:rate 0.5 0,05 Total Procedure: L 2. 3. 4. 100 10.0 Weighing: Alt the listed ingredients were accurately weighed into double lfne polybags, labelled and tagged.

Sifting; All the excipients anti Mi1rinone except magnesium stearate were sifted through ASTM40 mesh.

Magnesium stearate. is sifted through ASTM 60 mesh.

Blending: Milrinone. and other excipients except magnesium stearate wer.e added illto Tutbula shaker mixer and mixed for 15 min.

Magnesium $1earate was added into the blend and mixed for 5 min.

Compression: The lubricated blend was compressed using circular B tooling punches with 2 mm tips: In process checks: W.eight of Tablets: 10 mg Hardness: 30 N - 40 N 20 Thickness; 2Amm-2.S mm Fri.ability: 0.486 % Disintegration test : 4 to 5 min.

Example 5: pH solubility studies on Milrinone 2$ Aim: Tq perfpnn thesatu.rated. solubility ofMilrinone in <.iifferentbuffers.

Buffe.rs: l. pH 1.2 - Hydrochloric Acid Buffer 2. pH 4.5 - Acetate Buffer Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 3. pH 6.8 - Phosphate Buffer 4. pH 7.4 - Phosphate Buff er, Procedure: i. 2 mt. buffer solutio.rt is. placed into. a 8. mL USP Type I clear glass vi.al (with screw cap 5 and PTFE septa) 2. 10 mg of Milrinone is added in each vial and the vial is. shaken to dissolve the compound. 3.

The addition ofMilrinone is continued till th.efonnation of saturated solution. 4.

The pFf of the saturat(;'ld solution is measured after the addition. ofMilrinone. 10 5.

If there is any difference in pH more than 0.1 units is obsef\/ed when compared to the initial pH, the pH was adjusted with acid or base respectiv:elyto bring it to the initial pR 6.

The vials are closed with screw cap and kept for mixing using rotary tube shaker for 24h, 15 Note: The vials are observed at frequent .intervals and if the sotution is clear, further amount of Milrinone is added to make a saturated solution.

Results: The solubility oflvfilttnone atd~fferent pH buffers S:r.

No.

Buffer Saturated solubility .(mg/mL) 1 pH 1,2 Hydrochloric Aci cl Buff er 25.385 mg/mL 2 pH 4 .. 5 Acetate Buffer i.826 mg/mL 3 pH 6.8 Phosphate Buffer 0:742 mg/mL 4 pH 7.4 Phosphate Suffer 0.603 mg/mt 20 Con~Iusion: The solubility results indicate thit Milrinone is highly soluble in acidic pH. ~d the solubility Js deoreased gradually with increase in pR Thus the. discriminatory dissqlution media for Milrinone tabl.ets should be pH 6,8 ot 7A.

Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 Example 6: Dbsolution Profiles .qf .Formulations The following procedure was t1sed to determine if a sustained release formulation of a compound of Jommla (1) would achieve the desired release profile across the different .environments encountered durin~ tran$it through thei GI tract.

The desired sustained~release 5 formulation provides zero order kinetics of drug delivery (i,e; a linear delivery with respect to time).

Controlled release of drugfrom the dosage form relies upon two proce.sses: dissolution and release . .Reagents 1.

Potassium dihydrogen orthophosphate (AR grade) 1 O 2.

Hydtochlorlc acid (AR grade) 3.

Sodium hydroxide (AR grade) 4.

Methanol (HPLC grade) 5..

Water (HPLC grade) Dissolution p.aramete.rs (For Acid Stage) 15 Medium Tempeniture Apparatus.

Rotational speed Sampling time O. lNHydrochlorlc acid, 900 mL '37.0 ±: 0.59C USP Apparatos II (paddle) SO rpm 2.h 20 Preparation of O.lN Hydrochloric acid pH 1 Diluent and Dissolution Buffer 85mL of concentrated hydrochloric.acid in 1000 mL of water, mix well.

Preparation of pH 6.8 Dilq.ent Dissolve 6,8 g Potassium .dihydro,gen orthophosphate and 0.9 g of sodium hydroxide in 1000 rnL of water a:nd adjust the pH to 6..8 with sodium hydroxide solution or orthophosphoric 25 acid.

Preparatic:m 9f Standard ,olutionJor pH l @alysis of Milrinone Accurately weigh and transfer about 55 mg of Milrinone working standard into a 100 mL volumetric flask.

Add about 10 mL of methanol and sonicate to dissolve then make up to the mark with O. lN hydro.chloric acid.

Dilute 5 mL of above s.olution to 100 mL with O..lN 30 hydrochloric add.

Further dilute 5mL of above solution to 100 mLwith 0. lNHCl.

Preparation Qf sample solutio11 Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 Transfer the content of one capsule in each of the six dissolution vessels and start the dissolution test in (UN H.Cl Dissolution Buffer.

At the specified time withdraw about 10 mL of the aliquot from each. dissolution vesseL Further tlilμte 4 mL of above soluticm to 10 mL with OJN HCI.

Diluent, S Dissolution parameters (For 0.lN HO Buffer Stage) Medium Temperature Apparatus Rotational speed 10 Sampling time 0, lN HCl Buffer, 900 mL 37,0:±: os~c USP Apparatus II (paddle) 50 tpm 1 h,2h.

Where test article is to be .exposed to 0.IN HCI Dissohition Buffer for .2 hours and then expose<! to pH 6.S Buffer for 12 hoursj the test article is. removed from the diss.olution vessel, washed briefly with water and placed immediately into the required dissolution vessel .cantaining the pH 6.8 Buffer. 15 Procedure Measure the absorbance of standard (in duplicate) and sample solution using dissolution medium as blank at 265 :rm1.

Calculation 20 % of drug released Wherein: AT AS 25 DS OT p C AT OS p 100 AS OT 100 C Absorbance•of sample solution.

Average absorbance. of standard solution.

Dilution factor of the standard solution. .Dilution factor of the sample solution.

Percent potency ofMilrinoneworking standard, on as is basis.

Label claim of Milrinooe pet capsule (in tng).

Dissolution p.arameters. (For Buffer Stage) 30 Medium Temperature Date Rei;ue/Date Received 2022-08-23 pH 6.8 Buffer, 900 mL 3?.0± 0,5°C WO 2016/101024 USP Apparatus II (paddle) SO rpm PCTiAU2015/050820 Apparatus Rotational speed Sampling time l h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h and 12 h. 5 Preparation of pH 6.8 Dissolution Buffer an.d Diluent.

Dissolve 6,8 g Potassium dihydrogen orthophosphate.and 0.9 g.of sodium hydroxide in 1000 mL of water and adjust the pH to 6.8 with sodium hydroxide solution or orthophosphoric acid.

Preparation of.Standard solution 10 Accurately weigh and transfer about 55 mg of Milrinone working standard into a 100 mL volumetric .flask.

Add about 1 O mL of methanol and sonicate to dissolve., then make :up to the mark with pH 6.8 Diluent.

Dilute 5 mL of above solution to 200 mL with Diluent.

Preparation of sample solution Transfer the content of one capsule in each of the six dissolution vessels and start the 15 dissolution test in pH 6.8 Dissolution Buffer.

At the specified time withdraw about 10 mL of the aliquot from each dissolutionvesseL Further qilute 4 mL ofabove solution to 10 mL with diluent.

Procedure Measure the absorbance of standard (in duplicate) and sample Solution using dissolution 20 medium as blank at 265 nm.

Cal~ulatio.JJ. % of drug released 25 Wherein: AT AS DS DT 30 P C = AT DS p AS DT 100 Absorbance of sample solution ..

Average absofbance or standard solution.

Dilution factor ofthe standard solution.

Dilution factor of the sample solution. 100 C Percent potencyofMilrinone working standard, on as is basis, Label claim. of JMilrinone per ~apsule (in mg).

Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 Example 6 Table 1: Dissolution profile of Enteric coated minitablet of Example l in 0.1 N HCI followed by pH (i.$ phosphate buffer Example 1 Time (30°/o Enteric Coat) 0 0 I 0 2 0 3 8 4 19 5 29 6 42 7 55 g 67 9 76 10 85 11 93 12 97 13 102 14 104 R2 value 0.974 Assay l05.4% w/w 5 C011dusion: The fonnulatio11 of Etttmple 1 sh,owed zero order release profiJe and. !lhowed max:imu.m release up to 105% {the assay of this batch is 105%), The enteric coating of 30% was sufficient to prevent the drng release in stomach.

Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 This dissolution profile data demonstrates the in vitro zero order release of Milrinone from. a formulation of the invention over 12 hours, and a sustained release profile which is consistent with intravenously administered Milrinone.

In pa,rticular, dissolution profil\;:s show zero order release of Milrinone at pH 6:8 over about 12 hours (Le.

Ri > 0.9) tc, provide about 10:0% 5 release of the active pharmaceutical irtgredient.

This sustained release profile is consistent with providing a plasma exposure in patients that is similar to that achieved by a dosing regime with intravenous formulations ofMilrinone Example 7: Pba.rmacokinetic Study of HPMC ER Milrinone Formulation (Example 1) 10 versus IRMilr'inone Formulation (Example 4) in Dogs Experimental Materials Pentagastrin a:nd Ail1mo:nium formate were purchased from Sigma: (St.

Louis, Missouri).

Amrinone was purchased from. LKT Lab (St.

Paul, Minnesota).

Milrinone formulations 15 Example 1 (ER milrinone) and Example 4 (IR milrinone) were prepared as described.

Gelatin capsules were received from Torpac (Fairfield, New Jersey).

Dichloromethane and high performance liquid chromatography (HPLC) grade acetonitrile was purchased from Honeywel] (Muskegon, Michigan).

Water was obtained using a: Millipore system (Billerica,.

Massachusetts).

American Chemical Society grade fotmic acid was received from Aeros. 20 Organics (New Jersey).

Animals Purpose-bred female beagle dogs (Marshall Farms, North Rose, New York) weighing between 8 and 1 I kg were housed unrestrained in accordance with Association for Assessment and A:c.creditatioh of Laboratoiy Animal Care International (AAALAC) 25 guidelines.

Dogs were maintained on 300 g of 21% protein dog diet #2021 (Harlan Teklad, Madison, Wisconsin) once daily when not on study<Prior to each study, dogs were fasted overnight.

All studies were cooducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Counci111996). 30 Gastric Pb-modifying Treatment and Dosing Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 Pentagastrin was dissolved in saline (0.024 mL/kg) and administered via IM injection in the animaPs right or left hind leg (6ug/kg) 30 minutes prior to test article administration, Following dose administration, the area was gently m~saged.

The oral rnil'rinone doses were prepared by counting minita1'lets into size-3 gelatin capsules, 5 One or two milrinone filled capsule was orally administered to each dog, followed by water (10 mL}to assist.swallowing.

The experiments were conducted in two groups of three dogs each.

Asses.sing Pb-dependent Absorption B.eagle dogs (n = 3) wete dosed in a nonrandomized, crossover d.esign, with at least a. l week 10 washout between treatments.

All animals were fed their normal daily ration. of food the day prior to dose administration..

All animals were fasted at 17:40 the day prior to TR dose administration and at 18:22 the day prior to ER dose administration.

All l:lllimals were fed following the 3hr collections.

The IR and ER milrinone formulations were orally administered (5 mg/kg in gelatin. capsules) to pentagastrin-pretreated animals.

Serial blood 15 samples (2 mL) were collected from the jugular vein into potassium ethylenediaminetetraacetic acid tubes before dose and 0.5, 1; l.5, 3, 6, 9, 11, 12,. 14,. 18, 24., 30, 36, 42 and 48 h after dose, Blood samples were lcept on ice until processed for plasma.

Blood samples were centrifuged at 3200 RPM for 10 minutes at approximately 5°C.

Plasma s1;1.mples were directly transferred to 96-well plate tubes (I.I mL).

Ph1gs were'placed on the 20 tubes.

Plasma samples were stored at ~20 ± 5 °C until analyzed by liquid chromatographytandem mass spectrometry (LC/ MS/MS).

Sample Analysis Milrinone was extracted from dog plasma usiug elichloromethane protein precipitation. 25 Calibration curves were cQnstructed using commercial beagle dog plasma spiked with indiyidual test compounds over the analysis ran$e of 0.5-500 ng/mL.

Fifty mi~roliterS' of each plasma sample and .internal standard (amrinone, 2 ng) were added to .micro centrifuge tubes.

One volume (1.0 mL) of dichlommethane was added to each tube, and the rack was vortexed for approxima:tely 6 min to aid in the precipitation.

The tubes were centrifuged a:t 30 13,000 rp:m at. room temperatQrefor 6 min.

Supematants (800 uL) were transferred to a clean culture tubes and dri.ed down at room temperature using Turbovap, The recon solution (150 uL .of mobile phas.e A) was a:dded to the dried tubes and .subjected to LC/MS/MS. artalysis.

Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 Sample analysis was perfonned with 20 uL sample injection .on an AB Sciex API--4000 triple quadrupole mass spectrometer.

Analytes were separated using a Betasil C8 (100 x 2.1 nun) 5μ (Thermo Electron Co).

Chromatographic conditions were 10% mobile phase A (1/9, a:cetonitrilelld mM ammonium 5 formate, pH 3.0) and 90% mobile phase B (0.1% formic acid in acetonitrile) at 0.3 mi/min, ramped to 80% MP A in 1.5 min, then to 90% MP-A in 2 min.

The system was returned to initial over 10 secs., and the column was reequilibrated at initial conditions for IA rnin, LC/MS/MS analysis was carried out at positive ion. mode using multiple reaction monitoring (MRM) transitions for tnilrirtone (m/z 2l2-+ 140) and thf;'l internal standard (amrinone, m/z 10 188-+133).

Data analysis used linear fitting with 1/x:2 weighting: All analytical results were within acceptable specifications, including perfonnance of quality control samples, reprooucibility, linearity; accuracy, and precision, The lower limit of quantitation was established at 0.5 ng/mL using the predefined criteria for reproducibility; accuracy, and precision. 15 Pharmacokinetic Analysis The plasma concentrations versus time profiles obtained after oral administration of IR Milrinone and ER Milrinone were analyzed μsing noncomp.artmental analysis (WinNonlin'> Professional, Version 5.2 software; Pharsight Corp., Mountain View, CA).

Cmax was defined a:s the highest observed plasma concentra#ort, and Imax was. the tim.e at which Cmax 20 occurred.

The area u.nder the concentration-time curve from zero to the last quantifiable time point (AUCO-t) was calculated using the Linear Up/Log Down method. AUCO-t was extrapolated to infinity and reported as AUCO-oo.

RESULTS 25 Effect of Different Trea tnients on Gastric Ph Table 1 and Fig:ure 5; provides the pharmacokinetic values for the IR Milrinone and ER Milrinone formulation. dosing in groups of 3 dogs.

This data shows that the .ER .Milrinone achieved a reduced Cmax in comparison to the IR Milrinone (650rtg/mLvs ~180ng/tnL); that the ER Milrinone. a similar overall exposure as measured by theAUC in comparison to the IR 30 Milrirtone (675 lng•hr/mL vs 9478ng•hr/mL), artd that the ER Milrinone maintained stable milrinone plasma concentrations over a .12 hour period.

Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 Example 7 Table l.

Summary PharmacokineticParameters of Milrinone in the.PlaJma of Female Beagle Dogs Following 5 mg/kg PO Administration of lmmediate or E:dended Release Tablets in a ~Iatin Capsule Grou.pl Group2 Parameter (units) (Immediate Release) (Extended Release) Mean SD Mean SD C1riax (ng:/mL) 3180 1173 650 113 tmax{hr) LOO 0.50 7.67 4 .. 16 AUC(O-t) 9478 3695 6751 2150 .(ng•hr/mL) . .

AUCco-co) 9488 3696 6759 2152 (ng•fa/mL) h12 (hr) 4.97 0.77 l.71 0.83 Vz'-obs 4029 965 4143 1049 (mL/kg) Cl obs 586 234 802 298 (mL/hr/kg) 5 PK Paratneter Descriptions tmax: AUC<o-tf .AUC<o,,:,): 10 tm: Vz obs: Cl"""obs: Maximum Observed Concentration Time Point at Ctn.ax AUC to the last non-zero concentration (tis the corresponding time) AUC(O.c,;,) = AUCto-t} + AUC (t-,,:,) Half-life; time taken for dmg plasma concentration to fall by one- half, Observed Volume of Distribution Observed .Clearance Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 Example 7 Table 2, Individual Female Be1,1gle Dog Plas.ma Concentrations of Milrinone Following a Single .5 mg/kg PO .Administration as Immediate Release Ta.blets in a Gelatin Capsule Dog#l Dog#2 Dog#·3 Animal Animal Arthnal Weight 5.571 Weight 9.257 Weight 9.653 (kg) (kg) (kg) Dose Dose .Dose 28 46 48 {mg) (mg) (mg) Actual Actual Actual I;)osage 5.03 Posage 4.91 Dosage 4.97 (mg/kg) (mg/kg) (mg/kg) Sample Sample Sample Mean '.fime SD Cone.

Cone.

Cone.

Cone. (hr) (n.g/mL) (ng/mL) ( ng/.mL) (ng/mL) (ng/mL) 0 BLQ .BLQ BLQ BLQ NIA 0.5 1840 2440 3570 2617 878 1 1750 2590 3680 2673. 968 1.5 1210 4020 2420 2550 1410 3 717 1990 737 1148 729 6 142 574 331 349 217 9 214 217 71,0 174 89.6 11 110 71.3 118 99.8 25.0 12 73.0 87.8 81.2 80..7 7.4J 14 51.3 89 58.2 66.2 20.1 18 .30.6. 31.5 30.5 30..9 0..551 24 5.45 16.3 8.46 10.1 5 .. 60 Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 3() 1 .. 63 2.34 36 '2,10 4.22 42 1,55 1.74 48 0.795 0.964 BLQ = Below Limit of Quantitation NIA= NotApplicable PCTiAU2015/050820 2.85 2 . .27 0,.613 2.96 3.09 1.07 1 .. 64 1.64 0.095 2.00 1.25 0:652 Example 7 T,i,~le 3 ImUvidnal Female Beagle Dog Plasma CQncentrations of Milrin.one Following .a Single 5 mg/Jig :PO Administratfon as E:dended Release Tablets in a Gelatin 5 Capsule Dog#4 Dog#5 Dog#.6 Animal Animal Animal 9.6 Weight 7,682 Weight 9.083 Weight 57 (kg) (kg) {kg) Dose Dose Dose JS .46 48 (mg) (mg) (mg) Actual Actual Actu.aJ 4.9 Dosage 4 ... 95 ·oosage 5.06 Dosage 7 (trig/kg) (mg/kg) (mg/kg) $ample Sa~ple Sample Mean Time SD Cone.

Cone.

Cone.

Cone. (hr) (ng/mL) (ng/mL) (nglmL) (ng/mL) (nglmL) 9 BLQ BLQ B:LQ BLQ N/A 0.5 BLQ 1.19 BLQ BLQ NIA 1 .BLQ 15.6 0.57 5.39 .S.85 1.5 204 198 54.4 152 84.7 3 569 487 601 552 58.8 6 563 459 519 514 52.2 Date Rei;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 9 570 696 373 546 163 11 545 779 138 487 324 12. 457 597 109 388 251 14 264 456 42.1 254 207 18 50.2 149 IO.I 69.8 71.5 24 16.4 25.8 1.36 14.5 12.'.3 The above in vivo phannacokirtetic data shows plasma levels following administration of Milrinorie at a dosage of 5 mg/kg.

The plasma. concentrations versus time profiles obtained after oral administration of immediate release (IR) Milrinone and extended release 5 (ER) Milrinone (i.e. a composition of t;he present invention) were amtlysed.

Table 1 shows phatmacokinetic data for the IR Milrinone and ER Milrino:ne formulation dosin.g.

These data demonstrate that the ER Milrinone achieved a reduced Cmax irt comparison. to the IR Milrinone, and that the ER Milrinone maintained stable Milrinone plasma concentrations over a 12 hour period.

Thi.s tn vivo data. substantiates the In Vitro relea.se data obtained ab(>ve in ..

Exai::hple 6 .a.pd ptovides confirmation that the formulations of.the present inve.ntfon satisfy the r.eguitemen.ts of the desired release profile. 15 Example 8: To exemplify the therapeutic utility ormH:rinone in pa.dents.with BFpEF the following study was performed.

Patients with HFpEF underwent an invasive assessment of central hemodynamics by Swan Ganz catheterization under resting conditions and during symptom limited supine cycling.

In particul;u:, the pulmonary artery and pulmonary capilh1;ry wedge 20 pressure were measured.

It is well known that patients with HFpEF exhibit a rapid and excessive .rise in pulmonary artery and. pulmonary capillary wedge pressute due to left ventricular diastolic dysfunction.

Following this measurement patients were randomly allocated to receive an intravenous bolus of milrinone 50 μg/kg over 10 minutes or an infu$ion of saline.

Atthe end of this infusion :the measurements were repeated, Date Rei;ue/Date Received 2022-08-23 WO :2016/101024 PCT/AU:2015/0508:20 The resμlts are shown in the table below: Rest mercise Pu.Im.

Art Pres.s Wec;lge Press.

Pulm, Art,.

Press Wedge Press. mtnHg tnmHg mm.Hg tnmHg Base.line (h=4) 21±.J 12±.2 41 ±2 30±2 Placebo 20± l 12± l 37± 1 25± 1 Baseline (n=:;4) .21 :±3 11±1 49:1;;3 33±1 Milrinone 15 :±: 2* 4± l* 32.± 6* 19 :± 2** These data show that milrlnone improves the hemodynamic response in patients with BFpEF durln,g e&erciis:e IU'ldthis effect would be expeett').d tobebenefi<;ial in these patients.

The cita,tion Qf.any referepce herei.n 1$hould not. be constl'.tfed as a.o l,\dmiss.icm that ~uc,h reference ls ava:Uable as ''Prior Art" to the instant. appUcation. ] 0 Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or ·specific coUeetfon of features, Those of skm in the art wm therefore appreciate that, in Hght of the instant dlsclQsi,re,. various modifications and changes can be made in the particular embodiments el".emplifled without .. departing from the scope of the pres.ent invi:intion.

All such modifications ] 5 and changes ate intended to he i ac1uded within the scope of the appended claims.

Date Rer;ue/Date Received 2022-08-23 WO 2016/101024 PCTiAU2015/050820 BIBLIOGRAPHY I.

Edelmann F., et al., Effect of Spironolactone on Diastolic Function and Exercise Capacity in Patients with Heart Failure with .Pre5etv(:')d EJection Fraction; JAMA, 2013, 309(8):781-791. 5 2.

Komajda M. and Lam C.S.P., Heart Failure with PreServed Ejection.

Fraction: a Clinical Dilemma; European Heart Journal, 2014, 3:5:1022-1032. 3.

Loffredo F.S., et al., Heart Failure with Preserved Ejection Fraction, .Molecular Pathways or the Aging Myocardium; Circulation .Research, .2014, 1 l 5;97-101. 4.

Redfield M.M,, et al., Effect of Phosphodiesterase~5 Inhibition on Exercise Capacity 10 and Clinical Status in Heart Failure with Preserved Ejection Fraction; JAMA, 20B, 309(12): 1268-1277. 5.

Sharma K. and Kass D.A, Heart Failure with Preserved Ejection Fraction, Mechanisms, Clinical Features, and Therapies; Circulation Research, 2014, 115:79;. 96. 15 6.

Yancy C.W. f:lt al., 2013 ACCFYAHA Guideline for the Management of Heart Failure, A Report of the American College of Cardiology Foundation/American He.art Association Task Force onPractice Guidelines, Circulation,2013, 128:e240-e327.

Date Rei;ue/Date Received 2022-08-23

Claims

WHAT IS CLAIMED IS: 1. Use of an oral controlled-release formulation of l,2-dihydro-3-cyano-6- methyl-5-(4-pyridinyl)-2(1H)-pyridinone (milrinone) or a pharmaceutically acceptable salt thereof in treating a patient having heart failure with preserved ejection fraction 5 (HFpEF); and wherein the formulation comprises: i) a core comprising milrinone or a pharmaceutically acceptable salt thereof and one or more polymers and one or more excipients; and ii) a sustained-release coating. 10 2. The use according to claim 1, wherein the use is for oral administration of milrinone or a pharmaceutically acceptable salt thereof at 5 mg to 75 mg in a single dose per day or in divided doses up to four times per day. 3. The use according to claim 1 or claim 2, wherein the formulation comprises milrinone or a pharmaceutically acceptable salt thereof, 15 hydroxypropylmethylcellulose or hydroxypropylcellulose having a viscosity of 80,000 to 120,000 cps, hydroxypropylmethylcellulose having a viscosity of about 50 cps, and at least one pharmaceutically acceptable excipient; wherein the hydroxypropylmethylcellulose or hydroxypropylcellulose having a 20 viscosity of 80,000 to 120,000 cps and the hydroxypropylmethylcellulose having a viscosity of about 50 cps are in a ratio of 2:1 to 1:2, and the ratio of milrinone or a pharmaceutically acceptable salt thereof to total hydroxypropylmethylcellulose or hydroxypropylmethylcellulose and hydroxypropylcellulose is 1:2 to 1:6. 4. The use according to claim 3, wherein the ratio of 25 hydroxypropylmethylcellulose or hydroxypropylcellulose having a viscosity of 80,000 to 120,000 cps to hydroxypropylmethylcellulose having a viscosity of about 50 cps is about 1:1. 63 Date Re<;ue/Date Received 2024-05-22 5. The use according to claim 3 or claim 4, wherein the ratio of milrinone or a pharmaceutically acceptable salt thereof to total hydroxypropylmethylcellulose or hydroxypropylmethylcellulose and hydroxypropylcellulose is about 1:3. 6. The use according to any one of claims 3 to 5, wherein the at least one 5 pharmaceutically acceptable excipient is a binder or a lubricant or a mixture thereof. 7. The use according to claim 6, wherein the binder is microcrystalline cellulose. 8. The use according to any one of claims 3 to 7, wherein milrinone or a pharmaceutically acceptable salt thereof is present in an amount of 10-30% w/w of the 10 core. 9. The use according to any one of claims 3 to 8, wherein the hydroxypropylmethylcellulose or hydroxypropylcellulose having a viscosity of 80,000 to 120,000 cps is present in an amount of 20-40% w/w of the core. 10. The use according to any one of claims 3 to 8, wherein the 15 hydroxypropylmethylcellulose having a viscosity of about 50 cps is present in an amount of 20-40% w/w of the core. 11. The use according to claim 6 or claim 7, wherein the binder is present in an amount of 16-30% w/w of the core. 12. The use according to claim 1 or claim 2, wherein the core compnses 20 milrinone or a pharmaceutically acceptable salt thereof, a hydrophilic matrix comprising at least two natural gums, and at least one pharmaceutically acceptable excipient; wherein the two natural gums are in a ratio of 2: 1 to 1 :2; and the ratio of milrinone or a pharmaceutically acceptable salt thereof to the hydrophilic 25 matrix is 1:1 to 1:2.5. 13. The use according to claim 12, wherein the hydrophilic matrix comprises xanthan gum, locust bean gum or a mixture thereof. 14. The use according to claim 13, wherein the hydrophilic matrix comprises xanthan gum and locust bean gum. 64 Date Re<;ue/Date Received 2024-05-22 15. The use according to claim 14, wherein the xanthan gum and locust bean gum are in a ratio of 1: 1. 16. The use according to any one of claims 12 to 15, wherein the ratio of milrinone or a pharmaceutically acceptable salt thereof to the hydrophilic matrix is 5 about 1:1.5. 17. The use according to any one of claims 12 to 16, wherein the at least one pharmaceutically acceptable excipient is selected from a binder, a filler, a glidant, a lubricant and mixtures thereof. 18. The use according to any one of claims 12 to 17, wherein milrinone or a IO pharmaceutically acceptable salt thereof is present in an amount of 15-25% w/w of the core. 19. The use according to any one of claims 12 to 18, wherein the hydrophilic matrix is present in an amount of 20-40% w/w of the core. 20. The use according to claim 1 or claim 2, wherein the core comprises: (i) a coating composition comprising milrinone or a pharmaceutically acceptable salt thereof, one or more polymers, and one or more excipients, and (ii) inert spherical particles; wherein the coating composition is on coated on the surface of the spherical particles; 20 wherein the ratio of milrinone or a pharmaceutically acceptable salt thereof to the spherical particles is about 1:5 to 1:25; and wherein the coated particles further comprise a sealing coating. 21. The use according to claim 20, wherein the inert spherical particles are sugar or starch spherical particles. 25 22. The use according to claim 20 or claim 21, wherein the coating composition further includes a plasticiser. 23. The use according to any one of claims 20 to 22, wherein the ratio of milrinone or a pharmaceutically acceptable salt thereof to polymer and optionally plasticiser is 1.5: 1 to 2: I. 65 Date Re<;ue/Date Received 2024-05-22 24. The use according to any one of claims 20 to 23, wherein the polymer comprises hydroxypropy lmethy lcellulose. 25. The use according to claim 22, wherein the plasticiser is polyethylene glycol. 26. The use according to any one of claims 20 to 25, wherein the ratio of milrinone or a pharmaceutically acceptable salt thereof to spherical particles is about 1:15 to 1:20. 27. The use according to claim 20, wherein the sealing coating compnses hydroxypropylmethylcellulose and polyethylene glycol. IO 28. The use according to any one of claims 1 to 27, wherein the sustainedrelease coating comprises a cellulose derivative or a copolymer of acrylic acid, methacrylic acid and/or their esters. 29. The use according to claim 28, wherein the cellulose derivative 1s ethy lcellulose. 15 30. The use according to claim 28, wherein the sustained-release coating further comprises a low viscosity HPMC. 31. The use according to any one of claims 1 to 30, wherein the sustainedrelease coating comprises a copolymer of acrylic acid, acrylic ester, methacrylic acid, methacrylic ester or mixtures thereof, optionally with a methacrylic ester with 20 quaternary ammonium groups. 32. The use according to any one of claims 1 to 31, wherein the formulation comprises more than one sustained-release coating. 33. The use according to any one of claims 1 to 31, wherein the formulation further comprises one or more of a seal coating, a buffer coating and an enteric-release 25 coating. 34. The use according to any one of claims 1 to 33, wherein the patient is a human. 66 Date Re<;ue/Date Received 2024-05-22