Sustained release formulations using non-aqueous carriers

ES 2 809 178 T3

DESCRIPTION

Sustained release formulations using non-aqueous vehicles

Background

Sustained-release injectable formulations offer the opportunity to provide therapeutic amounts of active pharmaceutical ingredients over an extended period of time from a single injection, thus eliminating the need for once or twice daily injections. Currently available sustained release injectable formulations using, for example, microspheres and an aqueous vehicle, have several disadvantages. The formulations do not offer long-term stability in the aqueous vehicle, therefore separate packaging and storage is required for the microspheres and the aqueous vehicle, and the patient must take various steps to combine the microspheres and the aqueous vehicle before administer the injection.

Another disadvantage of currently available injectable microsphere formulations is a large immediate release after injection, which causes an undesirable in vivo release of active pharmaceutical ingredient in a single immediate release. When medications have harmful or toxic side effects, this is undesirable.

There is a need for formulations and methods to safely administer sustained release pharmaceutical formulations to patients so that the active ingredient is released in vivo over a prolonged period of time and without unacceptable initial immediate release. Ideally, the active ingredient is released to maintain levels within the therapeutic window, that is, in the concentration range higher than that necessary to elicit the desired clinical effect, but below that where the undesirable side effects outweigh the benefits of the drug. . It is also necessary that this active pharmaceutical ingredient be provided in a way that is easy and convenient for the patient to self-administer and that it be provided in a formulation that maintains stability over a long period of time in a liquid state. The disclosure is for these as well as other important purposes.

Summary

The disclosure provides formulations comprising microspheres containing active pharmaceutical ingredients, where the microspheres are suspended in a pharmaceutically acceptable non-aqueous vehicle. The formulations are one-component injectable microsphere formulations, so they do not require the patient to mix the formulation with a pharmaceutically acceptable carrier prior to injection. The disclosure offers distinct advantages over previous two-component formulations by providing a long shelf life of the composition in the vehicle, sustained release of the active pharmaceutical ingredient, a less complex vehicle, an easier to manufacture vehicle, an injection-administration device. less complex, a kit with fewer components and ease of use by patients.

The present invention provides a premixed manufactured formulation for injection comprising a suspension of (i) a pharmaceutically acceptable non-aqueous carrier comprising one or more C6-C12 fatty acid triglycerides and (ii) microspheres comprising a biodegradable and biocompatible polymer that is a poly (lactidac-glycolide) polymer and an active pharmaceutical ingredient that is exenatide.

Preferably, the microspheres have dispersed therein 1% to 10% (w / w) of exenatide and 0.1% to 5% (w / w) of sugar; particularly where the sugar is glucose, dextrose, galactose, maltose, fructose, mannose, sucrose, lactose, trehalose, raffinose, acarbose, glycol, glycerol, erythritol, treitol, arabitol, ribitol, sorbitol, dulcitol, iditol, isomalt, maltitol , lactitol, mannitol, xylitol, or a combination of two or more thereof.

Most preferably, the pharmaceutically acceptable non-aqueous vehicle is a fractionated or unfractionated coconut oil, a fractionated or unfractionated palm oil, a fractionated or unfractionated palm kernel oil, a fractionated or unfractionated sesame oil, a fractionated or unfractionated soybean, fractionated or unfractionated almond oil, fractionated or unfractionated rapeseed oil, fractionated or unfractionated corn oil, a fractionated or unfractionated sunflower oil, a fractionated or unfractionated peanut oil, a fractionated or unfractionated olive oil, a fractionated or unfractionated castor oil, a fractionated or unfractionated soybean oil, a fractionated safflower oil or unfractionated, a fractionated or unfractionated cottonseed oil, ethyl oleate, or a combination of two or more thereof.

In a preferred embodiment, the pharmaceutically acceptable non-aqueous carrier further comprises one or more monoglycerides, one or more diglycerides, one or more triglycerides, or a combination of two or more thereof.

In another preferred embodiment, the non-aqueous pharmaceutically acceptable carrier comprises

ES 2 809 178 T3 (i) a triglyceride comprising an ester of a C6 fatty acid;

(ii) a triglyceride comprising esters of a C8 fatty acid;

(iii) a triglyceride comprising an ester of a C10 fatty acid;

(iv) a triglyceride comprising an ester of a C12 fatty acid;

(v) a triglyceride comprising esters of three C8 fatty acids;

(vi) a triglyceride comprising esters of three C10 fatty acids;

(vii) a triglyceride comprising esters of two C8 fatty acids and one C10 fatty acid;

(viii) a triglyceride comprising esters of two C10 fatty acids and one C8 fatty acid;

(ix) a triglyceride comprising esters of two C8 fatty acids and one C6 fatty acid;

(x) a triglyceride comprising esters of two C10 fatty acids and one C6 fatty acid;

(xi) a triglyceride comprising esters of a Cs fatty acid, a C10 fatty acid and a C12 fatty acid;

(xii) a triglyceride comprising esters of a Cs fatty acid, a C10 fatty acid and a C6 fatty acid or (xiii) a combination of two or more thereof.

In a further preferred embodiment, the pharmaceutically acceptable non-aqueous carrier comprises a triglyceride comprising esters of (i) 0 to 2% by weight of Ce fatty acid, 65 to 80% by weight of Cs fatty acid, 20 to 35% by weight C10 fatty acid and 0 to 2% by weight C12 fatty acid;

(ii) 0 to 2% by weight of Ce fatty acid, 50 to 65% by weight of Cs fatty acid, 30 to 45% by weight of C10 fatty acid and 0 to 2% by weight of C12 fatty acid;

(iii) 0 to 2% by weight of Ca fatty acid, 45 to 65% by weight of Cs fatty acid, 30 to 45% by weight of C10 fatty acid, 0 to 3% by weight of C12 fatty acid; and 0 to 5% by weight of linoleic acid; or (iv) 0 to 2% by weight of C6 fatty acid, 45 to 55% by weight of Cs fatty acid, 30 to 40% by weight of C10 fatty acid, 0 to 3% by weight of C12 fatty acid and 10 to 20% by weight of succinic acid.

Preferably, in said formulation, the pharmaceutically acceptable non-aqueous vehicle comprises 0 to 2% by weight of C6 fatty acid, 50 to 65% by weight of Cs fatty acid, 30 to 45% by weight of C10 fatty acid and 0 to 2% by weight of C12 fatty acid.

In those embodiments, it is preferred that the triglycerides comprise up to 2% C14 fatty acids.

It is a preferred aspect of the invention that the formulation further comprises a pharmaceutically acceptable excipient; particularly, where the pharmaceutically acceptable excipient is a sugar, a sugar alcohol, an antioxidant, a preservative, or a combination of two or more thereof; and / or, where the pharmaceutically acceptable excipient is sucrose, glucose, dextrose, galactose, maltose, trehalose, fructose, maltodextrin, glycol, glycerol, erythritol, treitol, arabitol, ribitol, sorbitol, dulcitol, iditol, isomalt, maltitol, lactitol , mannitol, xylitol, benzoic acid, sorbic acid, meta cresol, sodium benzoate, potassium sorbate, methyl paraben, propyl paraben, butyl paraben, benzalkonium chloride, sodium metabisulphite, butylated hydroxyanisole, Butylated hydroxytoluene, sodium sulfite, tocopherol, thymol, ascorbate, propyl gallate, or a combination of two or more thereof.

It is also a preferred aspect of the invention that the formulation does not further comprise a gelling agent.

Also provided is the formulation of the invention for use in the treatment of diabetes, stimulation of insulin release; lowering of plasma glucagon; reduction of food intake; reduced appetite; decreased gastric motility; delayed gastric emptying; lowering of plasma lipid levels; treatment of impaired glucose intolerance; treatment of hyperglycemia; treatment of obesity; treatment of overweight; treatment of fatty liver disease; or treatment of nonalcoholic steatohepatitis in a patient in need thereof, which comprises administering the formulation of the invention to the patient.

Preferably, the formulation is for use in the treatment of diabetes; stimulation of insulin release; lowering of glucagon in plasma; reduction of food intake; reduced appetite; decreased gastric motility; or delayed gastric emptying.

The disclosure provides sustained release formulations comprising a pharmaceutically acceptable carrier consisting essentially of one or more triglycerides comprising C6-C12 fatty acids; and microspheres consisting essentially of a poly (lactide-co-glycolide) polymer that has dispersed therein about 1% to 10% (w / w) of exenatide and about 0.1% to 5% (w / w) of a sugar; wherein the lactide: glycolide ratio in the polymer is about 70:30 to 30:70, or about 1: 1. In one embodiment, exenatide is present in an amount of 1% to 5% (w / w) or 5% (w / w) and the sugar is present in an amount of 2% (w / w). The sugar can be, for example, glucose, dextrose, galactose, maltose, fructose, mannose, sucrose, lactose, trehalose, raffinose, acarbose, glycol, glycerol, erythritol, treitol, 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 vehicle. In

In one embodiment, the total pore volume of the microspheres is approximately 0.1 ml / g or less, as determined by mercury intrusion porosimetry, to provide a release profile having a concentration ratio Maximum serum exenatide during the release period (Cmax) to the average serum exenatide concentration during the release period (Cpro) of approximately 3 or less. Furthermore, although the microspheres are formulated in oil (i.e., a vehicle as disclosed herein), the microspheres do not necessarily have oil contained within the interior spaces or pores, or within a substantial number of interior spaces or pores. , of the microspheres, and still can achieve the amazing properties disclosed herein.

The disclosure provides sustained release formulations comprising a non-aqueous carrier and pharmaceutically acceptable microspheres comprising a biodegradable and biocompatible polymer and an active pharmaceutical ingredient. In one embodiment, the total pore volume of the microspheres is approximately 0.1 ml / g or less, as determined by mercury intrusion porosimetry, to provide a release profile that has a maximum serum concentration ratio of the beginning. active pharmaceutical during the release period (Cmax) to the average serum concentration of the active pharmaceutical ingredient during the release period (Cpro) of about 3 or less. Furthermore, although the microspheres are formulated in oil (i.e., a vehicle as disclosed herein), in some embodiments, the microspheres have no oil contained within the interior spaces or pores, or have no oil within a number of substantial space or pore interiors of the microspheres, yet still achieve the amazing properties disclosed herein. The formulation is a suspension whereby the microspheres are suspended in the vehicle. The nonaqueous vehicle can be an oil, such as fractionated oils, triglycerides, diglycerides, monoglycerides, diesters of propylene glycol and fatty acids, and the like.

In one embodiment, the active ingredient is not soluble in the vehicle. In various other embodiments, the active ingredient has a solubility in the vehicle 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. ml or less than 1 mg / ml. In still other embodiments, the active pharmaceutical ingredient has a solubility in 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 vehicle. In still other embodiments where it is desirable to have some active ingredient readily available, it can also be incorporated directly into the vehicle in a pharmaceutically effective amount.

Disclosure provides a kit, available to a patient or healthcare provider. The kit contains a container with 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 a single dose vial or a multiple dose vial. In another embodiment, the container is a cartridge, such as a cartridge for use in an injection apparatus. The cartridge can be a single or multi-dose cartridge. In different embodiments, the kit contains 1, 2, 3, 4 or even 5 or more of said packages that carry a formulation of the invention. A further advantage of the formulations is that, in one embodiment, the container is provided without preservatives. But in other embodiments, a preservative can be soluble in the selected carrier and provided in the formulation.

Also provided is a premixed formulation of the invention for injection consisting essentially of a suspension of:

(i) a pharmaceutically acceptable carrier comprising one or more C6-C12 fatty acid triglycerides; and (ii) microspheres consisting essentially of a poly (lactide-co-glycolide) polymer that has dispersed in it approximately 5% (w / w) of exenatide as an active pharmaceutical ingredient and approximately 2% (w / w) of saccharose; wherein the lactide: glycolide ratio in the polymer is approximately 1: 1.

Brief description of the drawings

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 vehicle is a medium chain triglyceride (MCT) commercially available as MIGLYOL® 812 (Sasol Germany GmbH, Witten, Germany).

Figure 1 provides a comparison of the pharmacokinetics of four different microsphere formulations. In three formulations, the vehicle was an oil (eg, 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 overlay) of extrapolated data from Figure 1 of the plasma concentration of exenatide over time for the microsphere formulation comprising the oil carrier and the microsphere formulation comprising the aqueous vehicle in male Sprague Dawley rats. The plasma concentration plateau of exenatide can be reached after approximately 5 doses.

Figure 3 illustrates in vitro release for a formulation comprising microspheres in an oil carrier

ES 2 809 178 T3 compared to formulations comprising microspheres in an aqueous vehicle.

Figure 4 illustrates the in vivo release profile in rats over 10 hours for a formulation comprising microspheres in an oil vehicle and a formulation comprising microspheres in an aqueous vehicle.

Figures 5A and B illustrate the purity of exenatide for 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 compared to the purity of exenatide that was stored in dry microspheres from 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.

Figure 6 illustrates the stability / potency of exenatide in a formulation in which the microspheres are suspended in an oil carrier, where a formulation is stored at 5 ° C and a formulation is stored at 25 ° C.

Detailed description

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 composed of a biodegradable and biocompatible polymer having an active pharmaceutical ingredient dispersed therein, where the microspheres are suspended in a non-aqueous vehicle. The formulations are one-component injectable formulations, compared to two-component formulations that require the microspheres to be stored dry in a container, while the liquid vehicle can be stored in a separate container, so the patient must mix the two before injection. The formulations offer the convenience of long-term stability of a pharmaceutical composition in a non-aqueous liquid carrier, thus eliminating the 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 needs to lightly shake the formulation before injecting it from the same container. When the provided container is also an injection device, even the step of injecting the formulation is eliminated. The formulations described herein offer the important additional advantage of substantially reducing the immediate release of the active pharmaceutical ingredient. Thus, even active pharmaceutical ingredients that have a toxic effect at higher concentrations can be safely administered using the formulations described herein.

The term "patient" refers to mammals, including humans, domestic animals, farm animals, zoo animals, and the like. In one embodiment, the patient is a human.

The terms "treat" or "treatment" refer to the administration of one or more active pharmaceutical ingredients to a patient who has a condition or disorder or a predisposition to a condition or disorder, for the purpose of alleviating, mitigating, remedying, recovering, improving, slowing down. or arresting the progression or worsening of the disease, or at least one symptom of the disease, condition or disorder, or the predisposition towards the condition or disorder.

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 Gila monster.

One-component formulation

The above injectable formulations contained at least two components. The first component can be dry microspheres and the second component can be a pharmaceutically acceptable aqueous carrier. The first component and the second component are stored in separate sealed packages (eg, vials, pen injection chambers). The patient receives the two-component formulation, and the patient or pharmacist must physically mix the two components prior to injection. In the case of an injection pen, the two components are mixed immediately prior to injection into the patient. Two component formulations are typically administered to the patient within a short time after being mixed with the pharmaceutically acceptable carrier. For example, the microsphere component and the pharmaceutically acceptable aqueous carrier are mixed and the formulation is then administered to the patient in about 30 or 60 minutes.

The formulations described herein are one-component injectable formulations. A one-component injectable formulation refers to a formulation that contains the microspheres and the pharmaceutically acceptable carrier provided in the same container, and that can 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 premixed formulation for injection. A one-component formulation provides great convenience for patient manufacturing, transportation, storage, and use.

In another embodiment, the one-component formulation described herein is provided in a sealed container. A sealed container is a container that has not been opened, punctured or does not have something inserted into it.

ES 2 809 178 T3 from the time of completion of manufacture. The time of completion of manufacture is the time when the container containing the formulation is initially sealed. Packages can include vials (single use or multiple use), syringes, injection pens (eg, single use or multiple use), and the like.

Vehicle

Carrier (or carrier) refers to a pharmaceutically acceptable non-aqueous liquid material. The vehicle is substantially inert so that it does not interact with the microspheres described herein and is non-toxic so that it does not adversely affect the patient. The vehicle is preferably approved or awaiting approval from a federal or state government regulatory agency or is listed in the US Pharmacopeia. or other generally recognized pharmacopoeia for use in mammals, such as humans. The term vehicle can include one or more compounds. The vehicle is a non-solubilizing vehicle, in that the vehicle does not solubilize the polymer (s) that form (s) the microspheres. In a further embodiment, the carrier does not solubilize the active pharmaceutical ingredient (s) within the microspheres. For example, the vehicle will not solubilize exenatide or other water soluble therapeutic peptides or proteins.

The term non-aqueous does not exclude small amounts of wastewater that do not have a demonstrated negative impact on the stability of sustained release compositions. Thus, a composition can 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) of water and is still considered non-aqueous. The vehicle does not solubilize the microspheres to the point of having a demonstrated negative impact on the stability of the microspheres or a demonstrated loss of immediate release control. In one embodiment, the vehicle does not enter or penetrate the biodegradable and biocompatible polymer and does not disperse within the biodegradable and biocompatible polymer. The vehicle 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 can occur to a less than 1% degree and is still considered a non-aqueous vehicle that is not swelling of the microspheres.

In one embodiment, the nonaqueous vehicle is a pharmaceutically acceptable oil. An oil is a substance that is in a viscous liquid state at room temperature or slightly warmer, and is hydrophobic (immiscible with water) and lipophilic (miscible with other oils, literally). Examples of pharmaceutically acceptable oil carriers include volatile vegetable oils and essential oils. Examples of pharmaceutically acceptable oil carriers include coconut oil, palm oil, palm kernel oil, sesame oil, soybean oil, almond oil, rapeseed oil, corn oil, sunflower oil, peanut oil. , olive oil, castor oil, soybean oil, safflower oil, cottonseed oil, ethyl oleate, and the like. The vehicle can comprise an oil or a combination of two or more oils.

In one embodiment, the vehicle is a fractionated oil or a combination of two or more fractionated oils. Exemplary pharmaceutically acceptable oil carriers include fractionated coconut oil, fractionated palm oil, fractionated palm kernel oil, fractionated sesame oil, fractionated soybean oil, fractionated almond oil, fractionated rapeseed oil, fractionated corn oil, Fractionated sunflower oil, fractionated peanut oil, fractionated olive oil, fractionated castor oil, fractionated soybean oil, fractionated safflower oil, fractionated cottonseed oil and the like. In one embodiment, the vehicle is fractionated coconut oil. In one embodiment, the vehicle is fractionated palm kernel oil. In one embodiment, the vehicle is a combination of fractionated coconut oil and fractionated palm kernel oil.

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. The person skilled in the art will appreciate that some long chain fatty acids can remain in the fractionated oil, but generally in amounts less than 5% by weight or less than 2% by weight of the total fatty acid content of the fractionated oil.

In the present invention, the carrier comprises one or more C6-C12 fatty acid triglycerides.

In one embodiment, the carrier further comprises a long chain triglyceride, a diglyceride, a monoglyceride, a propylene glycol diester of fatty acids, or a combination of two or more thereof.

In one embodiment, the vehicle is a medium chain triglyceride. Medium chain triglyceride can be synthetic or natural (for example, produced from fractionated oils, such as coconut oil and / or palm kernel oil). Medium chain triglyceride refers to glycerol esters having three fatty acid chains from Ce to CU, where the three fatty acid chains may be the same or different. Medium chain triglycerides are represented by the compound of Formula (I): where each x is in

ES 2 809 178 T3

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the chain is known as Ce fatty acid. When x is 6, the chain is called Cs fatty acid. When x is 8, the chain is known as the Cio fatty acid. When x is 10, the chain is known as C12 fatty acid. In various embodiments, each x is the same integer; two x's are the same integer and one x is a different integer; or each x is a different integer.

In various embodiments, the medium chain triglyceride comprises esters of (i) three Cs fatty acids; (Ii) three C10 fatty acids; (iii) two Cs fatty acids and one C10 fatty acid; (iv) two C10 fatty acids and one Cs fatty acid; (v) two Cs fatty acids and one Ce fatty acid; (vi) two C10 fatty acids and one Ce fatty acid; (vii) a Cs fatty acid, a C10 fatty acid and a Ce fatty acid; or (viii) any other combination of Ce, Cs, C10, and C12 fatty acids. In one embodiment, the medium chain triglyceride comprises two Cs fatty acids and one C10 fatty acid. In one embodiment, the medium chain triglyceride comprises two C10 fatty acids and one Cs fatty acid.

One skilled in the art will appreciate that a mixture of medium chain triglycerides can result from any process (eg fractionation, hydrogenation) to prepare medium chain triglycerides. For example, substantially all of the medium chain triglycerides obtained from fractionated coconut oil can comprise Cs and / or C10 fatty acids; however, there may be some medium chain triglycerides that contain Ce and / or C12 fatty acids.

In one embodiment, the medium chain triglycerides comprise esters of (i) 0 to 2% by weight Ce fatty acid, 65 to 80% by weight Cs fatty acid, 20 to 35% by weight C10 fatty acid, and 0 to 2% by weight of C12 fatty acid; (Ii) 0 to 2% by weight of Ce fatty acid, 50 to 65% by weight of Cs fatty acid, 30 to 45% by weight of C10 fatty acid and 0 to 2% by weight of C12 fatty acid; (iii) 0 to 2% by weight of Ce fatty acid, 45 to 65% by weight of Cs fatty acid, 30 to 45% by weight of C10 fatty acid, 0 to 3% by weight of C12 fatty acid; and 0 to 5% by weight of linoleic acid; or (iv) 0 to 2% by weight of Ce fatty acid, 45 to 55% by weight of Cg fatty acid, 30 to 40% by weight of C10 fatty acid, 0 to 3% by weight of C12 fatty acid and 10 to 20 succinic. In one embodiment, the medium chain triglyceride comprises 0 to 2% by weight of Ce fatty acid, 50 to 65% by weight of Cs fatty acid, 30 to 45% by weight of C10 fatty acid, and 0 to 2% by weight of C12 fatty acid, and which is commercially available as MIGLYOL® 812 (Sasol Germany GmbH, Witten, Germany). The% by weight is based on the total fatty acid content of the triglycerides. In one embodiment, the medium chain triglycerides can comprise up to 2% C14 fatty acids.

The vehicle can comprise one, two, three, four or more different medium chain triglycerides. In one embodiment, the carrier comprises a medium chain triglyceride comprising esters of two Cs fatty acids and one C10 fatty acid. In one embodiment, the carrier comprises a medium chain triglyceride comprising esters of one Cs fatty acid and two C10 fatty acids. In one embodiment, the vehicle comprises two different medium chain triglycerides, where a first medium chain triglyceride comprises esters of two Cs fatty acids and one C10 fatty acid and a second medium chain triglyceride comprises esters of one Cs fatty acid and two fatty acids.

C10. In one embodiment, the carrier comprises a medium chain triglyceride comprising 0 to 2% by weight of Ce fatty acid, 50 to 65% by weight of Cs fatty acid, 30 to 45% by weight of C10 fatty acid, 0 to 2 % by weight of C12 fatty acid, based on the total fatty acid content of the medium chain triglyceride.

Triglycerides can be prepared by methods known in the art and are commercially available as MIGLYOL® 810, 812, 818, 829 (Sasol Germany GmbH, Witten, Germany) or NEOBEE® 1053, 895, M-5 (Stepan Company, Northfield, IL ).

In another embodiment, the carrier is a propylene glycol diester of saturated vegetable fatty acids with chain lengths of Cs and C10 (caprylic and capric acid). An example of one of these commercially available vehicles is MIGLYOL® 840 (Sasol Germany GmbH, Witten, Germany). The non-aqueous, pharmaceutically acceptable carrier may optionally comprise other pharmaceutically acceptable excipients. Exemplary excipients include sugars (eg, sucrose, glucose, dextrose, galactose, maltose, trehalose, fructose, maltodextrin); sugar alcohols (eg, glycol, glycerol, erythritol, treitol, arabitol, ribitol, sorbitol, dulcitol, iditol, isomalt, maltitol, lactitol, mannitol, xylitol); preservatives (for example benzoic acid, sorbic acid, meta cresol, sodium benzoate, potassium sorbate, methyl paraben, propyl paraben, butyl paraben, benzalkonium chloride and the like, generally

ES 2 809 178 T3 soluble in oil, with some solubility in the selected vehicle); and antioxidants (eg, sodium metabisulfite, butylated hydroxyanisole, butylated hydroxytoluene, sodium sulfite, tocopherol, thymol, ascorbate, propyl gallate, and the like). In one embodiment, the carrier optionally comprises mannitol, maltodextrin, sorbitol, or a combination of two or more thereof.

The pharmaceutically acceptable carrier may contain a gelling agent; however, the gelling agent can only be present in an amount that does not cause a gel deposit to form at the site of administration of the formulation in vivo. In one embodiment, the pharmaceutically acceptable carrier does not contain a gelling agent. Examples of gelling agents include cellulose derivatives (eg, hydroxypropyl cellulose, carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose); polymers or copolymers (poloxamers) of polyoxyethylene and polyoxypropylene; chitosan acid and the like. One of skill in the art will understand that in vivo gel formation can be determined by methods known in the art, such as the use of histological sections and colored stains.

In certain embodiments, the non-aqueous, nonsolubilizing vehicle has a viscosity of 5 cP to 200 cP or 10 cP to 90 cP. In other embodiments, the viscosity of the non-solubilizing, non-aqueous vehicle is 20 cP to 80 cP or 30 cP to 70 cP. Thus, with reference to this disclosure, the ordinary skilled person will be able to identify other oils, triglycerides, or nonaqueous compounds that may also be present in the nonaqueous, nonsolubilizing vehicle.

Microspheres

The term microspheres includes microspheres, microparticles, nanoparticles, granules, 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 microparticle ranges can be from about one to about 180 microns in diameter. In still other embodiments, suitable release profiles are obtained when the 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 equal to about 50, 60, or 70 microns, and preferably less than about 80, 90, or 100 microns. In larger sizes, the microsphere is preferably substantially unaggregated to allow passage through a 25 gauge needle, or a 27 gauge needle, or a 30 gauge needle, or a 31 gauge needle.

Uniform and superior release profiles are obtained by controlling the size distribution. In one embodiment, a mean particle size is about 50 microns and the lower and upper range of particles is about 30 and 90 microns, respectively. Microsphere distribution can be described using a volume mean diameter. The mean diameter of the volume distribution represents the center of gravity of the distribution and is a type of average particle size. In various embodiments, the microspheres may have a mean volume distribution diameter of about 50 to 70 microns, about 50 to 60 microns, or about 50, 60, or 70 microns, with a volume distribution (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 volume distribution mean diameter of about 60 microns, with a volume distribution (DV) of less than or about 10% at 30 microns and a DV of greater than or about 90% at 90 microns. .

Microspheres can be prepared by processes known in the art and described, for example, 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.

In a further embodiment, the microspheres have a less porous outer layer and may further have a non-porous outer layer. Consequently, in the formulations disclosed herein, the oil does not have access to the interior spaces or pores, not even a substantial part of the interior spaces or pores. It is specifically contemplated that for each of the formulations disclosed herein, the microspheres may be additionally oil-free (or a carrier as disclosed herein) in the interior spaces of the microspheres. Therefore, the advantages of current formulations can be achieved without the presence of oil in the interior spaces of the microspheres when they are formulated.

Polymers

The microspheres comprise biodegradable and biocompatible polymers. A polymer is biocompatible if the polymer and any degradation products of the polymer are not toxic to the patient at administered levels and do not have any proven deleterious or adverse effects on the patient's body, for example, a substantial immune reaction at the injection site. . Biodegradable means that 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.

ES 2 809 178 T3

Exemplary biodegradable and biocompatible polymers include, for example, polylactides, polyglycolides, poly (lactide-co-glycolides), polylactic acids, polyglycolic acids, poly (lactic acid-co-glycolic acid) s, polycaprolactones, polycarbonates, polyesteramides, polyanhydrides , polyamino acids, polyorthoesters, polycyanoacrylates, poly (p-dioxanone), polyalkylene oxalates, biodegradable polyurethanes, mixtures thereof and copolymers thereof. In the present invention, the microspheres comprise a poly (lactide-coglycolide) polymer. A person of ordinary skill in the art can determine acceptable molecular weights for biodegradable and biocompatible polymers by taking into account factors such as the desired polymer degradation rate, physical properties such as mechanical strength, end group chemistry, and rate of polymer dissolution. Typically, an acceptable molecular weight range is from about 2,000 Daltons to about 2,000,000 Daltons. The biodegradable and biocompatible polymer can also be selected based on the inherent viscosity of the polymer. Suitable inherent viscosities are about 0.06 to 1.0 dl / g; from about 0.2 to 0.6 dl / g; or about 0.3 to 0.5 dl / g.

In one embodiment, the biodegradable and biocompatible polymer is a poly (lactide-co-glycolide) copolymer (also referred to as PLGA) having a lactide: glycolide ratio of 70:30 to 30:70, or 60:40 to 40: 60 or approximately 50:50. The molecular weight of the poly (lactide-co-glycolide) copolymer is from about 10,000 Daltons to about 90,000 Daltons. In another embodiment, the molecular weight of the poly (lactide-co-glycolide) copolymer is from about 30,000 Daltons to about 70,000, or from about 50,000 to about 60,000 Daltons.

The formulation can contain microspheres at a concentration of 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

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 that has 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 500 Da to 100 kDa; 1 kDa to 80 kDa; 1 kDa to 50 kDa; 1 kDa to 30 kDa; or 1 kDa to 20 kDa. In one embodiment, the peptide comprises from 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.

In the present invention, the microspheres comprise exenatide as an active pharmaceutical ingredient.

In one embodiment, the active pharmaceutical ingredient is a GLP-I receptor agonist compound, such as an exendin, an exendin analog, gLP-1 (7-37), a GLP-1 (7-37) analog, and the like. Examples of GLP-I receptor agonists include exendin-3, exenatide, GLP-1 (1-37), GLP-1 (7-37) -NH2, GLP-1 (7-36), GLP-I (7 -36) -NH2, Leu¹⁴-exendin-4, Leu¹⁴, Phe²⁵-exendin-4, exendin-4 (1-28), Leu¹⁴-exendin-4 (1-28), Leu¹⁴Phe²⁵-exendin-4 (1-28), exendin-4 (1-30), Leu¹⁴-exendin-4 (1-30), Leu¹⁴Phe²⁵-exendin-4 (1-30), liraglutide and the compounds described in, for example, US Patent No. 7,157,555, US Patent No. 7,220,721, US Patent No. United States No. 7,223,725, and WO 2007/139941.

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 (eg, pramlintide, davalintide, Val²⁷-davalintida); leptin, leptin agonists (eg, metreleptin); PYY (3-36) and its agonist analogs; glucagon, glucagon agonists, glucagon antagonists, peptide chimera of GLP-I receptor agonists and glucagon agonists, human amylin peptide chimera and salmon calcitonin, insulin, heparin, low molecular weight heparin, angiotensin, argipressin, argirelin , atosiban, bivalirudin, cetrorelix, desmopressin, enfuvirtide, deptifibatide, GHRP-2, GHRP-6, gonadorelin, leuprolide, lysipressin, melanotan, nesiritide, octreotide, oxytocin, PT 141, calcitonin, sermorelin, somatostatin, terlipressin, thymopentin, thymosinal, triptorelin, vapreotide, elcatonin, ziconotide, ghrelin, nafarelin and BNP-32.

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 (eg, atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin); beta blockers and / or non-selective alpha-1 blockers (eg, carvedilol, dilatrend, eucardic, carloc); PDE3 inhibitors (eg, cilostazol); antiplatelet drugs, antithrombotic drugs, anticoagulant drugs, glycoprotein llb / llla inhibitors (eg, abciximab, eptifibatide, tirofiban); antibacterial drugs (eg, ciprofloxacin, norfloxacin, levofloxacin, moxifloxacin, sparfloxacin, gemifloxacin, ecinofloxacin, delafloxacin); Factor Xa inhibitors (eg, glycosaminoglycans, oligosaccharides, heparinoids); direct Xa inhibitors (eg, xabanes); direct thrombin (II) inhibitors (eg, hirudin, argatroban, dabigatran, melagatran, ximelagatran, defibrotide, ramatroban, antithrombin III, protein C); thrombolytic drugs (eg, plasminogen activators, urokinase, streptokinase, serine endopipidases); ACE inhibitors (eg, lisinopril, aceon, acertil, armix, coverene, coverex, coversum,

ES 2 809 178 T3 prestaium, prexanil, Prexum, procaptan); ADP / P2Y12 receptor inhibitors (eg, clopidogrel, ticlopidine, prasugrel); prostaglandin analogs (eg, beraprost, prostacyclin, iloprost, treprostinil); anticoagulants (eg, coumarin, coumatetralyl, dicoumarol, ethyl biscoumacetate, fenprocoumon, warfarin, chlorindione, diphenadione, phenindione, thioclomarol); diuretics (eg, hydrochlorothiazide); macrolides (eg, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, telithromycin); NSAIDs and COX-3 inhibitors (eg, celecoxib, etoricoxib, parecoxib); and sulfonanilides (eg, nimesulide).

One skilled in the art 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 contains one peptide (eg, pramlintide) and another set of microspheres contains a different peptide (eg, metreleptin). In one embodiment, 1 to 99% of the microspheres comprise one active pharmaceutical ingredient and 99 to 1% of the microspheres comprise a different active drug 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 drug ingredient. The person skilled in the art 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 low-potency peptides can be provided in more microspheres and 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.

Formulations generally contain from about 0.01% (w / w) to about 50% (w / w) of the active pharmaceutical 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 pharmaceutical ingredient will vary depending on the desired effect, the potency of the agent, the planned release levels, and the period of time over which the polypeptide will be released. In certain embodiments, the loading range is between about 0.1% (w / w) and about 10% (w / w), for example, 0.5% (w / w) to about 5% (w / p), or 1% to 5% (w / w). When the active pharmaceutical ingredient is a GLP-1 receptor agonist, suitable release profiles can be obtained when the active pharmaceutical ingredient, for example exenatide, is loaded at about 2% w / w to about 7% w / w, including about 2% w / w /, about 3% w / w, about 4% w / w, about 5% w / w, about 6% w / w, or about 7% w / w.

Sugars

The microspheres can also comprise one or more sugars. A sugar is a monosaccharide, disaccharide or oligosaccharide or a derivative thereof. Monosaccharide sugar alcohols are suitable derivatives of sugar. Monosaccharides include, but are not limited to, glucose, fructose, and mannose. A disaccharide, as further defined herein, is a compound that upon hydrolysis produces two molecules of a monosaccharide. Suitable disaccharides include, but are not limited to, sucrose, lactose, and trehalose. Suitable oligosaccharides include, but are not limited to, raffinose and acarbose. The microspheres may further comprise glucose, dextrose, galactose, maltose, fructose, mannose, sucrose, lactose, trehalose, raffinose, acarbose, glycol, glycerol, erythritol, treitol, arabitol, ribitol, sorbitol, dulcitol, iditol, isomalt, lactitolitol , mannitol, xylitol, or a combination of two or more thereof. In one embodiment, the sugar is sucrose, glucose, mannose, or fructose. In one embodiment, the sugar is sucrose.

The amount of sugar present in the microspheres can range from about 0.01% (w / w) to about 50% (w / w), such as from about 0.01% (w / w) to about 10% (w / wt), such as from about 0.1% (w / w) to about 5% (w / w) of the total weight of the composition. In one embodiment, about 2% (w / w) sucrose is used.

Alternatively, reference can be made to the amount of sugar present in the microspheres in a weight ratio with the active pharmaceutical ingredient. For example, the active pharmaceutical ingredient and sugar may be present in a ratio of from about 10: 1 to about 1:10 weight: weight. In some particularly preferred embodiments, the ratio of active pharmaceutical ingredient (eg exenatide) to sugar (eg sucrose) is about 3: 2 (w / w), 4: 2 (w / w) or 5: 2 (p / p). Combinations of two or more sugars can also be used. The amount of sugar, when a combination is used, is the same as the ranges listed above.

Sustained release

The compositions are sustained release compositions, which means that the active pharmaceutical ingredient contained in the compositions will be released to 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,

ES 2 809 178 T3 two weeks, 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 the active pharmaceutical ingredient in the patient's body, as determined by the medical judgment of those skilled in the art.

Cmax as used herein is the maximum serum concentration of drug that appears during the release period that is monitored. Cpro as used herein is the average serum drug concentration obtained by dividing the area under the curve (AUC) of the release profile by the duration of release.

In one embodiment, the ratio of Cmax to Cpro is about 3 or less. This profile is particularly desirable for antidiabetic or glucoregulatory polypeptides, such as those described herein. A ratio of about 3 or less can provide a Cpro in a therapeutic window while avoiding adverse pharmacological side effects that can result from higher ratios. In addition, by controlling the physical aspects of the sustained release composition, as described herein, a higher desired release profile can be achieved and controlled, for example, by appropriate selection of vehicle properties, such as viscosity. Thus, a reduced immediate release (ie initial release; eg, Cmax at 0-1 day) is provided. In other embodiments, the Cmax to Cpro 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. In addition, a Cmax, if present, can go from immediate release or period from initial release to sustained phase of release. In one embodiment, the Cmax may appear at least 7, 14, 21, 28, 35, or 42 days after administration and may appear on any number of days in between. In a further embodiment, the Cmax appears about 21 to 35 days after administration, and in yet another embodiment it is about 28 to 31 days, and furthermore about 28 days after administration. In a further embodiment, the maximum drug concentration (eg plasma concentration) appears at least 7, 14, 21, 28, 35, or 42 days after administration and can appear on any number of days in between. In yet another embodiment, the maximum drug concentration appears approximately between 21 and 35 days after administration, particularly in the case of glucoregulatory agents such as exendin-4, GLP1, GIP or their analogues.

Longer lifespan

An advantage offered by the present formulations is a longer shelf life for the formulation. Sustained release compositions were unexpectedly found to retain remarkable stability when stored in a nonaqueous vehicle 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 that the formulation can be stored or maintained for that period of time under appropriate environmental conditions while retaining at least 90% of the desired activity of the active pharmaceutical ingredient relative to the activity in the initial formulation (such 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 compared to its activity immediately prior to storage. When the formulation contains microspheres, shelf life also refers to retention of the 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 those of ordinary skill in the art.

When formulated as disclosed herein, a peptide or protein as an active ingredient is less susceptible to oxidation and hydrolysis, either chemical or proteolytic, both during storage and during its sustained release period after injection. The addition of an antioxidant or other stabilizer is not required in these formulations, particularly those in which the carrier is a medium chain triglyceride.

Reduced immediate release

Another advantage of the present formulations is that the formulations according to the present disclosure offer a significantly reduced immediate release rate compared to other formulations. When previously available sustained release injectable formulations are injected into a patient, there is often an immediate release of active ingredient or agent associated with the injection. Without wishing to be bound by any specific theory, it is believed that this immediate release is caused by that amount of active pharmaceutical ingredient in the formulation that is not retained within the polymer that is released over time. By immediate release is meant the amount of active pharmaceutical ingredient released within the first 24 hours after injection. In other embodiments, it is the amount of active that is released 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 an immediate release after injection of less than 10% or less than 5%, or less than 3%, or less than 2.5%, or less than 2%, or less. 1% or less than 0.75% or less than 0.5% or less than 0.25% or less than 0.1%. The percentages refer to the percentage of the total amount of active pharmaceutical ingredient in the injected formulation. After injection of the formulation into the patient, immediate release can occur at any time up to approximately 24 hours, thereafter there may be a lag time in which substantially no active pharmaceutical ingredient is released from the microspheres, and then polymeric microspheres begin to degrade

ES 2 809 178 T3 and release the active pharmaceutical ingredient. The person skilled in the art will appreciate that the period of time in which immediate release occurs may vary from patient to patient.

Immediate release can be evaluated by measuring the proportion of the total area under the curve during a particular period of time after administration of a drug. The area under the curve (AUC) is a well-established measure in pharmaceutical sciences and measures the amount of drug or active ingredient that reaches the bloodstream in a given period of time. As is well known in the art, the selected period of time will vary depending on the period of time that the concentration of the drug in the blood is expected to be detectable or within the therapeutic window of the drug. AUC is calculated by plotting the drug concentration in the blood, eg plasma concentrations, at various times during the selected time period and then calculating the total area under the obtained curve. In an exemplary embodiment, the area under the curve is measured over a period of 42 days and using the formulations described herein, the release or immediate release measured in 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 an immediate discharge or proportion of the ABC that is 20% or less, 15% or less, 10% or less, 5% or less, or 2% or less than the obtained when the sustained release composition is contained in a vehicle in which the active pharmaceutical ingredient is soluble.

In another embodiment, the formulations described herein limit initial immediate release so that the upper limit of the therapeutic window for the active pharmaceutical ingredient is not exceeded. The therapeutic window is the concentration range of the active pharmaceutical ingredient in 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 ingredient are greater than the benefits, as is generally accepted among physicians. In an exemplary embodiment, the active pharmaceutical ingredient is an exendin, for example exenatide, or agonist analog thereof, and administration of the described formulations does not result in a circulating level of active drug ingredient greater than 400 pg / ml during the first 24 hours after administration. In another exemplary embodiment, the active pharmaceutical ingredient is an exendin, eg, exenatide, or agonist analog thereof, and administration of the disclosed formulations does not result in a circulating level of active drug ingredient greater than 350 pg / ml during the first 24 hours after administration.

The initial immediate release can also be evaluated by comparing the circulating concentrations of the active pharmaceutical ingredient in a period of time immediately after administration of the formulation with the circulating concentration of the drug in a second period immediately following the first. In one embodiment, use of the formulations of the present disclosure results in circulating concentrations of active pharmaceutical ingredient during the first 24 hours after administration that do not exceed the circulating concentration during the next 24 hours. In another embodiment, use of the formulations of the present disclosure results in an average circulating concentration of active pharmaceutical ingredient during the first 24 hours after administration that does not exceed the average circulating concentration during the next 24 hour period.

Storage methods

Another aspect provides storage methods for the sustained release formulations described herein. The storage methods of the formulations described herein may also be referred to as methods to prevent degradation of the microspheres. By storage it is meant that the formulation is retained for a period of time within its container without adding any additional components to the container and without removing the formulation from the container (for example, in the manufacturing facilities, during transport, in the pharmacy) . Storage time will typically be the amount of time between packaging the formulation and its use by the patient. After the storage time, the formulation is administered to the patient in need. Administration to the patient includes self-administration. The methods involve storing the 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 extended periods of time.

In another embodiment, the invention provides methods for maintaining the potency of (eg, avoiding loss of biological activity) and / or purity (eg, avoiding chemical changes in the molecule) of an active pharmaceutical ingredient. Therefore, a peptide or protein or other API that has undergone a chemical change (eg oxidation) can cause a loss of purity, but can still retain its potency. The methods involve storing a microsphere comprising an active pharmaceutical ingredient in a non-aqueous vehicle 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 vehicle. In the formulations described herein, at least 80%, at least 90%; at least 95%; at least 98%; o 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 less 18 months or at least 2 years.

ES 2 809 178 T3

Administration / Treatment

In another aspect, the present invention provides an active pharmaceutical ingredient for use in methods of administration 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 subcutaneously, intramuscularly, intraperitoneally, intraabdominally, intravenously, or any suitable form 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 previous step of combining the sustained release composition with a second vehicle.

In one embodiment, the administration does not comprise a mixing step. A mixing stage is a stage where the microspheres are combined with a vehicle prior to injection. In various embodiments, the mixing step is a step in which the microspheres are combined with a vehicle within the period of 1 week prior to injection into the patient. The vehicle can be a non-aqueous vehicle, such as those described herein. Formulation administration refers to the entire process of the user interacting with the formulation, including mixing, combining any principle that forms the formulation, and the actual injection or other way of providing the formulation to the patient,

The frequency of administration may vary depending on one or a combination of factors such as the amount of formulation administered, the release profile of the formulation, the amount of active pharmaceutical ingredient in the formulation and the level of circulation of the active pharmaceutical ingredient that must be attain. In particular embodiments, the formulations described herein can be administered once a day, once a 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 a year. In one embodiment, the formulation is administered once a week. In another embodiment, the formulation is administered once a month.

When the formulations comprise a GLP-I receptor agonist, such as GLP-I or an analog thereof, or an exendin (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 postprandial), obesity, overweight, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis (NASH) and the like. Formulations comprising a GLP-I receptor agonist (eg, exenatide) will also be useful for stimulating insulin release; lower plasma glucagon; reduce food intake, reduce appetite, decrease gastric motility, delay gastric emptying, and lower plasma lipids (eg, triglyceride levels, cholesterol). These treatment methods are described, for example, in United States Patent No. 5,424,286, United States Patent No. 6,858,576, United States Patent No. 6,872,700, US Patent No. 6,956,025, US Patent No. 6,956,025, and WO 2007/022518.

In certain embodiments, administration of any of the formulations provided herein that comprise a glucoregulatory peptide such as an exendin, eg, exenatide, results in a 2-hour plasma glucose of less than 300 mg / dl, less than 275 mg / dl, less than 250 mg / dl, or less than 225 mg / dl. In a particular embodiment, the administration of any of the formulations provided herein that comprise a glucoregulatory peptide such as an exendin, for example 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 that comprise a glucoregulatory peptide such as an exendin, eg, 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, administration of any of the formulations provided herein that comprise a glucoregulatory peptide such as an exendin, eg, exenatide, results in a 2-hour plasma glucose of less than 140 mg / dl. In additional embodiments, administration of any of the formulations provided herein comprising a glucoregulatory peptide such as an exendin, eg, exenatide, results in a fasting venous or capillary 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 of less than 110 mg / dl is reached, while in another embodiment a FBG level of less than 100 mg / dl is reached.

In additional embodiments, administration of any of the formulations provided herein comprising a glucoregulatory peptide such as an exendin, eg, exenatide, results in a 2-hour capillary or venous 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, the administration of any of the formulations provided herein that comprise a glucoregulatory peptide such as an exendin, for example, exenatide, results in a blood glucose level of 2

ES 2 809 178 T3 hours of less than 180 mg / dl. In additional embodiments, administration of any of the formulations provided herein comprising a glucoregulatory peptide such as an exendin, eg, exenatide, results 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 that comprise a glucoregulatory peptide such as an exendin, eg, exenatide, results in a 2-hour venous blood glucose level 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.

In one embodiment, glucose levels are average glucose levels calculated over a chosen period of time. Specific examples include, but are not limited to, average daily glucose levels, average weekly glucose levels, average monthly glucose levels, or average annual 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 over 5 minutes. In children, glucose is administered at a rate of 1.75 g / kg of body weight up to a maximum of 75 grams of glucose. A basal glucose level is obtained before ingestion and then generally every 30 minutes for 2 hours. For gestational diabetes, a 100 g, 3 hour test is often used.

Because glucose freely crosses the cell membrane of red blood cells, erythrocyte hemoglobin undergoes non-enzymatic glycosylation at amine residues. Hemoglobin A1c (HbAlc) refers to the percentage of hemoglobin molecules with glucose residues attached to the N-terminal valines of each of the two beta chains. Glycated hemoglobin includes HbAlc along with other forms of hemoglobin where glycosylation has occurred in other amino acids. The percentage of hemoglobin molecules subjected to glycosylation is proportional to the average ambient glucose concentrations during the previous period during the previous 60-90 days. HbAlc is a commonly used measure to assess the status of glycemic control in patients with diabetes.

In one embodiment, administration of any of the formulations provided herein comprising a glucoregulatory peptide such as an exendin, eg, exenatide, results in a reduction to, maintenance of, or both of HbAlc levels of less than 8 %. In another embodiment, HbAlc levels are lowered to, maintained at, or both less than 7.5%, while in yet another embodiment, HbAlc levels are lowered to, maintained at, or both less than 7%. In additional embodiments, administration of any of the formulations provided herein comprising a glucoregulatory peptide such as an exendin, eg, exenatide, results in a reduction to or maintenance of, or both, 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. In another embodiment, administration of any of the formulations provided herein comprising a glucoregulatory peptide such as an exendin, eg, exenatide, results in a reduction to, maintenance of, or both of glycated hemoglobin levels of less than 10%. In another embodiment, glycated hemoglobin levels are reduced to, maintained at, or both less than 9.5%; while in yet another embodiment, glycated hemoglobin levels are reduced to, maintained at, or both less than 9%. In additional embodiments, administration of any of the formulations provided herein comprising a glycoregulatory peptide such as an exendin, eg, exenatide, results in a reduction to, or maintenance of, or both of glycated 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, for example exenatide, results in a decrease in 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 1.8%, or at least 2%. Thus, the invention provides methods for reducing or maintaining glycated hemoglobin levels in the blood, the methods involving administering a composition described herein.

It should be noted that a subject needing to lower blood glucose is not limited to patients with diabetes mellitus, but may include any subject suffering from hyperglycemia for any reason, including, but not limited to, injury, trauma, surgery, stroke, and myocardial infarction. The amount of glucose drop will vary with the subject in question and will depend on factors such as the severity of the hyperglycemia and the severity of the disease, disorder, or condition in question.

Examples

The following examples provide additional illustrations of how to make and use 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).

ES 2 809 178 T3

Example 1

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

Example 2

The stability of the microspheres of Example 1 was investigated for their stability over an extended period of time while stored in a nonaqueous vehicle. The microspheres of Example 1 were stored for a period of 6 months at 5 ° C in a formulation comprising a non-aqueous vehicle (ie, sesame oil; MCT oil; and ethyl oleate, which is a monoglyceride). The control was an aqueous formulation comprising the microspheres of Example 1 in an aqueous vehicle containing carboxymethylcellulose and a surfactant.

The stability of the microspheres was determined by the morphology and size of the particles by microscopic examination. Purity, potency (by HPLC evaluation), and in vitro release of exenatide were also determined. As shown in Table 1, after 6 months of storage, the physical structure (ie size, morphology) of the microspheres did not change.

As shown in Table 2, microspheres stored in MCT oil showed no change in purity of exenatide according to HPLC analysis. Impurities can also be referred to as peptide degradation products. High purity means relatively little degradation of the peptide. Purity is relative to zero time formulation. Microspheres stored in sesame oil and ethyl oleate showed a slight decrease in exenatide purity. Impurities did not appear to be related to the oil or poly (lactide-co-glycolide) polymer (as a function of retention times), but appeared to be related to the stability of exenatide itself.

Table 3 shows that exenatide potency did not decrease significantly over the 6-month period regardless of the non-aqueous vehicle used.

<td colspan="5">Table 1: Particle size and morphology using microscope</td>

<td></td><td colspan="3">size (mm) (standard deviation (mm))</td><td>morphology</td>

<td></td><td>T = 0</td><td>1 month</td><td>6 months</td><td>0 to 6 months</td>

<td>Sesame oil</td><td> 64 (22)</td><td> 63 (23)</td><td> 64(12)</td><td>without changes</td>

<td>MCT oil</td><td> 65 (19)</td><td> 60 (22)</td><td> 61 (17)</td><td>without changes</td>

<td>ethyl oleate</td><td> 64 (16)</td><td> 62(16)</td><td> 59 (13)</td><td>without changes</td>

Table 2: Change in purity of formulation containing exenatide

<td rowspan="2"></td><td colspan="6">% purity exenatide</td>

<td>t = 0</td><td>1 month</td><td colspan="2">% change * 3 months% change *</td><td>6 months</td><td>% exchange*</td>

<td>Sesame oil</td><td> 95,93</td><td> 95,68</td><td> -0,25</td><td> 94,55 -1,38</td><td> 95,00</td><td> -0,93</td>

<td>MCT oil</td><td> 95,63</td><td> 95,56</td><td> -0,07</td><td> 94,67 -0,96</td><td> 95,50</td><td> -0,13</td>

<td>ethyl oleate</td><td> 95,60</td><td> 95,80</td><td> 0,20</td><td> 93,67 -1,93</td><td> 94,70</td><td> -0,90</td>

<td colspan="5">* Changes less than 0.5% are considered insignificant</td><td></td><td></td>

Table 3: Change in the potency of exenatide according to the vehicle in the formulation

<td>vehicle</td><td>zero time</td><td>1 month</td><td>3 months</td><td>6 months</td>

<td>Sesame oil</td><td> 97</td><td> 104</td><td> 98</td><td> 98</td>

<td>MCT oil</td><td> 94</td><td> 108</td><td> 99</td><td> 99</td>

<td>ethyl oleate</td><td> 95</td><td> 98</td><td> 99</td><td> 100</td>

ES 2 809 178 T3

Example 3

The pharmacokinetics of the formulations in Example 2 were determined, except that 2% (w / w) lecithin was added to the ethyl oleate vehicle. Individual injections with a dose of 53 mg / ml of microspheres per ml of non-aqueous vehicle were administered to 6 rats with a 21G needle. In the study, a comparison was also made with microspheres from Example 1 that were mixed with an aqueous vehicle just prior to injection.

Figure 1 provides a comparison of the pharmacokinetics of the four different microsphere formulations containing exenatide. In three formulations, the vehicle is an oil (eg, sesame oil; MCT oil; ethyl oleate). In a comparative formulation, the carrier is an aqueous diluent. As can be seen from the data, the formulations having an oil vehicle had reduced immediate release compared to the formulation having an aqueous vehicle.

Figure 2 is a graphical simulation of extrapolated data from Figure 1 of the plasma concentration of exenatide over time of the formulation comprising the MCT oil vehicle and the comparative formulation comprising the aqueous vehicle. The plasma concentration plateau of exenatide can be reached after approximately 5 doses.

Example 4

A formulation comprising the microspheres of Example 1 in an aqueous vehicle and a formulation comprising the microspheres of Example 1 in an MCT vehicle were prepared. Immediate release was assessed by adding approximately 0.75 ml of the formulations to a 10 mM HEPES release buffer. The mixture was shaken to ensure that the microspheres made 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 immediate release. The concentration of the dose tested for release was 150 mg / ml.

Figure 3 shows the lower immediate release of the formulation having the oil vehicle compared to the formulations having an aqueous vehicle. The graph shows that with an aqueous vehicle, approximately 0.6% exenatide was released on immediate release. With the formulation having the MCT oil vehicle, less than 0.1% exenatide was released in immediate release.

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 vehicle (saline). In the period of time after subcutaneous administration of the formulation, the entry of exenatide into the plasma was markedly less than the same microspheres administered in the aqueous vehicle. The formulation of the invention does not show immediate release, and a markedly more gradual entry into the blood plasma compared to the aqueous formulation. In contrast, the aqueous formulation showed immediate release followed by more acute entry into blood plasma.

Example 5

The microparticles were prepared in a manner similar to that described in the examples of US Patent No. 5,439,688, the disclosure of which is incorporated herein by reference. Eight samples were prepared by briefly mixing an active pharmaceutical ingredient (i.e. davalintide, pramlintide, metreleptin, bovine serum albumin, sodium salicylate, salicylic acid, minocycline HCl, insulin) and polymer (i.e. poly (lactide-copolymer) -glycolide) or polycaprolactone / PLGA copolymer) and then the mixture was put into a mill to obtain a well homogenized powder. The 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 required higher temperatures to produce a melt with good flow properties. The extruder contained two twin propellers that moved clockwise to produce efficient mixing. The material was extruded through a 1.5mm hole, collected, cooled to room temperature, and cut into short strands approximately 1-2 inches long. These strands were then fed to a 12 tooth rotor mill, followed by a sieving step to produce microparticles of approximately 20 to 100 microns. The microparticles were collected and stored at 5 ° C until later use.

Experimental samples were prepared by dispersing approximately 50 mg of the microparticles in 0.75 ml of an MCT oil vehicle. Samples were stored at 5 ° C and 25 ° C for two days, two weeks, or one month, at which time representative samples were analyzed. The fraction of drug that remained in the microparticles and the fraction of drug that cleaved in the MCT oil vehicle was determined. Briefly, the samples were centrifuged to separate the microparticles from the MCT oil vehicle. Each portion was independently treated to determine the amount of drug it contained. Results are reported on the basis of the percentage that resides in each independent portion.

ES 2 809 178 T3

Table 4: PLGA Copolymer; 2 days storage at 5 ° C

<td>Compound</td><td>Microparticles</td><td>MCT Vehicle</td>

<td>davalintida</td><td> 99,8 %</td><td> 0,2 %</td>

<td>pramlintide</td><td> 100,0 %</td><td> 0,0 %</td>

<td>Metreleptin</td><td> 100,0 %</td><td> 0,0 %</td>

<td>bovine serum albumin</td><td> 100,0 %</td><td> 0,0 %</td>

<td>sodium salicylate</td><td> 99,5 %</td><td> 0,5 %</td>

<td>salicylic acid</td><td> 98,9 %</td><td> 1,1 %</td>

<td>minocycline</td><td> 99,1 %</td><td> 0,9 %</td>

Table 5: PLGA Copolymer; 1 month storage at 5 ° C

<td>Compound</td><td>Microparticles</td><td>MCT Vehicle</td>

<td>davalintida</td><td> 99,4 %</td><td> 0,6 %</td>

<td>pramlintide</td><td> 99,7 %</td><td> 0,3 %</td>

<td>Metreleptin</td><td> 100,0 %</td><td> 0,0 %</td>

<td>bovine serum albumin</td><td> 100,0 %</td><td> 0,0 %</td>

<td>sodium salicylate</td><td> 98,7 %</td><td> 1,3 %</td>

<td>salicylic acid</td><td> 99,9 %</td><td> 0,1 %</td>

<td>minocycline</td><td> 99,9 %</td><td> 0,1 %</td>

<td>insulin</td><td> 99,5 %</td><td> 0,5 %</td>

Table 6: PLGA Copolymer; 2 days storage at 25 ° C

<td>Compound</td><td>Microparticles</td><td>MCT Vehicle</td>

<td>davalintida</td><td> 100,0 %</td><td> 0,0 %</td>

<td>pramlintide</td><td> 100,0 %</td><td> 0,0 %</td>

<td>Metreleptin</td><td> 100,0 %</td><td> 0,0 %</td>

<td>bovine serum albumin</td><td> 100,0 %</td><td> 0,0 %</td>

<td>sodium salicylate</td><td> 97,7 %</td><td> 2,3 %</td>

<td>salicylic acid</td><td> 99,1 %</td><td> 0,9 %</td>

<td>minocycline</td><td> 99,4 %</td><td> 0,6 %</td>

Table 7: PLGA Copolymer; 1 month storage at 25 ° C

<td colspan="3">PLGA polymer; 1 month storage at 25 ° C</td>

<td>Compound</td><td>Microparticles</td><td>MCT Vehicle</td>

<td>davalintida</td><td> 100,0 %</td><td> 0,0 %</td>

<td>pramlintide</td><td> 100,0 %</td><td> 0,0 %</td>

<td>Metreleptin</td><td> 100,0 %</td><td> 0,0 %</td>

<td>bovine serum albumin</td><td> 100,0 %</td><td> 0,0 %</td>

<td>sodium salicylate</td><td> 98,5 %</td><td> 1,5 %</td>

<td>salicylic acid</td><td> 99,8 %</td><td> 0,2 %</td>

<td>minocycline</td><td> 99,6 %</td><td> 0,4 %</td>

<td>insulin</td><td> 99,3 %</td><td> 0,7 %</td>

ES 2 809 178 T3

<td>Table: 8: co</td><td colspan="4">Polycaprolactone / PLGA Polymer; Two weeks of storage</td>

<td></td><td colspan="2">5 ° C</td><td colspan="2">25 ° C</td>

<td>Compound</td><td>Microparticles</td><td>MCT Vehicle</td><td>Microparticles</td><td>MCT Vehicle</td>

<td>pramlintide</td><td> 100,0 %</td><td> 0,0 %</td><td> 100,0 %</td><td> 0,0 %</td>

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 MCT carrier oil, even though its solubility in MCT oil is greater than 30 mg / ml. Therefore, the microparticles remain intact after storage in MCT even when the active pharmaceutical ingredient is soluble in MCT. The data further illustrates that the compositions can be successfully produced even using other polymer blends in the microparticles.

Example 6

The percent 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 MCT oil vehicle at 5 ° C; (ii) a formulation comprising the microspheres of Example 1 stored in an MCT oil vehicle at 25 ° C; (iii) dried microspheres from Example 1 that had been stored in a container for 9 months at 5 ° C without a liquid vehicle, and which were then mixed with an aqueous vehicle immediately prior to study; and (iv) dried microspheres from Example 1 which had been stored in a container for 9 months at 25 ° C without a liquid vehicle, and which were then mixed with an aqueous vehicle immediately prior to study.

Figures 5A and B show the following: (i) exenatide had a purity greater than 93% at 6 months and 9 months in the formulation with the oil vehicle 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 vehicle at a temperature of 25 ° C; (iii) exenatide had a purity greater than 94% at 6 months in which the microspheres had been stored dry at 5 ° C; and (iv) exenatide had a purity greater than 90% at 6 months in the formulation where the microspheres had been stored dry at a temperature of 25 ° C. 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.

Example 7

Formulations containing the microspheres of Example 1 and an MCT oil vehicle were stored at 5 ° and the potency of exenatide was measured at monthly intervals for 9 months. Additionally, the formulations containing the microspheres of Example 1 and an MCT oil vehicle were stored at 25 ° and the potency of exenatide was measured at monthly intervals for 6 months. Figure 6 presents the results showing that exenatide potency was preserved for at least 9 months.

Example 8

A formulation containing the microspheres of Example 1 was tested for physical integrity in an MCT oil vehicle. After storage for a period of 6 months at 5 ° C, the molecular weight of the poly (lactide-co-glycolide) copolymer did not change with respect 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 change in molecular weight of the dry microspheres (i.e., the microspheres stored for 6 months at 25 ° C without any vehicle). The mean diameter of the microspheres was measured after storage at 3, 6 and 9 months at 5 ° C or 25 ° C, and no change in mean diameter was detected from time zero.

Example 9

The lactide / glycolide ratio for the microparticles was also investigated for use with various APIs. The following table provides the various lactide / glycolide ratios used.

<td>Polymer</td><td>Drug</td><td>PM approx. polymer (kDa)</td><td>Lactide / glycolide ratio for PLGA</td>

<td>PLGA</td><td>davalintida</td><td> 10</td><td> 50/50</td>

<td>PLGA</td><td>pramlintide</td><td> 10</td><td> 50/50</td>

<td>PLGA</td><td>Leptin</td><td> 10</td><td> 75/25</td>

ES 2 809 178 T3 (continued)

Polymer Drug PM approx. polymer (kDa) Lactide / glycolide ratio for PLGA PLGA BSA 25 50/50 PLGA Sodium salicylate 25 50/50 PLGA Salicylic acid 25 50/50 PLGA Minocycline 10 75/25 PLGA Insulin 25 50/50 1.1: 1 PCL / PLGA pramlintide PCL = 150 50/50 PLGA