The present invention is a pharmaceutical composition for use in the treatment of diabetes to counteract postprandial blood glucose excursions and for acute treatment of hyperglycemia The composition, which includes human insulin or an insulin analog, citrate, treprostinil and stabilizing agents, has a faster uptake of insulin from injection sites than existing commercial insulin compositions and that is stable for commercial use. The composition is useful for providing meal-time insulin activity, for use in continuous subcutaneous insulin infusion (CSII) or as an acute treatment for hyperglycemia when insulin is needed.

The time-action profile of insulin is important for controlling postprandial blood glucose levels. In healthy individuals, the pancreas secretes a spike of insulin in response to absorbed food, which results in increased plasma insulin levels within several minutes. In individuals with type 1 diabetes and in certain individuals with type 2 diabetes, insulin must be administered; however, administered insulin enters blood slowly from the subcutaneous space. Delayed release of insulin and onset of action which is too slow at the beginning of a meal leads to hyperglycemia during or immediately after the meal. A protracted duration of action from the subcutaneous space leads to excessive insulin between meals which can cause postprandial hypoglycemia.

There have been previous efforts to accelerate the time action of insulin products. Early efforts to develop such products included the development of novel rapid-acting insulin analogs, like insulin lispro (HUMALOG®), insulin aspart (NOVOLOG®), and insulin glulisine (APIDRA®). Insulin lispro and insulin aspart achieve rapid action through changes in the amino acid sequences from human insulin that weaken the dimer-dimer interface and alter hexameric stability under subcutaneous conditions. Insulin glulisine also includes changes in the sequences of the amino acid chains in human insulin; however, its commercial formulation lacks zinc and does not form stabilizing hexamers. Another insulin analog stated to be rapid-acting, but which is still in development, Fluorolog, includes a single fluorine atom attached to the Phe residue at position 24 of the insulin B-chain.

The rapid-acting insulin analogs insulin lispro, aspart and glulisine became available in the 1990s and early 2000s. Even with so-called rapid-acting insulin

analogs, however, the maximum plasma insulin level is not reached until 50-90 minutes following a subcutaneous injection. This is slower than endogenous insulin release from a normally functioning pancreas and does not always match glucose absorption profiles.

Another avenue to achieve rapid action that has been explored is the use of ingredients or excipients which improve the time action profile of insulin when provided in combination with insulin. For example, U.S. 8,324,157 states that a faster onset of action compared with existing insulin therapies can be achieved by adding a nicotinic compound, such as nicotinamide, and the amino acid arginine, and optionally glutamic acid. US2013/0231281 discloses compositions comprising an insulin and oligosaccharides, either alone or in combination with a polyanionic compound and states that such compositions are fast acting. US2014/0113856 discloses compositions containing insulin in combination with a zinc chelator, such as ethyl enediaminetetraacetic acid (EDTA), another excipient described in the publication as a "dissolution/stabilization" agent, such as citric acid or sodium citrate, and a magnesium-containing compound, and states that such compositions have more rapid absorption rates and declines from peak concentrations. US2015/0065423 describes compositions comprising a peptide and a vasodilator}' agent, discloses lists of vasodilator}' agents from three different categories, and provides data on compositions containing insulin lispro and nitroglycerin and states that such formulations are rapid acting.

Despite these and other efforts, a need remains for insulin compositions mat have more rapid uptake of insulin into the blood from the injection site, more rapid onset and/or offset of action than existing insulin products, and chemical and physical stability during storage and use conditions. The present invention seeks to provide compositions which meet one or more of these needs.

It has surprisingly been found that compositions containing certain concentrations of both citrate and treprostinil have a more rapid time action profile man existing commercially available insulin compositions, and mat the chemical and physical stability of the compositions containing those concentrations of both citrate and treprostinil can be maintained, without eliminating the improvements in time action, by including in the compositions zinc and one or more additional stabilizing agents such as a surfactant, magnesium chloride or sodium chloride.

Accordingly, the present invention provides pharmaceutical compositions comprising: an insulin; citrate, in a concentration from about 5 to about 25 mM; treprostinil, in a concentration from about 0.04 to about 20 μg/mL; zinc, in a concentration sufficient to provide at least 2 zinc ions per six molecules of insulin; a preservative; and one or more additional stabilizing agents; and having a pH of about 7.0 to about 7.8 at room temperature.

In certain embodiments, the pharmaceutical composition comprises insulin lispro, in a concentration from about 100 to about 200 U/mL; citrate, in a

concentration from about IS to about 25 mM; treprostinil, in a concentration from about 0.5 to about 2 μg/mL; zinc, in a concentration from about 0.2 to about 1 mM; m-cresol, in a concentration from about 2.5 to about 3.8 mg/mL; poloxamer 188, in a concentration from about 0.03 to about 0.12 w/v; magnesium chloride, in a concentration resulting in a molar ratio of magnesium chloride to citrate of about 1:3; glycerol, in a concentration from about 1 to about 2 mg/mL; and has a pH from about 7.0 to about 7.8 at room temperature.

In certain embodiments, the pharmaceutical composition comprises: insulin lispro, in a concentration of about 100 U/mL; citrate, in a concentration of about 15 mM; treprostinil, in a concentration of about 1 μg/mL; zinc, in a concentration of about 0.6 mM; poloxamer 188, in a concentration of about 0.09 % w/v; magnesium chloride, in a concentration of about 5 mM; m-cresol, in a concentration of about 3.15 mg/mL; glycerol, in a concentration of about 1.61 mg/mL; and has a pH of about 7.4.

In certain embodiments, the pharmaceutical composition comprises: insulin lispro, in a concentration of about 2 ; citrate, in a concentration of about 15

mM; treprostinil, in a concentration of about zinc, in a concentration of

about 0.8 mM; poloxamer 188, in a concentration of about

magnesium chloride, in a concentration of about 5 mM; m-cresol, in a concentration of about 3.15 mg/mL; glycerol, in a concentration of about 1.61 mg/mL; and has a pH of about 7.4.

In certain embodiments, the pharmaceutical composition comprises insulin lispro, in a concentration from about 100 to about 200 U/mL; citrate, in a

concentration from about 15 to about 25 mM; treprostinil, in a concentration from about 0.5 to about 2 μg/mL; and zinc, in a concentration from about 0.2 to about 2 mM.

In certain embodiments, the pharmaceutical composition comprises insulin lispro, in a concentration from about 100 to about 200 U/mL; citrate, in a concentration from about IS to about 25 mM; treprostinil, in a concentration from about 0.5 to about 2 μg/mL; zinc, in a concentration from about 0.2 to about 2 mM; and magnesium, in a concentration resulting in a molar ratio of magnesium to citrate of about 1:1 to about 1:5.

In certain embodiments, the pharmaceutical composition comprises: insulin lispro, in a concentration of about 100 U/mL; citrate, in a concentration of about 15 mM; treprostinil, in a concentration of about 1 g mL; zinc, in a concentration of about 0.6 mM; and magnesium, in a concentration of about 5 mM.

In certain embodiments, the pharmaceutical composition comprises: insulin lispro, in a concentration of about 100 U/mL; citrate, in a concentration of about 15 mM; treprostinil, in a concentration of about 1 g/mL; zinc, in a concentration of about 0.6 mM; magnesium, in a concentration of about 5 mM; and m-cresol, in a concentration of about 3.15 mg/mL.

In certain embodiments, the pharmaceutical composition comprises: insulin lispro, in a concentration of about 100 U/mL; citrate, in a concentration of about 15 mM; treprostinil, in a concentration of about 1 μg/mL; zinc, in a concentration of about 0.6 mM; magnesium, in a concentration of about 5 mM; m-cresol, in a concentration of about 3.15 mg mL; and glycerol, in a concentration of about 12 mg/mL.

In certain embodiments, the pharmaceutical composition comprises: insulin lispro, in a concentration of about 100 U/mL; citrate, in a concentration of about 15 mM; treprostinil, in a concentration of about 1 μ^η^; zinc, in a concentration of about 0.6 mM; magnesium, in a concentration of about 5 mM; m-cresol, in a concentration of about 3.15 mg/mL; and a total chloride concentration of about 10 to about 50 mM; and has a pH of about 7.4

In certain embodiments, the pharmaceutical composition comprises: insulin lispro, in a concentration of about 100 U/mL; citrate, in a concentration of about 15 mM; treprostinil, in a concentration of about 1 g mL; zinc, in a concentration of about 0.6 mM; magnesium, in a concentration of about 5 mM; m-cresol, in a concentration of about 3.15 mg/mL; glycerol, in a concentration of about 12 mg/mL; and a total chloride concentration of about 10 to about 50 mM.

In certain embodiments, the pharmaceutical composition comprises: insulin lispro, in a concentration of about 100 U/mL; citrate, in a concentration of about 15 mM; treprostinil, in a concentration of about 1 μg/mL; zinc, in a concentration of about 0.6 mM; magnesium, in a concentration of about 5 mM; m-cresol, in a concentration of about 3.15 mg/mL; glycerol, in a concentration of about 12 mg/mL; and a total chloride concentration of about 10 to about 50 mM; and has a pH of about 7.4.

In certain embodiments, the pharmaceutical composition comprises insulin lispro, in a concentration of about 100 U/mL; citrate, in a concentration of 15 mM; treprostinil, in a concentration of 1 μg/mL; zinc, in a concentration of about 0.3 mM; phosphate, in a concentration of about 7mM; glycerol, in a concentration of about 16 mg/mL at pH 7.4.

In certain embodiments, the pharmaceutical composition comprises: insulin lispro, in a concentration of about 200 U/mL; citrate, in a concentration of about 15 mM; treprostinil, in a concentration of about 1 μg/mL; and zinc, in a concentration of about 0.7 to about 1.7 mM.

In certain embodiments, the pharmaceutical composition comprises: insulin lispro, in a concentration of about 200 U/mL; citrate, in a concentration of about 15 mM; treprostinil, in a concentration of about 1 μg/mL; zinc, in a concentration of about 0.8 mM; and magnesium in a concentration of about 5 to about 10 mM.

In certain embodiments, the pharmaceutical composition comprises: insulin lispro, in a concentration of about 200 U/mL; citrate, in a concentration of about 15 mM; treprostinil, in a concentration of about 1 μg/mL; zinc, in a concentration of about 0.8 mM; and magnesium in a concentration of about 5 mM.

In certain embodiments, the pharmaceutical composition comprises: insulin lispro, in a concentration of about 200 U/mL; citrate, in a concentration of about 15 mM; treprostinil, in a concentration of about 1 μg/mL; zinc, in a concentration of about 0.8 mM; magnesium in a concentration of about 5 to about 10 mM; and a total chloride concentration of about 10 to about 50 mM.

In certain embodiments, the pharmaceutical composition comprises: insulin lispro, in a concentration of about 200 U/mL; citrate, in a concentration of about 15 mM; treprostinil, in a concentration of about 1 μg/mL; zinc, in a concentration of about 0.8 mM; magnesium in a concentration of about 5 to about 10 mM; and glycerol in a concentration of about 12 mg mL.

In certain embodiments, the pharmaceutical composition comprises: insulin lispro, in a concentration of about 200 U/mL; citrate, in a concentration of about 15 mM; treprostinil, in a concentration of about 1 μg mL; zinc, in a concentration of about 0.8 mM; magnesium in a concentration of about 5 to about 10 mM; glycerol in a concentration of about 12 mg mL; and a total chloride concentration of about 10 to about 50 mM.

In certain embodiments, the pharmaceutical composition comprises: insulin lispro, in a concentration of about 200 U/mL; citrate, in a concentration of about 15 mM; treprostinil, in a concentration of about 1 μg mL; zinc, in a concentration of about 0.7 to about 1.7 mM; and m-cresol, in a concentration of about 3.15 mg/mL.

In certain embodiments, the pharmaceutical composition comprises: insulin lispro, in a concentration of about 200 U/mL; citrate, in a concentration of about 15 mM; treprostinil, in a concentration of about 1 μg mL; zinc, in a concentration of about 0.8 mM; magnesium in a concentration of about 5 to about 10 mM; and m-cresol in a concentration of about 3.15 mg/mL.

In certain embodiments, the pharmaceutical composition comprises: insulin lispro, in a concentration of about 200 U/mL; citrate, in a concentration of about 15 mM; treprostinil, in a concentration of about 1 μg mL; zinc, in a concentration of about 0.8 mM; magnesium in a concentration of about 5 to about 10 mM; a total chloride concentration of about 10 to about 50 mM; and m-cresol, in a concentration of about 3.15 mg/mL.

In certain embodiments, the pharmaceutical composition comprises: insulin lispro, in a concentration of about 200 U/mL; citrate, in a concentration of about 15 mM; treprostinil, in a concentration of about 1 μg mL; zinc, in a concentration of about 0.8 mM; magnesium in a concentration of about 5 to about 10 mM; glycerol in a concentration of about 12 mg/mL; and m-cresol, in a concentration of about 3.15 mg/mL.

In certain embodiments, the pharmaceutical composition comprises: insulin lispro, in a concentration of about 200 U/mL; citrate, in a concentration of about 15 mM; treprostinil, in a concentration of about 1 μg mL; zinc, in a concentration of about 0.8 mM; magnesium in a concentration of about 5 to about 10 mM; glycerol in a concentration of about 12 mg/mL; and a total chloride concentration of about 10 to about 50 mM; and m-cresol, in a concentration of about 3.15 mg/mL.

In certain embodiments, the pharmaceutical composition comprises: insulin lispro, in a concentration of about 200 U/mL; citrate, in a concentration of about 15 mM; treprostinil, in a concentration of about 1 μg/mL; zinc, in a concentration of about 0.8 mM; magnesium in a concentration of about 5 mM; glycerol in a concentration of about 12 mg/mL; and a total chloride concentration of about 10 to about 50 mM; and m-cresol, in a concentration of about 3.15 mg/mL.

In addition, the present invention also provides a method of treating diabetes comprising administering to a human in need thereof an effective dose of a pharmaceutical composition of the present invention.

In addition, the present invention provides a pharmaceutical composition for use in therapy. More particularly, the present invention provides a pharmaceutical composition for use in the treatment of diabetes. The present invention also provides the use of a pharmaceutical composition in the manufacture of a medicament for the treatment of diabetes.

In addition, the present invention provides an article of manufacture comprising a pharmaceutical composition. More particularly, in certain aspects the article of manufacture is a multi-use vial, a cartridge, a re-usable pen injector, a disposable pen device, a pump device for continuous subcutaneous insulin infusion therapy or a container closure system for use in a pump device for continuous subcutaneous insulin infusion therapy.

When used herein, the term "composition" refers to a combination of insulin and the other ingredients or excipients wherein the insulin and other ingredients or excipients are in a single combined formulation, typically an aqueous formulation.

When used herein, "insulin" means human insulin or a rapid-acting structural variant, mutein, or analog of human insulin that has the functional activity of but faster onset of action than human insulin. Particular rapid-acting analogs of human insulin are insulin lispro, insulin aspart, and insulin glulisine. Insulin for commercial products may be produced using recombinant DN A methods or by chemical synthesis. Recombinant methods are well-known and are strongly preferred. A molecule of human insulin (CAS No. 11061-68-0) consists of two amino acid chains, A and B, whose sequences are well-known.

The human insulin A-chain has the following sequence of amino acids:

Gly lie Val Glu Gin Cys Cys Thr Ser lie Cys Ser Leu Tyr Gin Leu Glu Asn Tyr Cys Asn (SEQ ID NO: 1).

The human insulin B-chain has the following sequence of amino acids:

Phe Val

The chains are joined by two disulfide bonds:

The A-chain has an intra-chain disulfide bond at CysA6-CysAl 1. Human insulin has the empirical formula and a molecular weight of 5808.

Insulin lispro, the drug substance in HUMALOG®, is identical to human insulin in terms of its primary amino acid sequence except for an inversion of the natural proline-lysine sequence on the B-chain at positions 28 and 29 (

Insulin lispro (CAS No. 133107-64-9) has been shown to be equipotent to human insulin on a molar basis but its effect after subcutaneous injection is more rapid and of shorter duration than that of injected soluble human insulin. HUMALOG® contains m-cresol as a preservative and a stabilizing agent, a tonicity modifier (glycerol), a buffering agent (dibasic sodium phosphate), a stabilizer (zinc oxide) and pH adjustment for the vehicle.

A molecule of insulin lispro consists of the human insulin A-chain (SEQ ID NO. 1) cross-linked with the insulin lispro B-chain, whose amino acid sequence is given by SEQ ID NO:3, below:

Phe Val Asn Gin His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly

Glu Arg Gly Phe Phe Tyr Thr Lys Pro Thr (SEQ ID NO: 3).

The chemical formula of insulin lispro is and its molecular

weight is approximately 5808. One unit of insulin lispro is equivalent to 0.0347 mg insulin lispro.

Insulin aspart (CAS No. 116094-23-6), the drug substance in NOVOLOG®, is another rapid-onset insulin analog. Its structure consists of the A-chain of human insulin (SEQ ID NO: 1) and a B-chain in which the Pro at B28 is replaced with Asp (Pro-B28-Asp human insulin), as reflected in the following amino acid sequence:

Phe Val Asn Gin His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Asp Lys Thr (SEQ ID NO: 4).

Insulin aspart (28^ aspartic acid-human insulin) has the empirical formula and a molecular weight of about 5826. One unit of insulin

aspart corresponds to 6 nmol, corresponding with 0.035 mg salt-free anhydrous insulin aspart.

Insulin glulisine (CAS No. 207748-29-6), the drug substance in APIDRA®, is yet another rapid-onset insulin analog. A molecule of insulin glulisine consists of human insulin A-chain (SEQ ID NO: 1) and a modified B-chain (Asn-B3-Lys, Lys- B29-Glu) compared with human insulin, as reflected in the following amino acid sequence:

Insulin glulisine
has the empirical formula
and a molecular weight of 5823. One unit of insulin glulisine corresponds approximately to 0.0349 mg of insulin glulisine.

The following scheme depicts the amino acid sequences and disulfide bonds of human insulin and of the rapid-acting insulin analogs that are presently approved for use in treating meal-time excursions of blood glucose:

In certain embodiments, the compositions of the present invention have concentrations of insulin from about 40 to about 500 U/mL. In certain embodiments, the compositions of the present invention have concentrations of insulin from about 100 to about 500 U/mL. In certain embodiments, the compositions of the present invention have concentrations of insulin from about 100 to about 300 U/mL. In certain embodiments, the compositions of the present invention have concentrations of insulin from about 100 to about 200 U/mL. In certain preferred embodiments, the compositions comprise about 100 U/mL or about 200 U/mL.

In an embodiment, the insulin is selected from the group consisting of human insulin, or a rapid-acting structural variant, mutein, or analog of human insulin, such as insulin lispro, insulin aspart or insulin glulisine. In a preferred embodiment, the insulin is insulin lispro.

The improvements in the time action profile of the insulin demonstrated in the present invention are achieved through the use of certain specific concentrations of citrate and treprostinil.

The term "citrate" refers to any compound containing the citrate ion, which has the chemical name 2-hyclroxypropane-l,2,3-tricarboxylate, molecular formula and molecular weight of 189. The citrate ion is widely distributed in plants

and animals and is a naturally occurring component of the diet. It is a common metabolite in oxidative metabolism and an important component of bone. A number of citrates are GRAS (generally regarded as safe) by the U.S. Food and Drug

Administration for use in foods, including the following:

Various citrate-containing compounds are also included in parenteral drug products according to the U.S. Food and Drug Administration Inactive Ingredients database, including for example, citric acid, citric acid monohydrate, citric acid anhydrous, sodium citrate, anhydrous trisodium citrate, trisodium citrate dihydrate. The particular citrate compound used in the compositions of the present invention may be the acidic form or various salt forms, especially the alkali (e.g., sodium and potassium) salts and/or mono or dihydrates thereof. Of these, sodium citrate is preferred.

It has been found mat the concentration of citrate which may be used in compositions that are both fast acting and stable ranges from about 5 to about 25 mM. Certain compositions have citrate concentrations of about 15, about 20 or about 25 mM. It has been found that higher concentrations of citrate may lead to greater improvements in time action, but also may lead to greater liabilities from a stability standpoint. Thus, compositions with citrate concentrations at the upper end of the range require additional stabilizing agents in order to have chemical and physical stability for long-term storage and use, as described in more detail below.

Treprostinil is a synthetic analog of prostacy clin, and has the chemical name

molecular weight of 390.52 and a molecular formula of
Treprostinil is the active ingredient in the commercial drug products sold under the trade names Remodulin®, Tyvaso® and Orenitran™, which are indicated for the treatment of pulmonary arterial hypertension to diminish symptoms associated with exercise (Remodulin®) and to improve exercise ability (Tyvaso® and Orenitran™). Tyvaso® and Orenitran™ are, respectively, inhalation and oral dosage forms, and Remodulin® is indicated for subcutaneous or intravenous use as a continuous infusion. Remodulin® is currently available in 1, 2.5, 5 and 10 mg/mL dosage strengths, and each mL contains 3 mg m- cresol, 6.3 mg sodium citrate, either 5.3 mg (1, 2.5 and 5 mg/mL strengths) or 4.0 mg (10 mg/mL strength strength) sodium chloride, and water for injection.

Like citrate, treprostinil is included to contribute to the improvement in the time action profile of the insulin. Unlike citrate, however, an increase in the concentration of treprostinil has not been found to have a negative impact on stability. Due to treprostiiuTs potent vasodilatory effects, however, the concentration of treprostinil in compositions of the present invention must not be so high as to cause undesired systemic effects.

Moreover, the amount of insulin, and thus the volume of the composition, administered to a given subject at a given time is titrated based upon the subject's blood glucose levels and/or anticipated carbohydrate intake. As a result, the total quantity of treprostinil provided will vary from injection to injection. For example, in some circumstances a diabetic may wish to have as little as 1 unit of insulin administered, which would be a total injection volume of just 10 μΐ, from a 100 U/mL insulin lispro composition. On the other hand, currently available injection devices provide for doses as high as 80 U in a single injection— the total volume of such a dose from a 100 U/mL composition would be over an order of magnitude higher than that of the 1 unit dose, and some type 2 diabetes patients may require a dose of more than 100 insulin units, usually requiring more than one injection.

Thus, in compositions wherein insulin and treprostinil are both present in a single combined formulation, the treprostinil concentration must be sufficient to contribute to improvements in time action, even when a relatively small dose of insulin is needed, but must not be so high as to cause undesired systemic effects when a relatively high dose of insulin is needed. In certain embodiments, the composition comprises treprostinil in a concentration from about 0.1 to about SO μΜ, or about 0.04 to about 20 μg mL. In certain embodiments, the composition comprises treprostinil in a concentration from about 0.04 to about 10 μg/mL. A preferred treprostinil concentration in insulin compositions having insulin concentrations ranging from about 100 to about 200 U/mL is from about 0.5 to about 2 μg/mL. In certain embodiments, the treprostinil concentration is about 1 μg/mL.

As described above, while the addition of citrate leads to improvements in time action, it may also contribute to greater liabilities from a stability standpoint.

Thus, the compositions of the present invention require one or more stabilizing agents above and beyond those included in currently available commercial formulations of rapid acting insulin analogs, such as excess zinc, surfactants, magnesium-containing compounds, such as magnesium chloride, and chloride-containing compounds, such as magnesium chloride and/or sodium chloride.

With regards to zinc, the compositions of the present invention must, at a minimum, include zinc in a concentration provides at least enough zinc ions for the insulin molecules to form stabilizing hexamers, which have 2 specific, high affinity zinc binding sites. See, e.g., BioMetals 18:295-303 (2005), available at

The zinc ions

incorporated into such insulin hexamers are sometimes referred to as "bound" zinc. Thus, the compositions of the present invention must include sufficient zinc to provide at least 2 ions of zinc per hexamer of insulin. In certain embodiments of the present invention having, for example, insulin concentrations of about 100 U/mL, about 200 U/mL, about 300 U/mL or about 500 U/mL, the minimum zinc

concentration necessary to provide 2 ions of zinc per insulin hexamer would be about 0.2 mM, about 0.4 mM, about 0.6 mM or about 1 mM, respectively.

The inclusion of excess zinc - i.e., more zinc than would be bound in the 2 specific, high affinity zinc binding sites in insulin hexamers described above -however may be used to further stabilize the composition. Such zinc is sometimes referred to as "free" or "unbound" zinc. Currently available zmc-containing formulations include between about 2 and 4 zinc ions per hexamer of insulin. For example, the 100 U/mL formulations of insulin lispro (HUMALOG®) and insulin aspart (NOVOLOG®) have about 3 ions of zinc per six molecules of insulin, which corresponds with a concentration of about 0.3 mM. The currently available 200 U/mL formulation of HUMALOG® has about 3.5 ions of zinc per six molecules of insulin, which corresponds with a zinc concentration of about 0.7 mM. The currently available 100 U/mL formulation of human insulin sold by Eli Lilly and Company (HUMULIN® R) contains about 2.3 ions of zinc per six molecules of insulin, which corresponds with a zinc concentration of about 0.23 mM.

In certain compositions of the present invention, the inclusion of excess free or unbound zinc - i.e., zinc which is not bound in the 2 specific, high affinity zinc binding sites in insulin hexamers described above - has been found to have a stabilizing effect Compositions having about 100 U/mL of insulin lispro and zinc concentrations up to about 1 mM - which would constitute about 0.2 mM bound and about 0.8 mM unbound or free zinc - have been found to be both fast acting and stable. The inclusion of too much free or unbound zinc, however, may attenuate the improvements in time action. For example, a composition having about 100 U/mL of insulin lispro with a zinc concentration of about 5 mM - which would constitute about 4.8 mM unbound zinc - was found to not have the improvements in time action seen in compositions with lower zinc concentrations. In certain embodiments, the concentration of zinc ranges from about 0.2 to about 2 mM, about 0.3 to about 1.7 mM, about 0.7 to about 1.7 mM, about 0.4 to about 1 mM, about 0.4 to about 0.8 mM or about 0.6 to about 0.9 mM. In certain embodiments the composition of zinc is about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.25 or about 1.7 mM. In certain embodiments comprising about 100 U/mL

insulin lispro, the concentration of zinc is about 0.6 mM. In certain embodiments comprising about 200 U/mL insulin lispro, the concentration of zinc is about 0.8 mM.

Another stabilizing agent which may be used is a surfactant. Examples of surfactants disclosed for use in parenteral pharmaceutical compositions include polysorbates, such as polysorbate 20 (TWEEN® 20), polyethylene glycols such as PEG 400, PEG 3000, TRITON™ X-100, polyethylene glycols such as

polyoxyethylene (23) lauryl ether (CAS Number: 9002-92-0, sold under trade name BRIJ®), alkoxylated fatty acids, such as MYRJ™, polypropylene glycols, block copolymers such as poloxamer 188 (CAS Number 9003-11-6, sold under trade name PLURONIC® F-68) and poloxamer 407 (PLURONIC® F127), sorbitan alkyl esters (e g., SPAN®), polyethoxylated castor oil (e.g., KOLLIPHOR®, CREMOPHOR®) and trehalose and derivatives thereof, such as trehalose laurate ester. In certain embodiments, the surfactant is selected from the group consisting of polyoxyethylene (23) lauryl ether, poloxamer 188 and trehalose laurate ester. Most preferred is poloxamer 188. In certain embodiments, the concentration of surfactant ranges from about 0.003 to about 2% w/v, about 0.003 to about 0.3% w/v or about 0.01 to about 0.2% w/v. In preferred embodiments wherein the surfactant is poloxamer 188, the concentration of poloxamer 188 ranges from about 0.06 to about 0.12 w/v. In certain embodiments, the concentration of poloxamer 188 is about 0.06% w/v. In other embodiments, the concentration of poloxamer 188 is about 0.09% w/v. In other embodiments, the concentration of poloxamer 188 is about 0.12% w/v.

Another stabilizing agent which may be used in compositions of the present invention is magnesium, which may be provided for example through the addition of a magnesium-containing compound such as magnesium chloride, which has a molecular formula of
and molecular weight of 95.211. While MgCl2 may have stabilizing effects in certain compositions, high chloride ion (CI") concentrations may result in insulin crystallization at low temperatures, and magnesium (Mg+2) concentrations which exceed the concentration of citrate will result in insulin precipitation. Thus, the maximum amount of magnesium chloride that may be included is limited by the amount of citrate that is included. In certain embodiments, when MgC.2 is used to provide magnesium as a stabilizing agent in the compositions of the present invention, the molar ratio of magnesium chloride to citrate ranges from about 1 :2 to about 1: 10. In certain embodiments the ratio of magnesium ranges from about 1 : 1 to about 1 :5. Preferably the molar ratio of magnesium chloride to citrate ranges from about 1:3 to about 1:5. In certain embodiments, the concentration of magnesium ranges from about 1 about 15 mM. In certain embodiments, the concentration of magnesium ranges from about 1 about 5 mM, about 5 to about 10 mM or about 10 to about 15 mM. In certain embodiments, the concentration of magnesium is about 2.5, about 5, about 7.5 or about 10 mM.

Another stabilizing agent which may be used in compositions of the present invention is a chloride-containing compound, such as sodium chloride, which has molecular formula NaCl and molecular weight of 58.44. Sodium chloride is used in some currently available formulations of rapid acting insulin analogs, such as APIDRA® (insulin glulisine), which comprises 5 mg/mL sodium chloride and NOVOLOG® (insulin aspart), which comprises 0.58 mg/mL sodium chloride. In certain embodiments of the present invention wherein sodium chloride is used as a stabilizing agent, the concentration of sodium chloride ranges from about 1 to about 50 mM. In certain embodiments of the present invention wherein sodium chloride is used as a stabilizing agent, the concentration of sodium chloride ranges from about 10 to about 40 mM. In certain embodiments, the concentration of sodium chloride ranges from about 15 to about 25 mM. In certain embodiments, the concentration of sodium chloride is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19 or about 20 mM.

Both MgCl2 and NaCl result in the addition of chloride (CI") ions, and if the total chloride content of the composition is too high, the insulin in the composition may crystallize at low temperatures and may also lead to instability at high temperatures. Thus, if MgCl2 and/or NaCl are used as stabilizing agents, the total chloride content of the composition must be taken into consideration. Determining the total chloride amount present in the composition if MgCl2 and/or NaCl are used as stabilizing agents must also take into consideration the fact that chloride ions may also be added to the composition through the addition of other components, for example with the insulin bulk active pharmaceutical ingredient (API), through the addition of small amounts of HC1 which may be necessary for pH adjustments, and/or in connection with the provision of Zn, which may be added in the form of a solution prepared by solubilizing zinc oxide (ZnO) with HC1. Thus, the total chloride concentration from all sources must be considered if MgCl2 and/or NaCl are to be used as stabilizing agents. In terms of concentrations, low temperature insulin crystallization has been observed in compositions containing about 100 mM NaCl, but such issues were not observed in compositions containing up to about 30 mM total chloride. In addition, the low temperature crystallization issues associated with relatively high chloride concentrations have also been found to be sensitive to citrate concentrations. Thus, compositions of the present invention having citrate concentrations at the lower end of the range provided for herein may be more tolerant of relatively higher chloride concentrations than compositions having citrate concentrations at the higher end of the range provided for herein. For example, the addition of sodium chloride concentrations as high as 50-75 mM to formulations containing 25 mM citrate have been observed to lead to low temperature

crystallization issues, but such issues are not consistently observed either when 50 mM sodium chloride is added to a 15 mM citrate formulation or when 25 mM sodium chloride is added to a 25 mM citrate formulation. The total chloride added through the use of NaCl and/or MgCl2 as stabilizing agents should not be more than about 50 mM. In certain embodiments of the present invention, the total chloride

concentration, from all sources, ranges from about 10 to about 50 mM. In certain embodiments, the total chloride concentration ranges from about 13 to about 45 mM. In certain embodiments, the total chloride concentration ranges from about 20 to about 25 mM. In certain embodiments, the total chloride concentration ranges from about 15 to about 35 mM. In certain embodiments, the total chloride concentration ranges from about 20 to about 25 mM. In certain embodiments, the total chloride concentration is about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM or about 25 mM.

In certain embodiments, the composition may include more than one additional stabilizing agent in order to ensure the composition maintains a

commercially acceptable stability profile. A preferred combination of stabilizing agents which may be used in compositions of the present invention includes excess zinc and magnesium Another preferred combination of stabilizing agents which may be used in compositions of the present invention includes zinc, a surfactant, such as poloxamer 188, and magnesium chloride or sodium chloride.

The compositions of the present invention include one or more preservatives, which provide anti-microbial properties and may further provide stability benefits. The compositions are sterile when first produced, however, when the composition is provided in a multi-use vial or cartridge, an anti-microbial preservative compound or mixture of compounds that is compatible with the other components of the formulation is typically added at sufficient strength to meet regulators' and pharmacopoeial anti-microbial preservative requirements. See U.S. Pharmacopeia Monographs. Insulin lispro injection. USP29-NF24; British Pharmacopeia

Monographs 2008 Volume III: Insulin aspart injection; U.S. Pharmacopeia

Monographs. Insulin assays; and U.S. Pharmacopeia general chapters. USP29-NF24. Rockville, MD: U.S. Pharmacopeial Convention; 2005. Antimicrobial effectiveness testing; pp. 2499-2500. Preferred preservatives are aryl acids and phenolic compounds, or mixtures of such compounds. Effective concentrations can be ascertained readily using the methods referenced above. Preservatives commonly used in insulin products include phenol (CAS No. 108-95-2, molecular formula

molecular weight 94.11,), and m-cresol (CAS No. 108-39-4, molecular

formula C7HgO, molecular weight 108.14). Present commercial compositions, for example, contain 3.15 mg/mL m-cresol (HUMALOG® and APIDRA®), 1.72 mg/mL m-cresol and 1.50 mg/mL phenol (NOVOLOGKD), and 2.5 mg/mL m-cresol
In an embodiment, the preservative is selected from the group consisting of phenol and m-cresol. Preferably the preservative is m-cresol. In certain embodiments the m-cresol concentration is from about 2.5 to about 3.8 mg/mL. Preferably the concentration of m-cresol is about 3.15 mg/mL.

It is desirable to approximately match the tonicity (i.e., osmolality) of body fluids at the injection site as closely as possible when administering the compositions because solutions that are not approximately isotonic with body fluids can produce a painful stinging sensation when administered. Thus, it is desirable that the compositions be approximately isotonic with body fluids at the sites of injection. If the osmolality of a composition in the absence of a tonicity agent is sufficiently less than the osmolality of the tissue (for blood, about 300 mOsmol/kg; the European

Pharmacopeial requirement for osmolality is > 240 mOsmol/kg), then a tonicity agent should generally be added to raise the tonicity of the composition to about 300 mOsmol/kg. The osmolality of the composition is determined by the identities and

concentrations of other excipients in the composition, including the stabilizing agent(s). Thus, the concentrations of all of the various excipients in a composition must be assessed in order to determine whether a tonicity agent must be added and such assessments and determinations are readily made using standard techniques. See Remington: The Science and Practice of Pharmacy, David B. Troy and Paul Beringer, eds., Lippincott Williams & Wilkins, 2006, pp. 257-259; Remington: Essentials of Pharmaceutics, Linda Ed Felton, Pharmaceutical Press, 2013, pp. 277-300. Typical tonicity agents include glycerol (glycerin), mannitol and sodium chloride. If the addition of a tonicity agent is required, glycerol is preferred. In certain embodiments the concentration of glycerol is from about 1 to about 16 mg/mL. In certain embodiments, the concentration of glycerol is from about 1 to about 2 mg/mL, about 3 to about 4 mg/mL, about 5 to about 6 mg/mL, about 7 to about 8 mg/mL, about 9 to about 10 mg/mL, about 11 to about 12 mg/mL, about 13 to about 14 mg/mL, or about 15 to about 16 mg/mL. In certain embodiments, the concentration of glycerol is about 5, about 12 or about 16 mg/mL.

Citrate, which as noted above is added to contribute to improvements in time action, is also known to also have buffering properties, but if desired an additional buffering compound may be included. Examples of such buffering compounds are phosphate buffers, such as dibasic sodium phosphate, sodium acetate and

or TR1S. If an additional buffering compound is

necessary, TRIS or phosphate buffers are preferred. The pH for commercial insulin compositions is usually in the range of 7.2 to 7.6, with 7.4 ± 0.1 as a common target pH. The pH of the present invention is typically from about 7.0 to about 7.8 and it is adjusted using physiologically appropriate acids and bases, typically hydrochloric acid 10% and sodium hydroxide 10%. Preferably, the pH is about 7.4.

The route of administration for the compositions of the present invention will typically be by self-administered subcutaneous injection, e.g., by use of a syringe or a pen device, or by continuous subcutaneous insulin infusion therapy with an insulin pump device, though intravenous, intradermal, or intraperitoneal routes may also be used.

WE CLAIM:

1. A pharmaceutical composition comprising:

a. an insulin;

b. citrate, in a concentration from about 5 to about 25 mM;

c. treprostinil, in a concentration from about 0.04 to about

d. zinc, in a concentration sufficient to provide at least 2 zinc ions per six

molecules of insulin;

e. a preservative; and

f. one or more additional stabilizing agents;

and having a pH of about 7.0 to about 7.8 at room temperature.

2. The pharmaceutical composition of claim 1 wherein the zinc concentration is from about 0.2 to about 2 mM.

3. The pharmaceutical composition of any of claims 1-2 wherein the zinc concentration is from about 0.2 to about 1 mM.

4. The pharmaceutical composition of any of claims 1-3, wherein the zinc concentration is from about 0.6 to about 0.8 mM.

5. The pharmaceutical composition of any of claims 1-4, wherein the one or more additional stabilizing agents are selected from the group consisting of a surfactant, magnesium and sodium chloride.

6.

The pharmaceutical composition of any of claims 1-5, wherein the one or more additional stabilizing agents comprise magnesium

7. The pharmaceutical composition of claim 6, wherein the magnesium is present in a concentration resulting in a molar ratio of magnesium to citrate from about 1 :2 to about 1:10.

8. The pharmaceutical composition of claim 7, wherein the molar ratio of magnesium to citrate is from about 1:3 to about 1 :5.

9. The pharmaceutical composition of any of claims 7-8 wherein the magnesium is provided as magnesium chloride.

10. 0. The pharmaceutical composition of any of claims 1-9, wherein the insulin

concentration is from about 100 to about 300 U/mL.

11. The pharmaceutical composition of any of claims 1-10, wherein the insulin

concentration is either about 100 U/mL or about 200 U/mL.

12. The pharmaceutical composition of any of claims 1-1 1 , wherein the insulin is insulin lispro.

13. The pharmaceutical composition of any of claims 1-12, wherein the concentration of citrate is from about 10 to about 25 mM.

14. The pharmaceutical composition of any of claims 1-13, wherein the concentration of treprostinii is from about 0.04 to about 10 με/ηιΤ.

15. The pharmaceutical composition of any of claims 1-14, wherein the concentration of treprostinii is from about 0.5 to about 2 pg/mL.

16. The pharmaceutical composition of any of cl aims 1-15, wherein the preservative is m~ cresol.

17. The pharmaceutical composition of claim 16, wherein the concentration of m-cresol is from about 2,5 to about 3.8 mg/niL,

18. The pharmaceutical composition of any of claims 1-17, wherein the one or more additional stabilizing agents comprise sodium chloride.

19. The pharmaceutical composition of claim 18 wherein the sodium chloride is present in a concentration from about 1 to about 50 mM.

20. The pharmaceutical composition of any of claims 1-19, wherein the total

concentration of chloride is from about 10 to about 50 mM.

21. The pharmaceutical composition of any of claims 1 -20, further comprising a tonicity agent.

22. The pharmaceutical composition of claim 21, wherein the tonicity agent is glycerol

23. The pharmaceutical composition of claim 22, wherein the concentration of glycerol is from about i to about 15 mg/mL.

24. A pharmaceutical composition comprising:

a. insulin lispro, in a concentration from about 100 to about 200 U/mL;

b. citrate, in a concentration from about 5 to about 25 mM;

c. treprostinii, in a concentration from about 0.5 to about 2 .ug/mL; and d. zinc, in a concentration from about 0.2 mM to about 2 mM.

25. The pharmaceutical composition of claim 24, wherein the concentration of citrate is from about 15 to about 25 mM.

26. The pharmaceutical composition of any of claims 24-25, wherein the concentration of zinc is from about 0.6 to about 0.9 mM.

27. The pharmaceutical composition of any of claims 24-26, further comprising magnesium, in a concentration resulting in a molar ratio of magnesium to citrate from about 1 :3 to about 1 :5.

28. The pharmaceutical composition of any of claims 1-27 having a pH of about 7.4. 29. The pharmaceutical composition of claim 24 wherein:

a. insulm iispro is in a concentration of about 100 U/mL;

b. citrate is in a concentration of about 15 mM;

c. treprostinil is in a concentration of about 1 ,u.g/mL; and

d. zinc is in a concentration of about 0.6 mM;

and further comprising:

e. magnesium chloride in a concentration of about 5 mM;

f. m-cresol, in a concentration of about 3. 15 mg/mL;

g. glycerol, in a concentration of about 12 mg/mL;

and having a pH of about 7.4.

30. The pharmaceutical composition of claim 24 wherein:

a. insulin lispro is in a concentration of about 200 U/mL:

b. citrate is in a concentration of about 15 mM;

c. treprostinil is in a concentration of about 1 μg/mL; and

d. zinc is in a concentration of about 0.8 mM;

and further comprising:

e. magnesium chloride in a concentration of about 5 mM;

f. m-cresol, in a concentration of about 3.15 mg/mL; and

g. glycerol, in a concentration of about 12 mg/mL;

and having a pH of about 7.4.

31. The pharmaceutical composition of any of claims 1-30, wherein the composition provides for an uptake of insulin into the blood that is at least 20% more rapid than for compositions which contain the same insulin but which do not contain citrate and treprostinil.

32. The pharmaceutical composition of any of claims 1-30, wherein the composition is stable to allow for storage of at least 24 months at 2-8 °C and up to 28 days in-use at temperatures of up to 30°C.

33. A method of treating diabetes comprising administering to a human in need thereof an effective dose of the pharmaceutical composition of any one of Claims 1-32.

34. The pharmaceutical composition of any one of Claims 1-32 for use in therapy.

35. The pharmaceutical composition of any one of Claims 1 -32 for use in the treatment of diabetes.

36. Use of the pharmaceutical composition of any one of Claims 1-32 in the manufacture of a medicament for the treatment of diabetes.

37. An article of manufacture comprising any one of the pharmaceutical compositions of Claims 1-32.

38. The article of manufacture of Claim 37 which is a multi-use vial.

39. The article of manufacture of Claim 37 which is a multi-use cartridge.

40. The article of manufacture of Claim 37 which is a re-usable pen injector.

41. The article of manufacture of Claim 37 which is a disposable pen device.

42. The article of manufacture of Claim 37 which is a pump device for continuous
subcutaneous insulin infusion therapy.
43. The article of manufacture of Claim 37 which is a container closure system for use in a pump device for continuous subcutaneous insulin infusion therapy.