(19)

*EP002341905B2*
(11) EP 2 341 905 B2
(12) NEW EUROPEAN PATENT SPECIFICATION
After opposition procedure

(45) Date of publication and mention (51) International Patent Classification (IPC):
of the opposition decision: A61P 5/50 (2006.01)
13.09.2023 Bulletin 2023/37
(52) Cooperative Patent Classification (CPC):
(45) Mention of the grant of the patent: (C-Sets available)
29.04.2020 Bulletin 2020/18 A61K 38/2278; A61K 9/0019; A61K 9/10;
A61K 9/5153; A61K 31/00; A61K 31/60;
(21) Application number: 09812292.2 A61K 31/65; A61K 38/26; A61K 38/28;
A61K 45/06; A61K 47/14; A61K 47/44; A61P 1/16;
(22) Date of filing: 04.09.2009 A61P 3/00; A61P 3/04; (Cont.)

(86) International application number:

PCT/US2009/056058

(87) International publication number:

WO 2010/028257 (11.03.2010 Gazette 2010/10)

(54) SUSTAINED RELEASE FORMULATIONS USING NON-AQUEOUS CARRIERS

FORMULIERUNGEN MIT NICHT WÄSSRIGEN TRÄGERN UND VERZÖGERTER FREISETZUNG

FORMULATIONS À LIBÉRATION PROLONGÉE À BASE DE SUPPORTS NON AQUEUX

(84) Designated Contracting States: • OEHRTMAN, Greg

AT BE BG CH CY CZ DE DK EE ES FI FR GB GR San Diego
HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL CA 92121 (US)
PT RO SE SI SK SM TR • JENNINGS, Robert, N.
San Diego
(30) Priority: 04.09.2008 US 94381 P CA 92121 (US)
• COLEMAN, Scott, H.
(43) Date of publication of application: San Diego
13.07.2011 Bulletin 2011/28 CA 92121 (US)

(60) Divisional application: (74) Representative: Hoffmann Eitle

20163108.2 / 3 685 837 Patent- und Rechtsanwälte PartmbB
Arabellastraße 30
(73) Proprietors: 81925 München (DE)
• Amylin Pharmaceuticals, LLC
San Diego, CA 92121 (US) (56) References cited:
• AstraZeneca Pharmaceuticals LP WO-A1-2005/102293 WO-A2-2007/024700
Wilmington, DE (US) US-A1- 2004 224 030 US-A1- 2004 224 030
US-A1- 2008 146 490
(72) Inventors:
• HOUCHIN, Mary, L. • JAIN R A ET AL: "Controlled release of drugs from
EP 2 341 905 B2

Rockville injectable in situ formed biodegradable PLGA

MD 20850 (US) microspheres: effect of various formulation
• LEE, Robin, H. variables", EUROPEAN JOURNAL OF
San Diego PHARMACEUTICS AND BIOPHARMACEUTICS,
CA 92128 (US) ELSEVIER SCIENCE PUBLISHERS B.V.,
• QI, Hong AMSTERDAM, NL, vol. 50, no. 2, 1 September
San Diego 2000 (2000-09-01), pages 257-262, XP004257199,
CA 92121 (US) ISSN: 0939-6411, DOI:
10.1016/S0939-6411(00)00062-X

Processed by Luminess, 75001 PARIS (FR) (Cont. next page)

 EP 2 341 905 B2

(52) Cooperative Patent Classification (CPC): (Cont.)

A61P 3/06; A61P 3/08; A61P 3/10; A61P 5/50;
A61P 43/00

C-Sets
A61K 31/60, A61K 2300/00;
A61K 31/65, A61K 2300/00

2
 EP 2 341 905 B2

Description

Background

5 [0001] Injectable sustained release formulations offer the opportunity to provide therapeutic amounts of active phar-
maceutical ingredients over an extended period of time from a single injection, thus eliminating the need for once or
twice daily injections. Presently available injectable sustained release formulations utilizing, for example, microspheres
and an aqueous carrier, carry several disadvantages. The formulations do not offer long term stability in the aqueous
carrier, thus necessitating separate packaging and storage for the microspheres and aqueous carrier, and the patient
10 must take several steps to combine the microspheres and aqueous carrier before administering the injection.
[0002] Another disadvantage of presently available injectable microsphere formulations is a large burst release fol-
lowing injection, which causes an undesirable in vivo release of active pharmaceutical ingredient in a single burst. When
medications have toxic or deleterious side effects, this is undesirable.
[0003] There is a need for formulations and methods of safely administering sustained release pharmaceutical for-
15 mulations to patients so that the active ingredient will be released in vivo over an extended period of time and without
an unacceptable initial burst release. Ideally the active ingredient is released so as to maintain levels within the therapeutic
window, i.e., in the concentration range above that needed to cause the desired clinical effect, but below that where
undesirable side effects outweigh the benefits of the drug. It is also necessary that this active pharmaceutical ingredient
be provided in a manner that is easy and convenient for the patient to self-administer and that is provided in a formulation
20 that maintains stability for a long period of time in a liquid state. The disclosure is directed to these as well as other
important ends.

Summary

25 [0004] The disclosure provides formulations comprising microspheres that contain active pharmaceutical ingredients,
where the microspheres are suspended in a non-aqueous pharmaceutically acceptable carrier. The formulations are
one-component injectable microsphere formulations, such that they do not require the patient to mix the formulation with
a pharmaceutically acceptable carrier prior to injection. The disclosure offers distinct advantages over prior two-compo-
nent formulations by providing for a long shelf life of the composition in the carrier, sustained release of the active
30 pharmaceutical ingredient, a less complex carrier, a more easily manufactured carrier, a less complex injection-delivery
apparatus, a kit with less components, and ease of use by patients.
[0005] The present invention provides a manufactured pre-mixed formulation for injection consisting essentially of a
suspension of

35 (i) a pharmaceutically acceptable non-aqueous carrier which comprises one or more triglycerides of C6-C12 fatty
acids and
(ii) microspheres which consist essentially of a poly(lactide-co-glycolide) polymer having dispersed therein about
5% (w/w) exenatide as active pharmaceutical ingredient and about 2% (w/w) sucrose; wherein the ratio of lactide:gly-
colide in the polymer is about 1:1, wherein the pharmaceutically acceptable non-aqueous carrier consists of one or
40 more triglycerides of C6-C12 fatty acids, wherein the triglycerides comprise 0 to 2 wt% C6 fatty acid, 50 to 65 wt%
C8 fatty acid, 30 to 45 wt% C10 fatty acid, and 0 to 2 wt% C12 fatty acid.

[0006] Also provided is the formulation of the invention for use in treating diabetes, stimulating insulin release; lowering
plasma glucagon; reducing food intake; reducing appetite; decreasing gastric motility; delaying gastric emptying; lowering
45 plasma lipid levels; treating impaired glucose tolerance; treating hyperglycemia; treating obesity; treating overweight;
treating fatty liver disease; or treating non-alcoholic steatohepatitis in a patient in need thereof comprising administering
to the patient the formulation of the invention.
[0007] Preferably, the formulation is for use in treating diabetes; stimulating insulin release; lowering plasma glucagon;
reducing food intake; reducing appetite; decreasing gastric motility; or delaying gastric emptying.
50 [0008] The disclosure provides sustained release formulations comprising a pharmaceutically acceptable carrier which
consists essentially of one or more triglycerides which comprise C6-C12 fatty acids; and microspheres which consist
essentially of a poly(lactide-co-glycolide) polymer having dispersed therein about 1% to 10% (w/w) exenatide and about
0.1% to 5% (w/w) of a sugar; wherein the ratio of lactide:glycolide in the polymer is about 70:30 to 30:70, or about 1:1.
In one embodiment, the exenatide is present in an amount of 1% to 5% (w/w) or 5% (w/w) and the sugar is present in
55 an amount of 2% (w/w). The sugar may be, e.g., glucose, dextrose, galactose, maltose, fructose, mannose, sucrose,
lactose, trehalose, raffinose, acarbose, glycol, glycerol, erythritol, threitol, arabitol, ribitol, sorbitol, dulcitol, iditol, isomalt,
maltitol, lactitol, mannitol, xylitol, or a combination of two or more thereof. In one embodiment, the sugar is sucrose. The
formulation is a suspension whereby the microspheres are suspended in the carrier. In one embodiment, the total pore

3
 EP 2 341 905 B2

volume of the microspheres is about 0.1 mL/g or less, as determined using mercury intrusion porosimetry, to provide a
release profile having a ratio of maximum serum concentration of exenatide during the period of release (Cmax) to average
serum concentration of exenatide during the period of release (Cave) of about 3 or less. Further, although the micro-
spheres are formulated in oil (i.e. a carrier as disclosed herein), the microspheres do not necessarily have oil contained
5 within the interior spaces or pores, or within a substantial number of interior spaces or pores, of the microspheres, and
yet can achieve the surprising properties disclosed herein.
[0009] The disclosure provides sustained release formulations comprising a pharmaceutically acceptable non-aqueous
carrier and microspheres which comprise a biocompatible, biodegradable polymer and an active pharmaceutical ingre-
dient. In one embodiment, the total pore volume of the microspheres is about 0.1 mL/g or less, as determined using
10 mercury intrusion porosimetry, to provide a release profile having a ratio of maximum serum concentration of the active
pharmaceutical ingredient during the period of release (Cmax) to average serum concentration of the active pharmaceutical
ingredient during the period of release (Cave) of about 3 or less. Further, although the microspheres are formulated in
oil (i.e. a carrier as disclosed herein), in some embodiments the microspheres do not have oil contained within the interior
spaces or pores, or do not have oil within a substantial number of interior spaces or pores of the microspheres, and yet
15 can achieve the surprising properties disclosed herein. The formulation is a suspension whereby the microspheres are
suspended in the carrier.
[0010] In one embodiment the active ingredient is not soluble in the carrier. In various other embodiments the active
ingredient has a solubility in the carrier of less than 0.01 mg/ml, or less than 0.05 mg/ml or less than 0.1 mg/ml or less
than 0.5 mg/ml or less than 1 mg/ml. In still other embodiments the active pharmaceutical ingredient has a solubility in
20 the carrier such that less than 10% of the active ingredient in the formulation is contained within the carrier with the
remaining 90% contained within the microparticles. In further embodiments less than 5% or less than 2% or less than
1% or less than 0.5% of the active ingredient is contained in the carrier. In still further embodiments where it is desirable
to have some active ingredient immediately available, it may also be directly incorporated into the carrier in a pharma-
ceutically effective amount.
25 [0011] The disclosure provides a kit, available to a patient or medical service provider. The kit contains a container
having a formulation of the invention, and instructions for use. In one embodiment the container is a pen injector. The
pen injector can be a single-dose pen injector or a multi-dose pen injector. In one embodiment the container is a vial,
which can be either a single-dose vial or a multi-dose vial. In another embodiment the container is a cartridge, such as
a cartridge for use in a injection apparatus. The cartridge can be either a single-dose or a multi- dose cartridge. In different
30 embodiments the kit contains 1, 2, 3, 4, or even 5 or more such containers carrying a formulation of the invention. One
further advantage of the formulations is that in one embodiment the container is provided preservative free. But in other
embodiments a preservative can be soluble in the selected carrier and provided in the formulation.

Brief Description of the Drawings

35
[0012] For each of Figures 1-6, the microspheres comprise a poly(lactide-co-glycolide) copolymer having exenatide
dispersed therein, as described in Example 1. For each of Figures 2-6, the oil carrier is a medium chain triglyceride
(MCT) commercially available as MIGLYOL® 812 (Sasol Germany GmbH, Witten, Germany).

40 Figure 1 provides a comparison of the pharmacokinetics of four different formulations of microspheres. In three
formulations, the carrier was an oil (e.g., sesame oil; MIGLYOL® 812; ethyl oleate). In the comparative formulation,
the carrier was an aqueous diluent.
Figure 2 is a graphical simulation (i.e., nanoparametric superposition) of data extrapolated from Figure 1 of the
plasma exenatide concentration over time for the microsphere formulation comprising the oil carrier and the micro-
45 sphere formulation comprising the aqueous carrier in male Sprague Dawley Rats. The plasma concentration plateau
of exenatide may be reached after about 5 dosings.
Figure 3 illustrates the in vitro release for a formulation comprising microspheres in an oil carrier compared to
formulations comprising microspheres in an aqueous carrier.
Figure 4 illustrates the in vivo release profile in rats over 10 hours for a formulation comprising microspheres in an
50 oil carrier and a formulation comprising microspheres in an aqueous carrier.
Figures 5A and B illustrate the purity of exenatide over 9 months at temperatures of 5°C and 6 months at 25°C when
stored in the formulations comprising the microspheres of Example 1 with an oil carrier as compared to the purity
of exenatide that was stored in dry microspheres of Example 1. In Figure 5A, the purity of exenatide was determined
by strong cation exchange HPLC. In Figure 5B, the purity of exenatide was determined by reverse-phase HPLC.
55 Figure 6 illustrates the stability/potency of exenatide in a formulation where the microspheres are suspended in an
oil carrier, where one formulation is stored at 5°C and one formulation is stored at 25°C.

4
 EP 2 341 905 B2

Detailed Description

[0013] The disclosure provides sustained release compositions provided in pharmaceutically acceptable carriers, for
the sustained release of an active pharmaceutical ingredient (API). The formulations may comprise microspheres com-
5 prised of a biocompatible, biodegradable polymer having an active pharmaceutical ingredient dispersed therein, where
the microspheres are suspended in a non-aqueous carrier. The formulations are one-component injectable formulations,
compared to two-component formulations which require that the microspheres be stored dry in one container while the
liquid carrier can be stored in a separate container, such that the patient must mix the two together prior to injection.
The formulations offer the convenience of long term stability of a pharmaceutical composition in a non-aqueous liquid
10 carrier, thus eliminating any need for the patient to add a pharmaceutically acceptable carrier to the pharmaceutical
composition prior to injection. The formulations are provided in a single container for easy use by the patient, who only
need to lightly agitate the formulation before injecting it from the same container. When the container provided is also
an injection device, even the step of syringing the formulation is eliminated. The formulations described herein offer the
additional important advantage of substantially reducing burst release of the active pharmaceutical ingredient. Thus,
15 even active pharmaceutical ingredients that have a toxic effect at higher concentrations can be safely administered using
the formulations described herein.
[0014] The term "patient" refers to mammals, including humans, animal pets, farm animals, zoo animals, and the like.
In one embodiment, the patient is a human.
[0015] The terms "treating" or "treatment" refer to the administration of one or more active pharmaceutical ingredients
20 to a patient who has a condition or disorder or a predisposition toward a condition or disorder, with the purpose to
alleviate, relieve, remedy, ameliorate, improve, slow or stop the progression or worsening of the disease, or at least one
symptom of the disease, condition or disorder, or the predisposition toward the condition or disorder.
[0016] "Exenatide" has the same meaning and amino acid sequence as exendin-4. More particularly, exenatide is a
synthetic peptide with the same amino acid sequence as exendin-4, which is a peptide isolated from the venom of the
25 Gila monster.

One Component Formulation

[0017] Previous injectable formulations contained at least two components. The first component may be dry micro-
30 spheres and the second component may be an aqueous pharmaceutically acceptable carrier. The first component and
second component are stored in separate sealed containers (e.g., vials, injection pen chambers). The patient receives
the two-component formulation, and the patient or pharmacist must physically mix the two components together prior
to injection. In the case of an injection pen, the two components are mixed together immediately prior to injection into
the patient. Two-component formulations typically are administered to the patient within a short time after being mixed
35 with the pharmaceutically acceptable carrier. For example, the microsphere component and the pharmaceutically ac-
ceptable aqueous carrier are mixed together and then the formulation is administered to the patient within about 30 or
60 minutes.
[0018] The formulations described herein are one component injectable formulations. A one component injectable
formulation refers to a formulation that contains both the microspheres and the pharmaceutically acceptable carrier
40 provided in the same container, and that may be administered to the patient without the need to first combine the
microspheres and the pharmaceutically acceptable carrier. Accordingly, the one component formulation is manufactured
as a pre-mixed formulation for injection. A one-component formulation provides significant convenience for manufac-
turing, transport, storage, and patient use.
[0019] In another embodiment the one-component formulation described herein is provided in a sealed container. A
45 "sealed container" is a container that has not been opened, punctured, or had anything introduced into it since its time
of completion of manufacture. The time of completion of manufacture is the time when the container holding the formulation
is initially sealed. Containers may include vials (single use or multi-use), syringes, injection pens (e.g., single use or
multi-use), and the like.

50 Carrier

[0020] "Carrier" (or vehicle) refers to a pharmaceutically acceptable non-aqueous liquid material. The carrier is sub-
stantially inert so that it does not interact with the microspheres described herein and is non-toxic so that it does not
negatively impact the patient. The carrier is preferably approved by or is awaiting approval by a regulatory agency of
55 the Federal or a state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia
for use in mammals, such as humans. The term "carrier" may include one or more compounds. The carrier is a non-
solubilizing carrier, in that the carrier does not solubilize the polymer(s) that forms the microspheres. In a further em-
bodiment, the carrier does not solubilize the active pharmaceutical ingredient(s) within the microspheres. For example,

5
 EP 2 341 905 B2

the carrier will not solubilize exenatide or other water-soluble therapeutic peptides or proteins.
[0021] The term "non-aqueous" does not exclude trace amounts of residual water that do not have a demonstrated
negative impact on the stability of the sustained release compositions. Thus, a composition may have about 0.1% (w/v)
water or even about 0.25% water or less than 0.1% (w/v) water or less than 0.25% (w/v) water and still be considered
5 non-aqueous. The carrier does not solubilize the microspheres to the extent of having a demonstrated negative impact
on the stability of the microspheres or demonstrated loss of burst release control. In one embodiment, the carrier does
not enter or permeate the biocompatible, biodegradable polymer and is not dispersed within the biocompatible, biode-
gradable polymer. The carrier also does not cause swelling of the microspheres to an extent that has a demonstrated
negative impact on the stability of the microspheres. For example swelling may occur to a degree of less than 1% and
10 still be considered a non-aqueous carrier that is non-swelling of the microspheres.
[0022] In one embodiment, the non-aqueous carrier is a pharmaceutically acceptable oil. An oil is a substance that is
in a viscous liquid state at ambient temperatures or slightly warmer, and is both hydrophobic (immiscible with water)
and lipophilic (miscible with other oils, literally). Exemplary pharmaceutically acceptable oil carriers include vegetable
oils and volatile essential oils. The carrier may comprise one oil or a combination of two or more oils.
15 [0023] In one embodiment, the carrier is a fractionated oil or a combination of two or more fractionated oils. In one
embodiment, the carrier is fractionated coconut oil. In one embodiment, the carrier is fractionated palm kernel oil. In one
embodiment, the carrier is a combination of fractionated coconut oil and fractionated palm kernel oil.
[0024] As used herein, fractionation is a process whereby long chain fatty acids are removed from the oil, such that
the resulting fractionated oil substantially comprises medium chain triglycerides.
20 [0025] In the present invention, the carrier is a medium chain triglyceride. The medium chain triglyceride may be
synthetic or natural (e.g., produced from fractionated oils, such as coconut oil and/or palm kernel oil). "Medium chain
triglyceride" refers to esters of glycerol having three C6-C12 fatty acid chains, where the three fatty acid chains may be
the same or different. Medium chain triglycerides are represented by the compound of Formula (I): wherein each x is in

25

30

35

the chain is referred to as a C 6 fatty acid. When x is 6, the chain is referred to as a Cs fatty acid. When x is 8, the chain
is referred to as a C10 fatty acid. When x is 10, the chain is referred to as a C12 fatty acid. In various embodiments, each
40 x is the same integer; two x are the same integer and one x is a different integer; or each x is a different integer.
[0026] The skilled artisan will appreciate that a mixture of medium chain triglycerides may result from any process
(e.g., fractionation, hydrogenation) used to prepare medium chain triglycerides. For example, substantially all of the
medium chain triglycerides obtained from fractionated coconut oil may comprise C8 and/or C10 fatty acids; however,
there may be some medium chain triglycerides containing C6 and/or C12 fatty acids.
45 [0027] In one embodiment, the medium chain triglyceride comprises 0 to 2 wt% C6 fatty acid, 50 to 65 wt% C8 fatty
acid, 30 to 45 wt% C10 fatty acid, and 0 to 2 wt% C12 fatty acid, and which is commercially available as MIGLYOL® 812
(Sasol Germany GmbH, Witten, Germany) The weight % is based of the total fatty acid content of the triglycerides.
[0028] In certain embodiments the non-aqueous, non-solubilizing carrier has a viscosity of from 5 cP to 200 cP or from
10 cP to 90 cP. In other embodiments the viscosity of the non-aqueous, non-solubilizing carrier is from 20 cP to 80 cP
50 or from 30 cP to 70 cP. Thus, with reference to this disclosure the person of ordinary skill will be able to identify other
oils, triglycerides, or non-aqueous compounds that also can be present in the non-aqueous, non-solubilizing carrier.

Microspheres

55 [0029] The term "microspheres" includes microspheres, microparticles, nanoparticles, pellets, cylinders, rods, discs,
and the like. A microsphere can have a spherical, non-spherical or irregular shape. The microsphere will be of a size
suitable for injection. A typical size range for microspheres is 1000 microns or less. In a particular embodiment, the
microsphere ranges from about one to about 180 microns in diameter. In yet further embodiments suitable release

6
 EP 2 341 905 B2

profiles are obtained when microspheres range from about 1 to 100 microns, from about 30 to 90 microns, or from about
50 to 70 microns. In one embodiment the mean microsphere size is not less than or is equal to about 50, 60 or 70
microns, and preferably less than about 80, 90, or 100 microns. At larger sizes, microsphere are preferably substantially
non-aggregated to allow passage through a 25 gauge needle, or a 27 gauge needle, or a 30 gauge needle, or a 31
5 gauge needle.
[0030] Consistent and superior release profiles are obtained by controlling size distribution. In one embodiment a
mean microsphere size is about 50 microns and the lower and upper range of microsphere are about 30 and 90 microns,
respectively. Distribution of microspheres can be described using a mean diameter of the volume. Mean diameter of the
volume distribution represents the center of gravity of the distribution and is a type of "average particle size." In various
10 embodiments, the microspheres have a mean diameter of the volume distribution of about 50 to 70 microns, about 50
to 60 microns or about 50, 60 or 70 microns, with a Distribution of Volume (DV) of less than or about 5%, 10%, or 15%
at 30 microns and a DV of greater than or about 80%, 85%, 90% or 95% at 90 microns. In one embodiment, the
microspheres have a mean diameter of the volume distribution of about 60 microns, with a Distribution of Volume (DV)
of less than or about 10% at 30 microns and a DV of greater than or about 90% at 90 microns.
15 [0031] Microspheres may be prepared by processes known in the art and described, e.g., in US Patent Nos. 7,563,871, ,
7,456,254, 7,223,440, 6,824,822, 6,667,061, 6,495,164, and 6,479,065.
[0032] In a further embodiment, the microspheres have a less porous outer layer, and further can have a non-porous
outer layer. Accordingly, in the formulations disclosed herein the oil does not have access to the interior spaces or pores
or even to a substantial portion of the interior spaces or pores. It is specifically, contemplated that for each of the
20 formulations disclosed herein the microspheres can additionally lack oil (or a carrier as disclosed herein) in the interior
spaces of the microspheres. Thus, the advantages of the present formulations can be achieved without the presence
of oil in the interior spaces of the microspheres when formulated.

Polymers
25
[0033] The microspheres consist essentially of a biocompatible, biodegradable polymer which is a poly(lactide-co-
glycolide) polymer having a ratio of lactide:glycolide of 1:1. A polymer is biocompatible if the polymer and any degradation
products of the polymer are non-toxic to the patient at administered levels and also possess no demonstrated deleterious
or untoward effects on the patient’s body, for example a substantial immunological reaction at the injection site. Biode-
30 gradable means the polymer will degrade or erode in vivo to form smaller units or Chemical species. Degradation can
result, for example, by enzymatic, Chemical and physical processes.
[0034] The molecular weight of the poly(lactide-co-glycolide) copolymer is about 10,000 Daltons to about 90,000
Daltons. In another embodiment, the molecular weight of the poly(lactide-co-glycolide) copolymer is about 30,000 Daltons
to about 70,000, or from about 50,000 to about 60,000 Daltons.
35 [0035] The formulation may contain microspheres at a concentration of from 1 mg/ml to 500 mg/ml; from 25 mg/ml to
300 mg/ml; or from 50 mg/ml to 200 mg/ml.

Active Pharmaceutical Ingredient

40 [0036] An active pharmaceutical ingredient is a biologically active compound that has a therapeutic, prophylactic, or
other beneficial pharmacological and/or physiological effect on the patient. The active pharmaceutical ingredient can
also be a mixture of two or more compounds. The term "peptide" refers to any compound having two or more consecutive
amino acids. As used herein, the term "peptide" is synonymous with peptide, polypeptide, and protein. In one embodiment,
the peptide has a molecular weight of from 500 Da to 100 kDa; from 1 kDa to 80 kDa; from 1 kDa to 50 kDa; from 1 kDa
45 to 30 kDa; or from 1 kDa to 20 kDa. In one embodiment, the peptide comprises 2 to 500 amino acid residues; 2 to 250
amino acid residues; 5 to 100 amino acid residues; or 5 to 50 amino acid residues.
[0037] In the present invention, the microspheres comprise about 5% (w/w) exenatide as an active pharmaceutical
ingredient.
[0038] In one embodiment, the active pharmaceutical ingredient is a GLP-I receptor agonist compound, such as an
50 exendin, an exendin analog, GLP- 1(7-37), a GLP- 1(7-37) analog, and the like. Exemplary GLP-I receptor agonist
compounds include exendin-3, exenatide, GLP-1(1-37), GLP-1(7-37)-NH2, GLP-1(7-36), GLP-I (7-36)-NH2, Leu14-ex-
endin-4, Leu14, Phe25-exendin-4, exendin-4(1-28), Leu14-exendin-4(1-28), Leu14,Phe25-exendin-4(1-28), exendin-
4(1-30), Leu14- exendin-4(1-30), Leu14,Phe25-exendin-4(1-30), liraglutide, and the compounds described in, e.g., US
Patent No. 7,157,555, US Patent No. 7,220,721, US Patent No. 7,223,725, and WO 2007/139941.
55 [0039] Other peptides known in the art can be used as the active pharmaceutical ingredient in the formulations described
herein. Exemplary peptides include amylin, amylin agonists (e.g., pramlintide, davalintide, Val27-davalintide); leptin,
leptin agonists (e.g., metreleptin); PYY(3-36) and agonist analogs thereof; glucagon, glucagon agonists, glucagon an-
tagonists, peptide chimera of GLP-I receptor agonists and glucagon agonists, peptide chimera of human amylin and

7
 EP 2 341 905 B2

salmon calcitonin, insulin, heparin, low-molecular weight heparin, angiotensin, argipressin, argireline, atosiban, bivaliru-
din, cetrorelix, desmopressin, enfuvirtide, deptifibatide, GHRP-2, GHRP-6, gonadorelin, leuprolide, lysipressin, melan-
otan, nesiritide, octreotide, oxytocin, PT 141, calcitonin, sermorelin, somatostatin, terlipressin, thymopentin, thymosin
al, triptorelin, vapreotide, elcatonin, ziconotide, ghrelin, nafarelin and BNP-32.
5 [0040] The active pharmaceutical ingredient can also be a small molecule. A "small molecule" is an organic molecule.
Exemplary small molecules include metformin, sulfonylureas, TZDs, statins (e.g., atorvastatin, cerivastatin, fluvastatin,
Lovastatin. mevastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin); non-selective beta blockers and/or alpha-
1 blockers (e.g., carvedilol, dilatrend, eucardic, carloc); PDE3 inhibitors (e.g., cilostazol); antiplatelet drugs, antithrombotic
drugs, anticoagulant drugs, glycoprotein IIb/IIIa inhibitors (e.g., abciximab, eptifibatide, tirofiban); antibacterial drugs
10 (e.g., ciprofloxacin, norfloxacin, levofloxacin, moxifloxacin, sparfloxacin, gemifloxacin, ecinofloxacin, delafloxacin); Factor
Xa inhibitors (e.g., glycosaminoglycans, oligosaccharides, heparinoid); direct Xa inhibitors (e.g., xabans); direct thrombin
(II) inhibitors (e.g., hirudin, argatroban, dabigatran, melagatran, ximelagatran, defibrotide, ramatroban, antithrombin III,
protein C); thrombolytic drugs (e.g., plasminogen activators, urokinase, streptokinase, serine endopiptidases); ACE
inhibitors (e.g., lisinopril, aceon, acertil, armix, coverene, coverex, coversum, prestarium, prexanil, Prexum, procaptan);
15 ADP receptor/P2Y12 inhibitors (e.g., clopidogrel, ticlopidine, prasugrel); prostaglandin analogs (e.g., beraprost, prosta-
cyclin, iloprost, treprostinil); anticoagulants (e.g., coumarin, coumatetralyl, dicoumarol, ethyl biscoumacetate, phenpro-
coumon, warfarin, clorindione, diphenadione, phenindione, tioclomarol); diuretics (e.g., hydrochlorothiazide); macrolides
(e.g., azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, telithromycin); NSAIDs and COX-3 inhib-
itors (e.g., celecoxib, etoricoxib, parecoxib); and sulphonanilides (e.g., nimesulide).
20 [0041] The skilled artisan will appreciate that the formulations described herein may contain two or more peptides;
two or more small molecules; or a combination of small molecules and peptides. For example, the formulation may
comprise two different sets of microspheres, where one set of microspheres contain one peptide (e.g., pramlintide) and
another set of microspheres contain a different peptide (e.g., metreleptin). In one embodiment, 1 to 99% of the micro-
spheres comprise one active pharmaceutical ingredient and 99 to 1% of the microspheres comprise a different active
25 pharmaceutical ingredient. In another embodiment 30 to 70% of the microspheres comprise one active pharmaceutical
ingredient and 70 to 30% of the microspheres comprise a different active pharmaceutical ingredient. The skilled artisan
will appreciate that the percentage of each type of peptide in the formulation will be determined by the relative potency
of the peptides. This formulation advantageously allows high potency peptides to be combined with low potency peptides
for simultaneous delivery to a patient because the low potency peptides can be provided in more microspheres and the
30 high potency peptides can be provided in fewer microspheres in the same formulation. Exemplary combinations of
peptides and/or small molecules that can be administered in different sets of microspheres and in the same formulation
include: pramlintide and insulin; pramlintide and metreleptin; davalintide and metreleptin; exenatide and metreleptin;
lovastatin and niacin; atorvastatin and amlodipine; simvastatin and ezetimibe; and exenatide and metformin.
[0042] The formulations generally contain from about 0.01% (w/w) to about 50% (w/w) of the active pharmaceutical
35 ingredient (based on the total weight of the composition). For example, the amount of active pharmaceutical ingredient
can be from about 0.1% (w/w) to about 30% (w/w) of the total weight of the composition. The amount of active pharma-
ceutical ingredient will vary depending upon the desired effect, potency of the agent, the planned release levels, and
the time span over which the peptide will be released. In certain embodiments, the range of loading is between about
0.1% (w/w) to about 10% (w/w), for example, from 0.5% (w/w) to about 5% (w/w), or from 1% to 5% (w/w).
40
Sugars

[0043] The microspheres contain about 2% (w/w) sucrose.

45 Sustained Release

[0044] The compositions are sustained release compositions, meaning that the active pharmaceutical ingredient con-
tained in the compositions will be released into the patient over an extended period of time such as, for example, a
period of two days, or three days, or at least two days, or at least three days, or over a period of one week, two weeks,
50 one month, three months, or one year. The release of the active pharmaceutical ingredient is considered complete when
there is no longer a therapeutic level of active pharmaceutical ingredient in the patient’s body, as determined by the
medical judgment of those of ordinary skill in the art.
[0045] Cmax as used herein is the maximum serum concentration of drug which occurs during the period of release
which is monitored. Cave as used herein, is the average serum concentration of drug derived by dividing the area under
55 the curve (AUC) of the release profile by the duration of the release.
[0046] In one embodiment the ratio of Cmax to Cave is about 3 or less. This profile is particularly desirable for anti-
diabetic or glucoregulatory polypeptides, such as those described herein. A ratio of about 3 or less can provide a Cave
in a therapeutic window while avoiding adverse drug side effects which can result from higher ratios. Further by controlling

8
 EP 2 341 905 B2

the physical aspects of the sustained release composition, as described herein, a superior desired release profile can
be achieved and controlled, for example, by appropriate selection of carrier properties, such as viscosity. There is thus
provided a reduced burst (i.e. initial release; e.g., Cmax at 0-1 day). In other embodiments the Cmax to Cave ratio is
from about 1 to about 3, or from 1 to 3, or from about 2 to about 3, or from 2 to 3. Further, a Cmax, if present, can be
5 shifted from the burst or initial release period into the "sustained phase" of release. In one embodiment the Cmax can
occur at at least 7, 14, 21, 28, 35 or 42 days post administration and can occur at any integer day in between. In a further
embodiment the Cmax occurs at about 21 to 35 days after administration, and in yet another embodiment is at about
28 to 31 days, and further at about 28 days after administration. In a further embodiment the maximal concentration of
drug (e.g. plasma concentration) occurs at at least 7, 14, 21, 28, 35 or 42 days post administration and can occur at any
10 integer day in between. In yet a further embodiment the maximal concentration of drug occurs at about 21 to 35 days
after administration, particularly in the case of glucoregulatory agents such as exendin-4, GLP-1, GIP or their analogs.

Longer Shelf Life

15 [0047] One advantage offered by the present formulations is a longer shelf life for the formulation. It was discovered
unexpectedly that sustained release compositions retain remarkable stability when stored in a non-aqueous carrier as
described herein. In one embodiment the formulation has a shelf life of at least 6 months. In other embodiments the
formulation has a shelf life of at least 1 year, or at least 18 months, or at least 2 years. By "shelf life" is meant the
formulation can be stored or maintained for that period of time under appropriate environmental conditions while retaining
20 at least 90% of the desired activity of the active pharmaceutical ingredient relative to the activity at initial formulation (as
100%). In another embodiment the active pharmaceutical ingredient retains at least 95%, or at least 98% or at least
99% of its desired activity as compared to its activity immediately before storage. When the formulation contains micro-
spheres, shelf life also refers to the retention of particle size and/or morphology of the microspheres. Retention of size
morphology can be determined by microscopic examination, the use of which is known to persons of ordinary skill in the art.
25 [0048] When formulated as disclosed herein a peptide or protein as active ingredient is less susceptible to oxidation
and to hydrolysis, either chemical or proteolytic, both during storage and during its sustained release period after injection.
The addition of an anti-oxidant or other stabilizer is not required in these formulations, particularly those where the carrier
is a medium chain triglyceride.

30 Reduced Burst Release

[0049] Another advantage of the present formulations is that formulations according to the present disclosure offer a
significantly reduced burst release rate compared with other formulations. When previously available injectable sustained
release formulations are injected into a patient there is often a "burst" of active ingredient or agent associated with the
35 injection. Without wanting to be bound by any specific theory, it is believed this burst is caused by that quantity of active
pharmaceutical ingredient in the formulation that is not retained within the polymer that is released over time. By "burst
release" is meant that quantity of active pharmaceutical ingredient released within the first 24 hours after injection. In
other embodiments it is that quantity of active that is release over 1 hour, or 2 hours, or 4 hours, or 8 hours, or 12 hours
after injection. In various embodiments the formulation of the invention has a burst release after injection of less than
40 10% or less than 5%, or less than 3%, or less than 2.5%, or less than 2%, or less than 1% or less than 0.75% or less
than 0.5% or less than 0.25% or less than 0.1%. Percentages refer to the percentage of the total amount of active
pharmaceutical ingredient in the injected formulation. Following injection of the formulation in the patient, the burst
release may occur at any time up to about 24 hours, thereafter there may be a lag time where substantially no active
pharmaceutical ingredient is released from the microspheres, and then the polymeric microspheres begin degrading
45 and releasing the active pharmaceutical ingredient. The skilled artisan will appreciate that the time period when the burst
release occurs may vary from patient to patient.
[0050] Burst can be assessed by measuring the proportion of the total area under the curve for a particular time period
following administration of a drug. Area under the curve (AUC) is a well established measurement in the pharmaceutical
sciences and measures the amount of drug or active ingredient that reaches the bloodstream in a set period of time. As
50 is well known in the art, the period of time selected will varying depending on the time period the concentration of the
drug in the blood is expected to be detectable or within the drug’s therapeutic window. AUC is calculated by plotting the
concentration of the drug in the blood, for example plasma concentrations, at various times during the selected time
period and then calculating the total area under the curve obtained. In one exemplary embodiment, the area under the
curve is measured for a 42 day period and using the formulations described herein, the release or burst as measured
55 within the first 24 hours is 5% or less, 2% or less, 1.5% or less, 1% or less, or 0.5% or less of the total AUC. In another
embodiment, the formulations described herein result in a burst or proportion of the AUC that is 20% or less, 15% or
less, 10% or less, 5% or less, or 2% or less than that obtained when the sustained release composition is contained in
a carrier in which the active pharmaceutical ingredient is soluble.

9
 EP 2 341 905 B2

[0051] In another embodiment, the formulations described herein limit initial burst such that the upper limit of the
therapeutic window for the active pharmaceutical ingredient is not exceeded. The therapeutic window is the range of
concentration of active pharmaceutical ingredient in the circulation, above which the active pharmaceutical ingredient
has its desired effect, but below the concentration at which the adverse effects associated with the active pharmaceutical
5 ingredient outweigh the benefits as would be generally accepted among physicians. In one exemplary embodiment, the
active pharmaceutical ingredient is an exendin, for example exenatide, or agonist analogue thereof, and administration
of the formulations described do not result in a circulating level of active pharmaceutical ingredient exceeding 400 pg/ml
during the first 24 hours following administration. In another exemplary embodiment the active pharmaceutical ingredient
is an exendin, for example exenatide, or agonist analogue thereof, and administration of the formulations described
10 does not result in a circulating level of active pharmaceutical ingredient exceeding 350 pg/ml during the first 24 hours
following administration.
[0052] Initial burst can also be assessed by comparing circulating concentrations of the active pharmaceutical ingredient
in a time period immediately following administration of the formulation with the circulating concentration of the drug in
a second time period that immediately follows the first. In one embodiment, use of the formulations of the present
15 disclosure result in circulating concentrations of active pharmaceutical ingredient during the first 24 hours following
administration that do not exceed the circulating concentration during the next 24 hour period. In another embodiment,
use of the formulations of the present disclosure result in average circulating concentration of active pharmaceutical
ingredient during the first 24 hours following administration do not exceed the average circulating concentration during
the next 24 hour period.
20
Methods of Storing

[0053] Another aspect provides methods of storing the sustained release formulations described herein. The methods
of storing the formulations described herein may also be referred to as methods of preventing the degradation of the
25 microspheres. By "storing" is meant that the formulation is retained for a period of time within its container without adding
any additional component to the container and without removing the formulation from the container (e.g., in the manu-
facturing facility, during transport, in the pharmacy). The storage time will typically be the amount of time between
packaging of the formulation and its use by the patient. After the storage time the formulation is administered to the
patient in need thereof. "Administering" to the patient includes self-administration. The methods involve storing the
30 sustained release formulations for a period of at least 1 week, at least 2 weeks, at least 1 month, at least 3 months, at
least 1 year, at least 18 months, or at least 2 years. In some embodiments, the formulations can be stored at 5°C or
25°C. There is minimal degradation of the microspheres when the formulations are stored for such extended periods of
time.
[0054] In another embodiment the invention provides methods of maintaining the potency of (e.g., preventing the loss
35 of biological activity) and/or purity (e.g. preventing chemical changes in the molecule) an active pharmaceutical ingredient.
Thus, a peptide or protein or other API that has undergone a chemical change (e.g. oxidation) may result in a loss of
purity, but may still retain its potency. The methods involve storing a microsphere comprising a active pharmaceutical
ingredient in a non-aqueous carrier as described herein for a period of time, whereby the potency and/or purity of the
active pharmaceutical ingredient is maintained by the microspheres and the non-aqueous carrier. In the formulations
40 described herein, at least 80%, at least 90%; at least 95%; at least 98%; or at least 99% of the potency and/or purity of
the active pharmaceutical ingredient is retained for a period of time of at least 1 week, at least 2 weeks, at least 1 month,
at least 3 months, at least 1 year, at least 18 months, or at least 2 years.

Administering/Treatment
45
[0055] In another aspect the present invention provides an active pharmaceutical ingredient for use in methods of
administering to a patient in need thereof. The methods involve administering to the patient a formulation or composition
as described herein. Any of the formulations described herein can be administered by parenteral administration, using
any of the methods described herein. For example, the formulations can be administered by subcutaneous, intra-mus-
50 cular, intra-peritoneal, intra-abdominal, intravenous, or any suitable manner of administration. In one embodiment, the
formulations described herein are administered subcutaneously. In one embodiment the methods involve injecting the
formulation without the patient performing a prior step of combining the sustained release composition with a second
carrier.
[0056] In one embodiment the administration does not comprise a mixing step. A mixing step is a step where the
55 microspheres are combined with a carrier prior to injection. In various embodiments the mixing step is a step where the
microspheres are combined with a carrier within the 1 week period prior to injection in the patient. The carrier can be a
non-aqueous carrier, such as those described herein. Administration of the formulation refers to the complete process
of the user interacting with the formulation, including mixing, combining any ingredients forming the formulation, and the

10
 EP 2 341 905 B2

actual injection or other form of providing the formulation to the patient,

[0057] The frequency of administration can vary depending on any one or a combination of factors such as the amount
of the formulation administered, the release profile of the formulation, the amount of active pharmaceutical ingredient
in the formulation, and the circulating level of active pharmaceutical ingredient to be achieved. In particular embodiments,
5 the formulations described herein can be administered once daily, once per week, once every two weeks, once a month,
once every two months, once every three months, once every four months, once every six months or once per year. In
one embodiment, the formulation is administered once a week. In another embodiment, the formulation is administered
once a month.
[0058] When the formulations comprise a GLP-I receptor agonist, such as GLP-I or an analog thereof, or an exendin
10 (e.g., exenatide) or an analog thereof, they can be used to treat numerous diseases, such as diabetes (e.g., Type 1
diabetes, Type II diabetes, gestational diabetes), impaired glucose tolerance, hyperglycemia (e.g., fasting and postpran-
dial), obesity, overweight, non- alcoholic fatty liver disease, non-alcoholic steatohepatitis (NASH), and the like. The
formulations comprising a GLP-I receptor agonist (e.g., exenatide) will also be useful to stimulate insulin release; lower
plasma glucagon; reduce food intake, reduce appetite, decrease gastric motility, delay gastric emptying, and lower
15 plasma lipid (e.g., triglycerides, cholesterol) levels. These methods of treatment are described, for example, in US Patent
No. 5,424,286, US Patent No. 6,858,576, US Patent No. 6,872,700, US Patent No. 6,956,025, and US Patent No.
6,956,025, and WO 2007/022518.
[0059] In certain embodiments, administration of any of the formulations provided herein comprising a glucoregulatory
peptide such as an exendin, e.g. exenatide, result in a 2 hour plasma glucose of less than 300 mg/dl, less than 275
20 mg/dl, less than 250 mg/dl, or less than 225 mg/dl. In a particular embodiment administration of any of the formulations
provided herein comprising a glucoregulatory peptide such as an exendin, e.g. exenatide, results in a 2 hour plasma
glucose of less than 200 mg/dl. In other embodiments, administration of any of the formulations provided herein comprising
a glucoregulatory peptide such as an exendin, e.g. exenatide, results in a 2 hour plasma glucose of less than 190 mg/dl,
less than 180 mg/dl, less than 170 mg/dl, less than 160 mg/dl, or less than 150 mg/dl. In certain embodiments, admin-
25 istration of any of the formulations provided herein comprising a glucoregulatory peptide such as an exendin, e.g.
exenatide, results in a 2 hour plasma glucose less than 140 mg/dl. In further embodiments, administration of any of the
formulations provided herein comprising a glucoregulatory peptide such as an exendin, e.g. exenatide, results in a
venous or capillary fasting blood glucose (FBG) level of less than 200 mg/dl, less than 175 mg/dl, less than 150 mg/dl,
less than 140 mg/dl, less than 130 mg/dl, less than 120 mg/dl, or less than 115 mg/dl. In one embodiment, a FBG level
30 of less than 110 mg/dl is achieved, while in another embodiment a FBG level of less than 100 mg/dl is achieved.
[0060] In additional embodiments, administration of any of the formulations provided herein comprising a glucoregu-
latory peptide such as an exendin, e.g. exenatide, results in a 2 hour venous or capillary blood glucose level of less than
300 mg/dl, less than 275 mg/dl, less than 250 mg/dl, less than 225 mg/dl, or less than 200 mg/dl. In a particular embodiment
administration of any of the formulations provided herein comprising a glucoregulatory peptide such as an exendin, e.g.
35 exenatide, results in a 2 hour blood glucose level of less than 180 mg/dl. In further embodiments, administration of any
of the formulations provided herein comprising a glucoregulatory peptide such as an exendin, e.g. exenatide, result in
blood glucose levels of less than 170 mg/dl, less than 160 mg/dl, less than 150 mg/dl, less than 140 mg/dl, less than
130 mg/dl, or less than 120 mg/dl. In particular embodiments, administration of any of the formulations provided herein
comprising a glucoregulatory peptide such as an exendin, e.g. exenatide, result in a 2 hour venous blood glucose level
40 of less than 120 mg/dl, while in other embodiments, a 2 hour capillary blood glucose level of less than 140 mg/dl is achieved.
[0061] In one embodiment, glucose levels are average glucose levels calculated over a chosen time period. Specific
examples include, but are not limited to, daily average glucose levels, weekly average glucose levels, monthly average
glucose levels or yearly average glucose levels. Two hour circulating glucose levels are determined after an oral glucose
tolerance test (OGTT). In the standard test, 75 g of anhydrous glucose is dissolved in 250-300 ml of water and administered
45 over 5 minutes. In children, glucose is administered at a rate of 1.75 g/kg body weight up to a maximum of 75 grams of
glucose. A baseline glucose level is obtained prior to ingestion and then typically every 30 minutes for 2 hours. For
gestational diabetes, a 100 g, 3 hour test is often used.
[0062] Because glucose freely crosses the cell membrane of red blood cells, erythrocyte hemoglobin undergoes a
nonenzymatic glycosylation at the amine residues. Hemoglobin A1c (HbAlc) refers to the percentage of hemoglobin
50 molecules with glucose moieties attached to the N-terminal valines of each of the two beta-chains. Glycohemoglobin
includes HbAlc along with other forms of hemoglobin where glycosylation has occurred at other amino acids. The
percentage of hemoglobin molecules undergoing glycosylation is proportional to the average ambient glucose concen-
trations during the previous during the previous 60-90 days. HbAlc is a commonly used measure to assess the state of
glycemic control in patients with diabetes.
55 [0063] In one embodiment, administration of any of the formulations provided herein comprising a glucoregulatory
peptide such as an exendin, e.g. exenatide, results in a reduction to, maintenance of, or both of HbAlc levels of less
than 8%. In another embodiment HbAlc levels are reduced to, maintained at, or both to less than 7.5%, while in yet
another embodiment, HbAlc levels are reduced to, maintained at, or both at less than 7%. In further embodiments,

11
 EP 2 341 905 B2

administration of any of the formulations provided herein comprising a glucoregulatory peptide such as an exendin, e.g.
exenatide, results in a reduction to or maintenance of, or both of HbAlc levels to less than 6.5%, less than 6%, less than
5.5%, less than 5% less than 4.5% or less than 4%. Thus, the compositions disclosed herein are useful in a method of
reducing or maintaining HbAlc levels in the blood, the methods comprising administering a composition disclosed herein.
5 In another embodiment, administration of any of the formulations provided herein comprising a glucoregulatory peptide
such as an exendin, e.g. exenatide, results in a reduction to, maintenance of, or both of glycosylated hemoglobin levels
of less than 10%. In another embodiment, glycosylated hemoglobin levels are reduced to, maintained at, or both to less
than 9.5%; while in yet another embodiment, glycosylated hemoglobin levels are reduced to, maintained at, or both at
less than 9%. In further embodiments administration of any of the formulations provided herein comprising a glucoreg-
10 ulatory peptide such as an exendin, e.g. exenatide, results in a reduction to, or maintenance of, or both of glycosylated
hemoglobin levels to less than 8.5%, less than 8%, less than 7.5%, less than 7% less than 6.5%, less than 6%, less
than 5.5%, less than 5%, less than 4.5% or less than 4%. In other aspects administration of any of the formulations
provided herein comprising a glucoregulatory peptide such as an exendin, e.g. exenatide, results in a lower of HbAlc by
at least 0.2%, at least 0.4%, at least 0.6%, at least 0.8%, at least 1%, at least 1.2%, at least 1.4%, at least 1.6%, at least
15 1.8%, or at least 2%. Thus, the invention provides methods of reducing or maintaining glycosylated hemoglobin levels
in the blood, the methods involving administering a composition described herein.
[0064] It should be realized that a subject in need of lowering of blood glucose is not limited to patients having diabetes
mellitus, but may include any subject suffering from hyperglycemia for whatever reason including, but not limited to,
injury, trauma, surgery, stroke and myocardial infarction. The amount of glucose lowering will vary with the subject in
20 question and depend on factors such as the severity of the hyperglycemia and the severity of the disease, disorder or
condition in question.

Examples

25 [0065] The following examples provide further illustrations of making and using the formulations described herein.
With respect to the Examples herein, MCT oil refers to medium chain triglyceride oil which is commercially available as
MIGLYOL® 812 (Sasol Germany GmbH, Witten, Germany).

Example 1
30
[0066] Microspheres may be prepared by processes known in the art and described, e.g., in US Patent No. 7,563,871
and US Patent No. 7,456,254. Microspheres comprising a poly(lactide-co-glycolide) copolymer having dispersed therein
5% (w/w) exenatide and 2% (w/w) sucrose were obtained. The poly(lactide-co-glycolide) copolymer had a ratio of
lactide:glycolide of 1:1. These microspheres are currently being developed by Amylin Pharmaceuticals, Inc. (San Diego,
35 CA), Alkermes, Inc. (Cambridge, MA), and Eli Lilly and Company (Indianapolis, IN) for a once-weekly formulation for
treating diabetes. Gedulin et al, Diabetologia, 48:1380-1385 (2004).

Example 2

40 [0067] The stability of the microspheres from Example 1 was investigated to determine their stability over an extended
period of time while stored in a non-aqueous carrier. Microspheres from Example 1 were stored for a period of 6 months
at 5°C in a formulation comprising a non-aqueous carrier (i.e., sesame oil; MCT oil; and ethyl oleate, which is a monoglyc-
eride). The control was an aqueous formulation comprising the microspheres from Example 1 in an aqueous carrier
containing carboxymethylcellulose and a surfactant.
45 [0068] The stability of the microspheres was determined by morphology and particle size via examination under a
microscope. Exenatide purity, potency (by HPLC evaluation), and in vitro release were also determined. As shown in
Table 1, after 6 months of storage the physical structure (i.e., size, morphology) of the microspheres did not change.
[0069] As shown in Table 2, the microspheres stored in a MCT oil showed no change in the purity of exenatide based
on an HPLC analysis. Impurities might also be referred to as degradation products from the peptide. High purity means
50 relatively little degradation of the peptide. The purity is relative to the formulation at time zero. The microspheres stored
in sesame oil and ethyl oleate showed a slight decrease in the purity of exenatide. The impurities did not appear to be
oil or poly(lactide-co-glycolide) polymer related (based on retention times), but appeared to be related to the stability of
exenatide itself.
[0070] Table 3 shows that the potency of exenatide did not significantly decrease over the 6 month period regardless
55 of the non-aqueous carrier that was used.

12
 EP 2 341 905 B2

Table 1: Particle size and morphology using microscope

size (mm) (standard deviation (mm)) morphology
T=0 1 month 6 months 0 to 6 months
5
sesame oil 64 (22) 63 (23) 64(12) no change
MCT oil 65 (19) 60 (22) 61 (17) no change
ethyl oleate 64 (16) 62(16) 59 (13) no change

10

Table 2: Change in Purity of Exenatide Containing Formulation

% purity of exenatide
t=0 1 month % change* 3 month % change* 6 month % change*
15
sesame oil 95.93 95.68 -0.25 94.55 -1.38 95.00 -0.93
MCT oil 95.63 95.56 -0.07 94.67 -0.96 95.50 -0.13
ethyl oleate 95.60 95.80 0.20 93.67 -1.93 94.70 -0.90
*Changes less than 0.5% are considered to be insignificant
20

Table 3: Change in Potency of Exenatide Based on Carrier in Formulation

carrier time zero 1 month 3 months 6 months
25
sesame oil 97 104 98 98
MCT oil 94 108 99 99
ethyl oleate 95 98 99 100
30

Example 3

[0071] The pharmacokinetics of the formulations in Example 2 were determined, except that 2% (w/w) lecithin was
added to the ethyl oleate carrier. Single injections with a dose of 53 mg/ml of microspheres per ml of non-aqueous carrier
35
were administered to 6 rats with a 21G needle. In the study, a comparison was also made to the microspheres from
Example 1 that were mixed with an aqueous carrier just before injection.
[0072] Figure 1 provides a comparison of the pharmacokinetics of the four different formulations of microspheres
containing exenatide. In three formulations, the carrier is an oil (e.g., sesame oil; MCT oil; ethyl oleate). In one comparative
formulation, the carrier is an aqueous diluent. As can be seen from the data, the formulations having an oil carrier had
40
reduced burst when compared to the formulation having an aqueous carrier.
[0073] Figure 2 is a graphical simulation of data extrapolated from Figure 1 of the plasma exenatide concentration
over time of the formulation comprising the MCT oil carrier and the comparative formulation comprising the aqueous
carrier. The plasma concentration plateau of exenatide may be reached after about 5 dosings.

45
Example 4

[0074] A formulation comprising the microspheres of Example 1 in an aqueous carrier and a formulation comprising
the microspheres of Example 1 in an MCT carrier were prepared. The burst release was evaluated by adding about 0.75
mL of the formulations to a 10 mM HEPES release buffer. The mixture was agitated to ensure that the microspheres
50
achieved full contact with the HEPES release buffer. After incubation at 37°C for one hour, the mixture was centrifuged
and the aqueous phase was analyzed by HPLC to determine the burst release. The concentration of the dose tested
for release was 150 mg/mL.
[0075] Figure 3 shows the lower burst release of the formulation having the oil carrier compared to the formulations
having an aqueous carrier. The graph shows that with an aqueous carrier, about 0.6% of exenatide was released in the
55
burst. With the formulation having the MCT oil carrier, less than 0.1% of exenatide was released in the burst.
[0076] Figure 4 illustrates the in vivo release profile in rats over 10 hours for the formulation of Example 1 in MCT oil
compared to a formulation comprising the same microspheres in an aqueous (saline) carrier. In the time period following

13
 EP 2 341 905 B2

sub-cutaneous administration of the formulation, the entrance of exenatide into the plasma was markedly lower than
the same microspheres administered in the aqueous carrier. The formulation of the invention shows no burst release,
and a markedly more gradual entrance into the blood plasma versus the aqueous formulation. In contrast, the aqueous
formulation showed a burst release followed by a sharper entrance into the blood plasma.
5
Example 5

[0077] Microparticles were prepared in a manner similar to that described in the examples in US Patent No. 5,439,688,
the disclosure of which is incorporated by reference herein. Eight samples were prepared by briefly mixing an active
10 pharmaceutical ingredient (i.e., davalintide, pramlintide, metreleptin, bovine serum albumin, sodium salicylate, salicylic
acid, minocycline HCl, insulin) and polymer (i.e, poly(lactide-co-glycolide) copolymer or polycaprolactone/PLGA copol-
ymer) and then the mixture was placed in a grinder to obtain a well-homogenized powder. Mixtures ranged from 2% to
10% w/w of the active pharmaceutical ingredient. The mixed powder was transferred to an extruder where the temperature
was adjusted according to the chosen polymer. Some polymers needed higher temperatures to produce a melt with
15 good flow properties. The extruder contained twin screws that moved clockwise to produce efficient mixing. The material
was extruded through a 1.5 mm orifice, collected, cooled at room temperature, and cut into short strands about 1-2
inches long. These strands were then fed into a 12-tooth rotor mill, followed by a sieving step to produce microparticles
of about 20 to 100 microns. The microparticles were collected and stored at 5°C until further use.
[0078] Experimental samples were prepared by dispersing about 50 mg of the microparticles into 0.75 mL of a MCT
20 oil carrier. The samples were stored at 5°C and 25°C for two days, two weeks, or one month, at which times representative
samples were tested. The fraction of drug that remained in the microparticles and the fraction of drug that partitioned
into the MCT oil carrier were determined. Briefly, the samples were centrifuged to separate the microparticles from the
MCT oil carrier. Each portion was treated independently to determine the amount of drug it contained. Results are
reported on the basis of the percent residing in each independent portion.
25
Table 4: PLGA copolymer; 2 Days Storage at 5°C
Compound Microparticles MCT Carrier
davalintide 99.8% 0.2%
30 pramlintide 100.0% 0.0%
metreleptin 100.0% 0.0%
bovine serum albumin 100.0% 0.0%
sodium salicylate 99.5% 0.5%
35
salicylic acid 98.9% 1.1%
minocycline 99.1% 0.9%

40
Table 5: PLGA copolymer; 1 Month Storage at 5°C
Compound Microparticles MCT Carrier
davalintide 99.4% 0.6%
45 pramlintide 99.7% 0.3%
metreleptin 100.0% 0.0%
bovine serum albumin 100.0% 0.0%
sodium salicylate 98.7% 1.3%
50
salicylic acid 99.9% 0.1%
minocycline 99.9% 0.1%
insulin 99.5% 0.5%
55

14
 EP 2 341 905 B2

Table 6: PLGA copolymer; 2 Days Storage at 25°C

Compound Microparticles MCT Carrier
davalintide 100.0% 0.0%
5
pramlintide 100.0% 0.0%
metreleptin 100.0% 0.0%
bovine serum albumin 100.0% 0.0%

10 sodium salicylate 97.7% 2.3%

salicylic acid 99.1% 0.9%
minocycline 99.4% 0.6%

15
Table 7: PLGA copolymer; 1 Month Storage at 25°C
PLGA Polymer; 1 Month Storage at 25°C
Compound Microparticles MCT Carrier
20 davalintide 100.0% 0.0%
pramlintide 100.0% 0.0%
metreleptin 100.0% 0.0%
bovine serum albumin 100.0% 0.0%
25
sodium salicylate 98.5% 1.5%
salicylic acid 99.8% 0.2%
minocycline 99.6% 0.4%
30
insulin 99.3% 0.7%

Table: 8: polycaprolactone/PLGA copolymer; Two Weeks Storage

35 5°C 25°C
Compound Microparticles MCT Carrier Microparticles MCT Carrier
pramlintide 100.0% 0.0% 100.0% 0.0%

40
[0079] The data in Tables 4-8 illustrate the broad applicability of the sustained release formulations described herein
to a variety of different active pharmaceutical ingredients, including peptides and small molecules. The compositions
have been successfully produced using a variety of peptides, bovine serum albumin, and even a selection of small
molecules. Surprisingly salicylic acid, which is oil soluble, did not migrate into the MCT carrier oil, despite that its solubility
45 in the MCT oil is greater than 30 mg/ml. Thus, the microparticles remain intact upon storage in MCT even when the
active pharmaceutical ingredient is soluble in MCT. The data further illustrate that the compositions can be successfully
produced even using other polymer mixtures in the microparticles.

Example 6
50
[0080] The percentage purity of exenatide was measured by HPLC at one month intervals over a 9 month period in
the following four formulations: (i) a formulation comprising the microspheres of Example 1 stored in an oil MCT oil carrier
at 5°C; (ii) a formulation comprising the microspheres of Example 1 stored in an MCT oil carrier at 25°C; (iii) dry micro-
spheres of Example 1 that had been stored in a container for 9 months at 5°C without a liquid carrier, and that were then
55 admixed with an aqueous carrier immediately prior to the study; and (iv) dry microspheres of Example 1 that had been
stored in a container for 9 months at 25°C without a liquid carrier, and that were then admixed with an aqueous carrier
immediately prior to the study.
[0081] Figures 5A and B show the following: (i) exenatide had a purity greater than 93% at 6 months and 9 months

15
 EP 2 341 905 B2

in the formulation with the oil carrier at a temperature of 5°C; (ii) exenatide had a purity greater than 86% at 6 months
and 9 months in the formulation with the oil carrier at a temperature of 25°C; (iii) exenatide had a purity of greater than
94% at 6 months where the microspheres had been stored dry at 5 °C; and (iv) exenatide had a purity of greater than
90% at 6 months in the formulation where the microspheres had been stored dry at a temperature of 25°C. In Figure
5 5A, the purity of exenatide was determined by strong cation exchange HPLC. In Figure 5B, the purity of exenatide was
determined by reverse-phase HPLC.

Example 7

10 [0082] Formulations containing the microspheres from Example 1 and an MCT oil carrier were stored at 5° and the
potency of exenatide was measured at monthly intervals for 9 months. Additionally, formulations containing the micro-
spheres from Example 1 and an MCT oil carrier were stored at 25° and the potency of exenatide was measured at
monthly intervals for 6 months. Figure 6 presents the results which show that the potency of exenatide was preserved
for at least 9 months.
15
Example 8

[0083] The physical integrity of a formulation containing the microspheres from Example 1 in an MCT oil carrier was
analyzed. After storage for a period of 6 months at 5°C, the molecular weight of the poly(lactide-co-glycolide) copolymer
20 did not change relative to time zero. After storage for a period of 6 months at 25°C, the molecular weight of the poly(lactide-
co-glycolide) copolymer decreased by 6 kDaltons, which was comparable to the molecular weight change of dry micro-
spheres (i.e., microspheres stored for 6 months at 25°C not in any carrier). The mean diameter of the microspheres was
measured after storage at 3, 6, and 9 months at either 5°C or 25°C, and no change in mean diameter was detected
relative to time zero.
25
Example 9

[0084] The ratio of lactide/glycolide for the microparticles was also investigated for use with various APIs. The Table
below provides the various lactide/glycolide ratios used.
30
Polymer Drug Approx polymer MW (kDa) Lactide/Glycolide ratio for PLGA
PLGA davalintide 10 50/50
PLGA pramlintide 10 50/50
PLGA Leptin 10 75/25
35
PLGA BSA 25 50/50
PLGA Na Salicylate 25 50/50
PLGA Salicylic acid 25 50/50
PLGA Minocycline 10 75/25
40 PLGA Insulin 25 50/50
1.1:1 PCL/PLGA pramlintide PCL = 150 50/50
PLGA = 10

45 Claims

1. A manufactured pre-mixed formulation for injection consisting essentially of a suspension of:

(i) a pharmaceutically acceptable carrier which comprises one or more triglycerides of C6 -C12 fatty acids; and
50 (ii) microspheres which consist essentially of a poly(lactide-co-glycolide) polymer having dispersed therein about
5% (w/w) exenatide as active pharmaceutical ingredient and about 2% (w/w) sucrose; wherein the ratio of
lactide:glycolide in the polymer is about 1:1
wherein the pharmaceutically acceptable non-aqueous carrier consists of one or more triglycerides of C6 -C12
fatty acids,
55 wherein the triglycerides comprise 0 to 2 wt% C6 fatty acid, 50 to 65 wt% C8 fatty acid, 30 to 45 wt% C10 fatty
acid, and 0 to 2 wt% C12 fatty acid.

16
 EP 2 341 905 B2

Patentansprüche

1. Hergestellte vorgemischte Formulierung zur Injektion, die im Wesentlichen aus einer Suspension von Folgendem
besteht:
5
(i) einem pharmazeutisch annehmbaren Träger, der ein oder mehrere Triglyceride von C 6-C12-Fettsäuren um-
fasst; und
(ii) Mikrokugeln, die im Wesentlichen aus einem Poly(lactid-co-glycolid)-Polymer bestehen, in dem etwa 5%
(Gew./Gew.) Exenatid als aktiver pharmazeutischer Bestandteil und etwa 2% (Gew./Gew.) Saccharose disper-
10 giert sind; wobei das Verhältnis von Lactid:Glycolid in dem Polymer etwa 1:1 beträgt,

wobei der pharmazeutisch annehmbare nicht-wässrige Träger aus einem oder mehreren Triglyceriden von
C6-C12-Fettsäuren besteht,
wobei die Triglyceride 0 bis 2 Gew.-% C6-Fettsäure, 50 bis 65 Gew.-% C8-Fettsäure, 30 bis 45 Gew.-%
15 C10-Fettsäure und 0 bis 2 Gew.-% C12-Fettsäure umfassen.

Revendications

20 1. Formulation pré-mélangée manufacturée pour injection essentiellement constituée d’une suspension de :

(i) un vecteur pharmaceutiquement acceptable qui comprend un ou plusieurs triglycérides d’acides gras en
C6-C 12 ; et
(ii) des microsphères qui sont essentiellement constituées d’un polymère de poly(lactide-co-glycolide) à l’inté-
25 rieur duquel sont dispersés environ 5 % (p/p) d’exénatide en tant que principe pharmaceutique actif et environ
2 % (p/p) de saccharose ; dans lequel le rapport lactide/glycolide dans le polymère est d’environ 1:1,

dans lequel le vecteur non aqueux pharmaceutiquement acceptable est constitué d’un ou plusieurs trigly-
cérides d’acides gras en C6-C12,
30 dans lequel les triglycérides comprennent de 0 à 2 % en poids d’un acide gras en C6, de 50 à 65 % en
poids d’un acide gras en C8, de 30 à 45 % en poids d’un acide gras en C10, et de 0 à 2 % en poids d’un
acide gras en C12.

35

40

45

50

55

17
EP 2 341 905 B2

18
EP 2 341 905 B2

19
EP 2 341 905 B2

20
EP 2 341 905 B2

21
EP 2 341 905 B2

22
EP 2 341 905 B2

23
EP 2 341 905 B2

24
 EP 2 341 905 B2

REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European
patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be
excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description

• US 7563871 B [0031] [0066] • US 7223725 B [0038]

• US 7456254 B [0031] [0066] • WO 2007139941 A [0038]
• US 7223440 B [0031] • US 5424286 A [0058]
• US 6824822 B [0031] • US 6858576 B [0058]
• US 6667061 B [0031] • US 6872700 B [0058]
• US 6495164 B [0031] • US 6956025 B [0058]
• US 6479065 B [0031] • WO 2007022518 A [0058]
• US 7157555 B [0038] • US 5439688 A [0077]
• US 7220721 B [0038]

Non-patent literature cited in the description

• GEDULIN et al. Diabetologia, 2004, vol. 48,

1380-1385 [0066]

25