Compositions and methods for modulating peripheral and hepatic lipid metabolism in HDV-based insulin therapy

Abstract

The disclosure provides methods, compositions, and / or algorithms for treating a subject having diabetes mellitus and / or a metabolic derangement.

Description

[0001] Atorney Docket No. 047589-5026WO1 (00244)

[0002] TITLE

[0003] Compositions and Methods for Modulating Peripheral and Hepatic Lipid Metabolism in HDV-Based Insulin Therapy

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 749,039, filed January 24, 2025, which is incorporated herein by reference in its entirety.

[0005] BACKGROUND

[0006] In a normal individual, insulin is secreted by the pancreas in response to elevated blood glucose levels. Endogenous insulin is released from the pancreas via the pancreatic vein, which flows into the portal vein and ultimately directs the hormone to the liver. Sufficient insulin passes through the liver to serve peripheral insulin-requiring tissues such as fat and muscle.

[0007] Insulin replacement is typically via subcutaneous injections for insulin-requiring patients with Type 1 or 2 diabetes. Insulin administered by this route is readily distributed to fat and muscle tissues, but it reaches the liver in significantly lower proportion as compared to endogenously secreted insulin. As a result, the liver fails to store glycogen following a meal and also converts excessive protein to glucose. The net effect of this situation in the patient is hyperglycemia due to inadequate control of hepatic glucose uptake and release. This exacerbates the early rise in glucose following ingestion of a meal and can also lead to frequent episodes of hypoglycemia in response to periods of inadvertent insulin overdosing attributable to “chasing” untoward high glucose values.

[0008] There is thus an unmet need in the art for compositions and methods for administering insulin to a subject, such that the insulin is delivered to peripheral tissues as well as to the liver of the subject. Such compositions and methods can be used to manage blood glucose levels in diabetic patients, as well as patients with metabolic derangements, such as but not limited to metabolic syndrome with elevated insulin levels, steatosis, and / or steatohepatitis. Further, such compositions and methods should allow for modulation of hepatic and peripheral lipid metabolism in the subject. The present disclosure meets this need.

[0009] BRIEF SUMMARY OF THE INVENTIONAtorney Docket No. 047589-5026WO1 (00244)

[0010] The invention provides in one aspect a pharmaceutical composition comprising an insulin and a lipid-based nanoparticle. In certain embodiments, the lipid-based nanoparticle is a Hepatic Directed Vesicle (HDV). In certain embodiments, at least a fraction of the insulin is dispersed within the HDV. In certain embodiments, the percentage of HDV-dispersed insulin as compared to the total insulin in the pharmaceutical composition ranges from about 0.1% to about 100%. In certain embodiments, the lipid nanoparticle is enclosed by a bipolar lipid membrane comprising cholesterol, dicetyl phosphate, an amphipathic lipid, and a hepatocyte receptor binding molecule. In certain embodiments, the amphipathic lipid comprises at least one selected from the group consisting of l,2-distearoyl-sn-glycero-3-phosphocholine, l,2-dipalmitoyl-sn-glycerol-[3-phospho-rac-(l-glycerol)], 1,2-distearoyl-sn-glycero-3 -phosphoethanolamine, l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-( succinyl), 1 ,2-dimyri stoyl-sn-gly cero-3 -phosphate, 1 ,2-dimyri stoyl-sn-gly cero-3 -phosphocholine, l,2-distearoyl-sn-glycero-3-phosphate, l,2-dipalmitoyl-sn-glycero-3-phosphate, and l,2-dipalmitoyl-sn-glycero-3-phosphocholine. In certain embodiments, the at least one hepatocyte receptor binding molecule extends outward from the nanoparticle. In certain embodiments, the size of the nanoparticle ranges from about 10 nm to about 200 nm.

[0011] The invention provides in one aspect a method of treating or ameliorating diabetes in a subject suffering therefrom.

[0012] The invention provides in one aspect a method of modulating peripheral lipid metabolism and hepatic lipid metabolism in a subject suffering from diabetes.

[0013] The invention provides in one aspect a method of preventing, ameliorating, minimizing, and / or reversing hepatic accumulation of lipids (fatty liver) in a subject suffering from diabetes.

[0014] In certain embodiments, the method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition of the disclosure.

[0015] BRIEF DESCRIPTION OF THE DRAWINGS

[0016] For the purposes of illustrating the disclosure, depicted in the drawings are certain embodiments of the disclosure. However, the disclosure is not limited to the precise arrangements and instrumentalities of the embodiments depicted in the drawings.

[0017] FIG. 1 illustrates a non-limiting study design as contemplated in certain embodiments herein.

[0018] FIG. 2 illustrates a non-limiting schedule of activities as contemplated in certain embodiments herein.Atorney Docket No. 047589-5026WO1 (00244)

[0019] FIG. 3 illustrates non-limiting objectives and endpoints as contemplated in certain embodiments herein.

[0020] FIG. 4 illustrates a non-limiting insulin infusion algorithm as contemplated in certain embodiments herein.

[0021] FIG. 5 illustrates a non-limiting insulin infusion algorithm as contemplated in certain embodiments herein.

[0022] DETAILED DESCRIPTION

[0023] Insulin replacement therapy in diabetes mellitus is intrinsically non-physiologic due to the subcutaneous route of delivery for treatment, as opposed to the intraportal delivery of insulin released from the functioning pancreatic islet. Thus, insulin replacement therapy leads to peripheral hyperinsulinemia and importantly under-insulinization of the liver.

[0024] Subcutaneously delivered hepatic-directed vesicle insulin (HDV-Insulin or HDV-I) has been developed to correct this latter shortcoming of standard insulin treatment.

[0025] In certain embodiments, hepatic directed vesicle-insulin (HDV-Insulin) is a nano-sized (<100 nanometers in diameter) insulin delivery system that uses any insulin or analogue and / or derivative thereof, such as but not limited to a commercially approved fast-acting insulin analog (Humalog®). HDV-Insulin further contains a hepatocyte targeting ligand, such as but not limited to biotin-phosphatidylethanolamine (Biotin-PE), embedded in the lipid bilayer of the carrier; the ligand selectively targets the delivery of the attached insulin to the hepatocytes in the liver.

[0026] When administered to animals subcutaneously following a meal, HDV-I delivers the non HDV associated insulin directly to peripheral tissues (primarily adipose and muscle) as with conventional insulin formulations, but the HDV-associated insulin is delivered to liver hepatocytes. This results in the activation of mealtime hepatic glycogen storage and a more physiologically normal control of glucose distribution than that achieved with conventional insulin therapy.

[0027] Previous studies of HDV-I using HDV-insulin lispro (HDV-L) have demonstrated an interaction between baseline A1C and subsequent risk of hypoglycemia: high A1C subjects (8.5% and above) had the same A1C reduction as control subject but with lower insulin doses and lower hypoglycemia risk, whereas better controlled subjects (A1C <8.5%) had similar A1C outcomes as control subjects using the same insulin doses but with greater hypoglycemia risk.Atorney Docket No. 047589-5026WO1 (00244)

[0028] An additional study (OPTI-1) was performed in well controlled Type 1 diabetes (T1D), with the goal of identifying optimum treatment algorithms for HDV-L when used in conjunction with basal insulin degludec. In OPTI-1, basal insulin dosage was decreased by either 10% or 40% at the time of changing bolus insulin from LIS to HDV-L. The results demonstrated that these acute reductions in basal dosage were safe and well tolerated. In addition, the basal dosage reduction allowed for an increase in bolus / basal ratio and total insulin dose which was accompanied by a progressive decrease in hypoglycemia risk, without a meaningful change in mean daily glucose or A1C following stabilization of insulin dosing.

[0029] In certain embodiments, the observed reductions in hypoglycemia risk observed in ISLE-1 and OPTI-1 result from the hepato-preferential effects of HDV-L as compared to LIS alone. By providing adequate insulin to hepatocytes, the liver is able to take up and store mealtime carbohydrate intake as glycogen, which is then more readily available for the prevention of hypoglycemia that may occur in the post-absorptive state.

[0030] In this present study, which is a HDV Insulin Phase 2 clinical study, the HDV-Insulin is administered via intravenous infusion. As shown herein, in certain embodiments, the present study supports this hypothesis by demonstrating a hepato-preferential metabolic effect (i.e. suppression of endogenous glucose production) of HDV-L as compared to LIS alone during a euglycemic clamp procedure in persons with T1D.

[0031] Compositions

[0032] The present disclosure provides in one aspect a pharmaceutical composition comprising an insulin and a lipid-based nanoparticle, wherein the lipid-based nanoparticle is a Hepatic Directed Vesicle (HDV).

[0033] In certain embodiments, the lipid-based nanoparticle of the disclosure and compositions comprising the same help deliver the insulin dispersed therewithin to the hepatocytes in the liver.

[0034] In certain embodiments, the size of the lipid-based nanoparticle ranges from about 10 nm to about 200 nm. In other embodiments, the size of the lipid-based nanoparticle is about 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, or 200 nm. In other embodiments, the size of the lipid-based nanoparticle is equal to or greater than about 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, or 200 nm. In other embodiments, the size of the lipid-based nanoparticle is equal to or lower than about 10 nm, 20 nm, 30 nm,Atorney Docket No. 047589-5026WO1 (00244)

[0035] 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, or 200 nm.

[0036] In certain embodiments, at least a fraction of the insulin is dispersed within the HDV. In certain embodiments, the percentage of HDV-dispersed insulin as compared to the total insulin in the pharmaceutical composition ranges from about 0.1% to about 100%. In certain embodiments, the nanoparticle comprises, and / or is defined by, a bipolar lipid membrane. In certain embodiments, the lipid nanoparticle is enclosed by a bipolar lipid membrane comprising cholesterol, dicetyl phosphate, an amphipathic lipid, and a hepatocyte receptor binding molecule. In certain embodiments, the membrane comprises cholesterol. In other embodiments, the membrane comprises dicetyl phosphate. In yet other embodiments, the membrane comprises an amphipathic lipid. In yet other embodiments, the membrane comprises l,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC). In yet other embodiments, the membrane comprises cholesterol, dicetyl phosphate, and DSPC. In yet other embodiments, the membrane comprises a hepatocyte receptor binding molecule.

[0037] In certain embodiments, the amphipathic lipid comprises at least one selected from the group consisting of l,2-distearoyl-sn-glycero-3 -phosphocholine, 1,2-dipalmitoyl-sn-glycerol-[3-phospho-rac-(l-glycerol)], l,2-distearoyl-sn-glycero-3-phosphoethanolamine, l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl), 1,2-dimyristoyl-sn-glycero-3 -phosphate, l,2-dimyristoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3 -phosphate, l,2-dipalmitoyl-sn-glycero-3 -phosphate, and 1,2-dipalmitoyl-sn-glycero-3 -phosphocholine. In other embodiments, the amphipathic lipid comprises at least one selected from the group consisting of l,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, l,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(l-glycerol)], l,2-distearoyl-sn-glycero-3-phosphoethanolamine, and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl).

[0038] In certain embodiments, the at least one hepatocyte receptor binding molecule extends outward from the nanoparticle.

[0039] In certain embodiments, the percentage of HDV-dispersed insulin as compared to the total insulin in the pharmaceutical composition is about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 20.5%, 21%, 21.5%, 22%, 22.5%, 23%, 23.5%, 24%, 24.5%, 25%, 25.5%, 26%, 26.5%, 27%, 27.5%, 28%, 28.5%, 29%, 29.5%, 30%, 30.5%, 31%, 31.5%, 32%, 32.5%, 33%, 33.5%,Atorney Docket No. 047589-5026WO1 (00244)

[0040] 34%, 34.5%, 35%, 35.5%, 36%, 36.5%, 37%, 37.5%, 38%, 38.5%, 39%, 39.5%, 40%, 40.5%, 41%, 41.5%, 42%, 42.5%, 43%, 43.5%, 44%, 44.5%, 45%, 45.5%, 46%, 46.5%, 47%, 47.5%, 48%, 48.5%, 49%, 49.5%, 50%, 50.5%, 51%, 51.5%, 52%, 52.5%, 53%, 53.5%, 54%, 54.5%, 55%, 55.5%, 56%, 56.5%, 57%, 57.5%, 58%, 58.5%, 59%, 59.5%, 60%, 60.5%, 61%, 61.5%, 62%, 62.5%, 63%, 63.5%, 64%, 64.5%, 65%, 65.5%, 66%, 66.5%, 67%, 67.5%, 68%, 68.5%, 69%, 69.5%, 70%, 70.5%, 71%, 71.5%, 72%, 72.5%, 73%, 73.5%, 74%, 74.5%, 75%, 75.5%, 76%, 76.5%, 77%, 77.5%, 78%, 78.5%, 79%, 79.5%, 80%, 80.5%, 81%, 81.5%, 82%, 82.5%, 83%, 83.5%, 84%, 84.5%, 85%, 85.5%, 86%, 86.5%, 87%, 87.5%, 88%, 88.5%, 89%, 89.5%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100%.

[0041] In certain embodiments, the percentage of HDV-dispersed insulin as compared to the total insulin in the pharmaceutical composition is equal to or greater than about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 20.5%, 21%, 21.5%, 22%, 22.5%, 23%, 23.5%, 24%, 24.5%, 25%, 25.5%, 26%, 26.5%, 27%, 27.5%, 28%, 28.5%, 29%, 29.5%, 30%, 30.5%, 31%, 31.5%, 32%, 32.5%, 33%, 33.5%, 34%, 34.5%, 35%, 35.5%, 36%, 36.5%, 37%, 37.5%, 38%, 38.5%, 39%, 39.5%, 40%, 40.5%, 41%, 41.5%, 42%, 42.5%, 43%, 43.5%, 44%, 44.5%, 45%, 45.5%, 46%, 46.5%, 47%, 47.5%, 48%, 48.5%, 49%, 49.5%, 50%, 50.5%, 51%, 51.5%, 52%, 52.5%, 53%, 53.5%, 54%, 54.5%, 55%, 55.5%, 56%, 56.5%, 57%, 57.5%, 58%, 58.5%, 59%, 59.5%, 60%, 60.5%, 61%, 61.5%, 62%, 62.5%, 63%, 63.5%, 64%, 64.5%, 65%, 65.5%, 66%, 66.5%, 67%, 67.5%, 68%, 68.5%, 69%, 69.5%, 70%, 70.5%, 71%, 71.5%, 72%, 72.5%, 73%, 73.5%, 74%, 74.5%, 75%, 75.5%, 76%, 76.5%, 77%, 77.5%, 78%, 78.5%, 79%, 79.5%, 80%, 80.5%, 81%, 81.5%, 82%, 82.5%, 83%, 83.5%, 84%, 84.5%, 85%, 85.5%, 86%, 86.5%, 87%, 87.5%, 88%, 88.5%, 89%, 89.5%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100%.

[0042] In certain embodiments, the percentage of HDV-dispersed insulin as compared to the total insulin in the pharmaceutical composition is equal to or lower than about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%,Atorney Docket No. 047589-5026WO1 (00244)

[0043] 19.5%, 20%, 20.5%, 21%, 21.5%, 22%, 22.5%, 23%, 23.5%, 24%, 24.5%, 25%, 25.5%, 26%, 26.5%, 27%, 27.5%, 28%, 28.5%, 29%, 29.5%, 30%, 30.5%, 31%, 31.5%, 32%, 32.5%, 33%, 33.5%, 34%, 34.5%, 35%, 35.5%, 36%, 36.5%, 37%, 37.5%, 38%, 38.5%, 39%, 39.5%, 40%, 40.5%, 41%, 41.5%, 42%, 42.5%, 43%, 43.5%, 44%, 44.5%, 45%, 45.5%, 46%, 46.5%, 47%, 47.5%, 48%, 48.5%, 49%, 49.5%, 50%, 50.5%, 51%, 51.5%, 52%, 52.5%, 53%, 53.5%, 54%, 54.5%, 55%, 55.5%, 56%, 56.5%, 57%, 57.5%, 58%, 58.5%, 59%, 59.5%, 60%, 60.5%, 61%, 61.5%, 62%, 62.5%, 63%, 63.5%, 64%, 64.5%, 65%, 65.5%, 66%, 66.5%, 67%, 67.5%, 68%, 68.5%, 69%, 69.5%, 70%, 70.5%, 71%, 71.5%, 72%, 72.5%, 73%, 73.5%, 74%, 74.5%, 75%, 75.5%, 76%, 76.5%, 77%, 77.5%, 78%, 78.5%, 79%, 79.5%, 80%, 80.5%, 81%, 81.5%, 82%, 82.5%, 83%, 83.5%, 84%, 84.5%, 85%, 85.5%, 86%, 86.5%, 87%, 87.5%, 88%, 88.5%, 89%, 89.5%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100%.

[0044] In certain embodiments, the membrane comprises at least one compound selected from the group consisting of a stabilizer and a C12-C24 acyl lysophosphatidylcholine.

[0045] In certain embodiments, the stabilizer is selected from the group consisting of m-cresol, benzyl alcohol, methyl 4-hydroxybenzoate, thiomersal, and butylated hydroxytoluene (2,6-di- / c / 7-butyl-4-methylphenol).

[0046] In certain embodiments, the stabilizer ranges from about 0.5% to about 25 % (w / w) in the membrane. In certain embodiments, the stabilizer ranges from about 10% to about 25 % (w / w) in the membrane. In other embodiments, the stabilizer is present in the membrane at a concentration of about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% (w / w). In other embodiments, the stabilizer is present in the membrane at a concentration equal to or greater than about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% (w / w). In other embodiments, the stabilizer is present in the membrane at a concentration equal to or lower than about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% (w / w).

[0047] In certain embodiments, the m-cresol ranges from about 0.5% to about 25% (w / w) in the membrane. In certain embodiments, the m-cresol ranges from about 10% to about 25% (w / w) in the membrane. In other embodiments, the m-cresol is present in the membrane at a concentration of about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,Atorney Docket No. 047589-5026WO1 (00244)

[0048] 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% (w / w). In other embodiments, the m-cresol is present in the membrane at a concentration equal to or greater than about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% (w / w). In other embodiments, the m-cresol is present in the membrane at a concentration equal to or lower than about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% (w / w).

[0049] In certain embodiments, the membrane further comprises a C12-C24 acyl lysophosphatidylcholine. In other embodiments, the membrane further comprises stearoyl lysophosphatidylcholine.

[0050] In certain embodiments, the C12-C24 lysophosphatidylcholine ranges from about 0.5% to about 30% (w / w) in the membrane. In other embodiments, the C12-C24 lysophosphatidylcholine ranges from about 1% to about 30% (w / w) in the membrane. In yet other embodiments, the C12-C24 lysophosphatidylcholine is present in the membrane at a concentration of about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30% (w / w). In yet other embodiments, the C12-C24 lysophosphatidylcholine is present in the membrane at a concentration equal to or greater than about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30% (w / w). In yet other embodiments, the C12-C24 lysophosphatidylcholine is present in the membrane at a concentration equal to or lower than about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30% (w / w).

[0051] In certain embodiments, the stearoyl lysophosphatidylcholine ranges from about 0.5% to about 30% (w / w) in the membrane. In other embodiments, the stearoyl lysophosphatidylcholine ranges from about 1% to about 30% (w / w) in the membrane. In other embodiments, the stearoyl lysophosphatidylcholine ranges from about 0.5% to about 30% (w / w) in the membrane. In yet other embodiments, the stearoyl lysophosphatidylcholine is present in the membrane at a concentration of about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30% (w / w). In yet otherAtorney Docket No. 047589-5026WO1 (00244)

[0052] embodiments, the stearoyl lysophosphatidylcholine is present in the membrane at a concentration equal to or greater than about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30% (w / w). In yet other embodiments, the stearoyl lysophosphatidylcholine is present in the membrane at a concentration equal to or lower than about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30% (w / w).

[0053] In certain embodiments, the amount of the C12-C24 lysophosphatidylcholine in the membrane is about l%-30% (w / w) of the amount of the at least one amphipathic lipid in the membrane. In yet other embodiments, the amount of the C12-C24 lysophosphatidylcholine in the membrane is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30% (w / w) of the amount of the at least one amphipathic lipid in the membrane. In yet other embodiments, the amount of the C12-C24 lysophosphatidylcholine in the membrane is equal to or greater than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30% (w / w) of the amount of the at least one amphipathic lipid in the membrane. In yet other embodiments, the amount of the C12-C24 lysophosphatidylcholine in the membrane is equal to or lower than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30% (w / w) of the amount of the at least one amphipathic lipid in the membrane.

[0054] In certain embodiments, the amount of the C12-C24 lysophosphatidylcholine in the membrane is about 1% to about 30% (w / w) of the amount of DSPC in the membrane. In yet other embodiments, the amount of the C12-C24 lysophosphatidylcholine in the membrane is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% (w / w) or 30% (w / w) of the amount of DSPC in the membrane. In yet other embodiments, the amount of the C12-C24 lysophosphatidylcholine in the membrane is equal to or greater than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% (w / w) or 30% (w / w) of the amount of DSPC in the membrane. In yet other embodiments, the amount of the C12-C24 lysophosphatidylcholine in the membrane is equal to or lower than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,Atorney Docket No. 047589-5026WO1 (00244)

[0055] 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% (w / w) or 30% (w / w) of the amount of DSPC in the membrane.

[0056] In certain embodiments, the amount of the C12-C24 lysophosphatidylcholine in the membrane is about 1 mole % to about 50 mole % of the amount of the at least one amphipathic lipid in the membrane. In yet other embodiments, the amount of the C12-C24 lysophosphatidylcholine in the membrane is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mole % of the amount of the at least one amphipathic lipid in the membrane. In yet other embodiments, the amount of the C12-C24 lysophosphatidylcholine in the membrane is equal to or greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mole % of the amount of the at least one amphipathic lipid in the membrane. In yet other embodiments, the amount of the C12-C24 lysophosphatidylcholine in the membrane is equal to or lower than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mole % of the amount of the at least one amphipathic lipid in the membrane.

[0057] In certain embodiments, the amount of the C12-C24 lysophosphatidylcholine in the membrane is about 1 mole % to about 50 mole % of the amount of DSPC in the membrane. In yet other embodiments, the amount of the C12-C24 lysophosphatidylcholine in the membrane is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mole % of the amount of DSPC in the membrane. In yet other embodiments, the amount of the C12-C24 lysophosphatidylcholine in the membrane is equal to or greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mole % of the amount of DSPC in the membrane. In yet other embodiments, the amount of the C12-C24 lysophosphatidylcholine in the membrane is equal to or lower than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mole % of the amount of DSPC in the membrane.

[0058] In certain embodiments, the amount of the stearoyl lysophosphatidylcholine in the membrane is about 1% to about 30% (w / w) of the amount of DSPC in the membrane. In yet other embodiments, the amount of the stearoyl lysophosphatidylcholine in the membrane isAtorney Docket No. 047589-5026WO1 (00244)

[0059] about 1%, 6%, 7%, 8%, 9%, 10%„ 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30% (w / w) of the amount of DSPC in the membrane. In yet other embodiments, the amount of the stearoyl lysophosphatidylcholine in the membrane is equal to or greater than about 1%, 6%, 7%, 8%, 9%, 10%„ 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30% (w / w) of the amount of DSPC in the membrane. In yet other embodiments, the amount of the stearoyl lysophosphatidylcholine in the membrane is equal to or lower than about 1%, 6%, 7%, 8%, 9%, 10%„ 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30% (w / w) of the amount of DSPC in the membrane.

[0060] In certain embodiments, the amount of the stearoyl lysophosphatidylcholine in the membrane is about 1 mole % to about 50 mole % of the amount of DSPC in the membrane. In yet other embodiments, the amount of the stearoyl lysophosphatidylcholine in the membrane is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mole % of the amount of DSPC in the membrane. In yet other embodiments, the amount of the stearoyl lysophosphatidylcholine in the membrane is equal to or greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mole % of the amount of DSPC in the membrane. In yet other embodiments, the amount of the stearoyl lysophosphatidylcholine in the membrane is equal to or lower than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mole % of the amount of DSPC in the membrane.

[0061] In certain embodiments, the insulin is covalently bound to the lipid-based nanoparticle.

[0062] In certain embodiments, the insulin is not covalently bound to the lipid-based nanoparticle.

[0063] In certain embodiments, the insulin is suspended in an aqueous solution comprising a free dissolved insulin that is not dispersed within the lipid-based nanoparticle.

[0064] In certain embodiments, the HDV-dispersed insulin and the free dissolved insulin are independently selected from the group consisting of insulin lispro, insulin aspart (including FIASP®, Novo Nordisk), regular insulin, insulin glargine, insulin zinc, extended human insulin zinc suspension, isophane insulin, human buffered regular insulin, insulin glulisine,Atorney Docket No. 047589-5026WO1 (00244)

[0065] recombinant human regular insulin, recombinant human insulin isophane, insulin detemir, biphasic human insulin, and insulin degludec (including TRESIBA®, Novo Nordisk), and any combinations thereof.

[0066] In certain embodiments, the amphipathic lipid comprises at least one selected from the group consisting of l,2-distearoyl-sn-glycero-3 -phosphocholine, 1,2-dipalmitoyl-sn-glycero-3 -phosphocholine, l,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(l-glycerol)], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl).

[0067] In certain embodiments, the hepatocyte receptor binding molecule comprises biotin. In certain embodiments, the biotin-containing hepatocyte receptor binding molecule comprises at least one selected from the group consisting of N-hydroxy succinimide (NHS) biotin; sulfo-NHS-biotin; N-hydroxysuccinimide long chain biotin; sulfo-N-hydroxysuccinimide long chain biotin; D-biotin; biocytin; sulfo-N-hydroxysuccinimide-S-S-biotin; biotin-BMCC; biotin-HPDP; iodoacetyl-LC-biotin; biotin-hydrazide; biotin-LC-hydrazide; biocytin hydrazide; biotin cadaverine; carboxybiotin; photobiotin; p-aminobenzoyl biocytin trifluoroacetate; p-diazobenzoyl biocytin; biotin DHPE (2,3-di acetoxy propyl 2-(5-((3aS,6aR)-2-oxohexahydro-lH-thieno[3,4-d]imidazol-4-yl)pentanamido)ethyl phosphate); biotin-X-DHPE (2,3 -diacetoxypropyl 2-(6-(5-((3aS,6aR)-2-oxohexahydro-lH-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido) ethyl phosphate); 12-((biotinyl)amino)dodecanoic acid; 12-((biotinyl)amino)dodecanoic acid succinimidyl ester; S-biotinyl homocysteine; biocytin-X; biocytin x-hydrazide; biotinethylenediamine; biotin-XL; biotin-X-ethylenediamine; biotin-XX hydrazide; biotin-XX-SE; biotin-XX, SSE; biotin-X-cadaverine; a-(t-BOC)biocytin; N-(biotinyl)-N’ -(iodoacetyl) ethylenediamine; DNP-X-biocytin-X-SE; biotin-X-hydrazide; norbiotinamine hydrochloride; 3-(N-maleimidylpropionyl)biocytin; ARP; biotin-l-sulfoxide; biotin methyl ester; biotin-maleimide; biotin-poly(ethyleneglycol) amine; (+) biotin 4-amidobenzoic acid sodium salt; Biotin 2-N-acetylamino-2-deoxy-P-D-glucopyranoside; Biotin-a-D-N-acetylneuraminide; Biotin-a-L-fucoside; Biotin lacto-N-bioside; Biotin-Lewis-A trisaccharide; Biotin-Lewis-Y tetrasaccharide; Biotin-a-D-mannopyranoside; and biotin 6-O-phospho-a-D-mannopyranoside.

[0068] In certain embodiments, the biotin-containing hepatocyte receptor binding molecule is selected from the group consisting of 2,3-diacetoxypropyl 2-(5-((3aS,6aR)-2-oxohexahydro-lH-thieno[3,4-d] imidazol-4-yl)pentanamido)ethyl phosphate (biotin DHPE) and biotin-X-Atorney Docket No. 047589-5026WO1 (00244)

[0069] DHPE (2,3 -diacetoxy propyl 2-(6-(5-((3aS,6aR)-2-oxohexahydro-lH-thieno[3,4-d]imidazol-4-yl)pentanamido) hexanamido)ethyl phosphate).

[0070] In certain embodiments, the composition further comprises cellulose acetate phthalate, which is at least partially bound to the insulin dispersed within the lipid-based nanoparticle.

[0071] In certain embodiments, the composition further comprises at least one charged organic molecule bound to the insulin dispersed within the lipid-based nanoparticle, wherein the charged organic molecule is at least one selected from the group consisting of protamines, polylysine, poly (arg-pro-thr)nin a mole ratio of 1 : 1 : 1, poly (DL-Ala-poly-L-lys)n in a mole ratio of 6:1, histones, sugar polymers comprising a primary amino group, polynucleotides with primary amino groups, proteins comprising amino acid residues with carboxyl (COO ) or sulfhydral (S") functional groups, and acidic polymers.

[0072] In certain embodiments, the cholesterol ranges from about 5% to about 25% (w / w) in the membrane. In other embodiments, the cholesterol is present in the membrane at a concentration of about 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 20.5%, 21%, 21.5%, 22%, 22.5%, 23%, 23.5%, 24%, 24.5%, or 25% (w / w). In other embodiments, the cholesterol is present in the membrane at a concentration equal to or greater than about 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 20.5%, 21%, 21.5%, 22%, 22.5%, 23%, 23.5%, 24%, 24.5%, or 25% (w / w). In other embodiments, the cholesterol is present in the membrane at a concentration equal to or lower than about 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 20.5%, 21%, 21.5%, 22%, 22.5%, 23%, 23.5%, 24%, 24.5%, or 25% (w / w).

[0073] In certain embodiments, the dicetyl phosphate ranges from about 2% to about 25% (w / w) in the membrane. In certain embodiments, the dicetyl phosphate ranges from about 10% to about 25% (w / w) in the membrane. In other embodiments, the dicetyl phosphate is present in the membrane at a concentration of about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 20.5%, 21%, 21.5%, 22%, 22.5%, 23%, 23.5%, 24%, 24.5%, or 25% (w / w). In other embodiments, the dicetyl phosphate is present in the membrane at a concentration equal to or greater than about 2%, 3%, 4%, 5%,Atorney Docket No. 047589-5026WO1 (00244)

[0074] 6%, 7%, 8%, 9%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 20.5%, 21%, 21.5%, 22%, 22.5%, 23%, 23.5%, 24%, 24.5%, or 25% (w / w). In other embodiments, the dicetyl phosphate is present in the membrane at a concentration equal to or lower than about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 20.5%, 21%, 21.5%, 22%, 22.5%, 23%, 23.5%, 24%, 24.5%, or 25% (w / w).

[0075] In certain embodiments, the DSPC ranges from about 40% to about 75% (w / w) in the membrane. In other embodiments, the DSPC is present in the membrane at a concentration of about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, or 75% (w / w). In other embodiments, the DSPC is present in the membrane at a concentration equal to or greater than about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, or 75% (w / w). In other embodiments, the DSPC is present in the membrane at a concentration equal to or lower than about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, or 75% (w / w).

[0076] In certain embodiments, the hepatocyte receptor binding molecule ranges from about 0.5% to about 10% (w / w) in the membrane. In other embodiments, the hepatocyte receptor binding molecule is present in the membrane at a concentration of about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.5 %, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% (w / w). In other embodiments, the hepatocyte receptor binding molecule is present in the membrane at a concentration equal to or greater than about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.5 %, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% (w / w). In other embodiments, the hepatocyte receptor binding molecule is present in the membrane at a concentration equal to or lower than about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%,Atorney Docket No. 047589-5026WO1 (00244)

[0077] 3.8%, 3.9%, 4.0%, 4.5 %, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% (w / w).

[0078] In certain embodiments, the amount of the stearoyl lysophosphatidylcholine in the membrane is about 5%-30% (w / w) of the amount of DSPC in the membrane.

[0079] In certain embodiments, the membrane comprises cholesterol, dicetyl phosphate, DSPC, stearoyl lysophosphatidylcholine, m-cresol, and at least one selected from the group consisting of biotin DHPE and biotin-X-DHPE. In other embodiments, the membrane comprises cholesterol, dicetyl phosphate, DSPC, stearoyl lysophosphatidylcholine, m-cresol, and biotin DHPE.

[0080] In certain embodiments, the membrane comprises cholesterol, dicetyl phosphate, DSPC, m-cresol, and at least one selected from the group consisting of biotin DHPE and biotin-X-DHPE. In other embodiments, the membrane comprises cholesterol, dicetyl phosphate, DSPC, m-cresol, and biotin DHPE.

[0081] In certain embodiments, the membrane comprises cholesterol, dicetyl phosphate, DSPC, stearoyl lysophosphatidylcholine, and at least one selected from the group consisting of biotin DHPE and biotin-X-DHPE. In other embodiments, the membrane comprises cholesterol, dicetyl phosphate, DSPC, stearoyl lysophosphatidylcholine, and biotin DHPE.

[0082] In certain embodiments, the membrane comprises cholesterol, dicetyl phosphate, DSPC, stearoyl lysophosphatidylcholine, m-cresol, and biotin DHPE in a % (w / w) ratio selected from the group consisting of: (a) about 9.4 : 18.1 : 56.8 : 14.1 : 0.0 : 1.5; (b) about 7.7 : 15.0 : 58.6 : 0.0 : 17.4 : 1.3; and (c) about 8.4 : 16.2 : 47.5 : 7.6 : 19.0 : 1.3.

[0083] In certain embodiments, the lipid-based nanoparticles useful within the disclosure are described in U.S. Patent Application Nos. US20110135725 and US20090087479 and PCT Patent Application Publication No. WO 2018 / 169954, all of which are incorporated herein in their entireties by reference. In certain embodiments, the reduced or minimal aggregation properties of the nanoparticle of the disclosure improves its stability and pharmaceutical developability as compared to nanoparticles of the prior art.

[0084] In certain embodiments, the lipid-based nanoparticle of the disclosure is defined and / or enclosed by a bipolar lipid membrane. In other embodiments, the nanoparticle of the disclosure comprises a hepatocyte-targeting compound, which helps deliver the therapeutic agent (such as, but not limited to, insulin) associated with, and / or dispersed within, the nanoparticle to a hepatocyte. In yet other embodiments, the nanoparticle of the disclosure is part of a composition further comprising a “free” therapeutic agent, which is not associated with, and / or dispersed within, the nanoparticle. The nanoparticle, and any compositionsAtorney Docket No. 047589-5026WO1 (00244)

[0085] comprising the same, can be administered by any compatible and / or feasible routes, such as but not limited to by injection (such as, for example, subcutaneously and / or transdermally), inhalationally, buccally and / or orally, so as to treat a subject that benefits from administration of the therapeutic agent associated with, and / or dispersed within, the nanoparticle, and / or of the “free” therapeutic agent, which is not associated with, and / or dispersed within, the nanoparticle.

[0086] Liposomes usually comprise amphipathic phospholipid materials that form bilayer membranes that define and / or enclose the liposomes. They can have a single membrane (unilamellar), or multiple bilayers with a microscopic onion-like appearance. Liposomes can be rather large, measuring several microns in diameter. Liposomes generally have a spherical (or nearly spherical) shape, wherein the intact surface has no available “open” edges and thus cannot interact with other available “open” edge liposome(s) to undergo particle aggregation.

[0087] In contrast, phospholipid nanoparticles with diameters equal to or lower than about 200 nm have a restricted ability to bend into a spherical configuration, which should in principle be their thermodynamically stable structure. As a result, these low-diameter nanoparticles do not form a perfectly spherical particle, but rather a nearly planar sheet.

[0088] Without wishing to be limited by any theory, those nearly planar sheets can be described as “nanodiscs” or “nanodisks” or “nanoFrisbees” or “bicelles.” Such nanoparticles have “open” edges in their membranes, and these “edges” promote nanoparticle aggregation. As a result, in many instances the nanoparticles are generated as discrete particles, which then proceed to aggregate into larger, easily visible (wispy or feather-like) floating particles. This phenomenon may hamper the developability of the low-diameter nanoparticles as drug delivery agents. In certain embodiments, unlike in the case of liposomes, the API is not carried in the core volume of (or within) the bicelles. In other embodiments, the API is attached and / or bound to the membrane surface of the bicelles, either through a purely physical interaction or a covalent linkage. In one aspect, the present disclosure addresses this issue, providing compositions and methods that allow for closing the “open” edges of the nearly planar sheets (nanodiscs and / or nanoFrisbees) and thus minimizing or suppressing their tendency to self-aggregate.

[0089] As described herein, in certain embodiments, the lipid-based nanoparticles of the disclosure are useful as pharmaceutical carriers, and do not form the wispy, feathery-like structures described elsewhere herein. In certain embodiments, the nanoparticles of the disclosure comprise certain amphipathic lipids and / or certain organic molecules that enableAtorney Docket No. 047589-5026WO1 (00244)

[0090] the “open” edges of the planar nanoparticle membranes to be changed in a way that prevents aggregation of the nanoparticles.

[0091] In certain embodiments, appropriate closing of the “open” edges of the lipid-based nanoparticle is promoted by replacing a portion of distearoyl phosphatidylcholine [also known as (S)-2,3-bis(stearoyloxy)propyl (2-(trimethylammonio)ethyl) phosphate orDSPC, which comprises two Cis acyl groups covalently linked to a glycerol backbone] with a C12-C24 acyl lysophosphatidylcholine [also known as C12-C24 acyl lysolecithin, or 1-(C12-C24 acyl)-.s / 7-glycero-3-phosphocholine, or (S)-2-hydroxy-3-(Ci2-C24 acyl oxy )propyl (2-(trimethylammonio)ethyl) phosphate, which comprises a single C12-C24 acyl group covalently linked to a glycerol backbone]:

[0092]

[0093] distearoyl phosphatidylcholine (DSPC)

[0094]

[0095] C12-C24 acyl lysophosphatidylcholine In certain embodiments, appropriate closing of the “open” edges of the lipid-based nanoparticle is promoted by replacing a portion of distearoyl phosphatidylcholine [also known as (S)-2,3-bis(stearoyloxy)propyl (2-(trimethylammonio)ethyl) phosphate orDSPC, which comprises two Cis acyl groups covalently linked to a glycerol backbone] with stearoyl lysophosphatidylcholine [also known as l-steroyl-sw-glycero-3 -phosphocholine, or (S)-2-hydroxy-3-(stearoyloxy)propyl (2-(trimethylammonio)ethyl) phosphate, which comprises a single Cis acyl group covalently linked to a glycerol backbone]:

[0096]

[0097] distearoyl phosphatidylcholine (DSPC)

[0098]

[0099] stearoyl lysophosphatidylcholine (SLPC)Atorney Docket No. 047589-5026WO1 (00244)

[0100] In certain embodiments, when incorporated into the membrane, a C12-C24 acyl lysophosphatidylcholine (such as but not limited to stearoyl lysophosphatidylcholine) prevents and / or minimizes the aggregation that occurs when that compound is omitted from the membrane. In other embodiments, the C12-C24 acyl lysophosphatidylcholine (such as but not limited to stearoyl lysophosphatidylcholine), with its single aliphatic chain, enables closure of any existing membrane “edge” in the nanoparticle.

[0101] In certain embodiments, when incorporated into the membrane, any of certain small molecule stabilizers or any salts and / or solvates thereof, such as but not limited to m-cresol, benzyl alcohol, methyl 4-hydroxybenzoate, thiomersal, and butylated hydroxytoluene (also known as 2,6-di-tert-butyl-4-methylphenol), prevents and / or minimizes the aggregation that occurs when that compound is omitted from the membrane. In other embodiments, the small molecule stabilizers or any salts and / or solvates thereof enable closure of any existing membrane “edges” in the nanoparticle.

[0102] In certain embodiments, when incorporated into the membrane, any combinations of any of certain small molecule stabilizers or any salts and / or solvates thereof, and the C12-C24 acyl lysophosphatidylcholine, prevents and / or minimizes the aggregation that occurs when that compound is omitted from the membrane.

[0103] The disclosure should not be construed to be limited to the constructs described and / or exemplified herein. Rather, the disclosure provides methods of stabilizing and / or preventing aggregation of liposomes and other lipid-based nanoparticles, wherein the membrane is contacted with at least one selected from the group consisting of a stabilizer and a C12-C24 acyl lysophosphatidylcholine. In certain embodiments, the contacting removes or minimizes any “free” edges in the membrane that lead to aggregation of the liposomes and other lipid-based nanoparticles.

[0104] In certain embodiments, the compositions of the disclosure comprise an effective dose of a hepatocyte targeted pharmaceutical composition that combines free therapeutic drug (such as, but not limited to, insulin) and therapeutic drug associated with the lipid-based nanoparticle of the disclosure. The combination of free therapeutic drug and therapeutic drug associated with the lipid-based nanoparticle creates a dynamic equilibrium process between the two forms of therapeutic drug that occurs in vivo to help control the movement of free therapeutic drug to the receptor sites of hormonal action. In the case of insulin as the therapeutic drug, those receptor sites are the muscle and adipose tissues of a diabetic patient. Hepatocyte targeted therapeutic drug is also delivered to the liver of a patient over a different designated time period than free therapeutic drug, thereby introducing new pharmacodynamicAtorney Docket No. 047589-5026WO1 (00244)

[0105] profiles of therapeutic drug when the therapeutic drug remains associated with the nanoparticle and / or when free therapeutic drug is released from the nanoparticle. In addition, a portion of therapeutic drug that is associated with the nanoparticle is targeted to the liver. In the case of insulin as the therapeutic drug, the new pharmacodynamic profile of the product provides not only basal insulin for peripheral tissues, but also meal-time hepatic therapeutic drug stimulation for the management of hepatic glucose storage during a meal. Free insulin is released from the site of administration and is distributed throughout the body. Insulin associated with the lipid-based nanoparticle is delivered to the liver. The rate of release of insulin associated with the nanoparticle is different than the rate of release of free insulin from the site of administration. These different release rates of insulin delivery, combined with the targeted delivery of insulin associated with the nanoparticle to the liver, provide for the normalization of glucose concentrations in patients with Type 1 and Type 2 diabetes mellitus, as well as patients with metabolic derangements, such as but not limited to metabolic syndrome with elevated insulin levels, steatosis, and / or steatohepatitis. In certain embodiments, the hepatocyte targeted composition comprises any therapeutically effective insulin or insulin derivative or analog, or any combination of two or more types of insulin or insulin derivative or analog.

[0106] Compounds described herein also include isotopically labeled compounds wherein one or more atoms is replaced by an atom having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes suitable for inclusion in the compounds described herein include and are not limited to2H,3H,nC,13C,14C,36C1,18F,1231,125I,13N,15N,150,17O,18O,32P, and35S. In certain embodiments, isotopically labeled compounds are useful in drug and / or substrate tissue distribution studies. In other embodiments, substitution with heavier isotopes such as deuterium affords greater metabolic stability (for example, increased in vivo half-life or reduced dosage requirements). In yet other embodiments, substitution with positron emitting isotopes, such asnC,18F,15O and13N, is useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy. Isotopically-labeled compounds are prepared by any suitable method or by processes using an appropriate isotopically-labeled reagent in place of the non-labeled reagent otherwise employed.

[0107] In certain embodiments, the compounds described herein are labeled by other means, including, but not limited to, the use of chromophores or fluorescent moieties, bioluminescent labels, or chemiluminescent labels.Atorney Docket No. 047589-5026WO1 (00244)

[0108] Compounds of the disclosure can in certain embodiments form acids or bases. In certain embodiments, the disclosure contemplates acid addition salts. In other embodiments, the disclosure contemplates base addition salts. In yet other embodiments, the disclosure contemplates pharmaceutically acceptable acid addition salts. In yet other embodiments, the disclosure contemplates pharmaceutically acceptable base addition salts. Pharmaceutically acceptable salts refer to salts of those bases or acids that are not toxic or otherwise biologically undesirable.

[0109] Suitable pharmaceutically acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid. Examples of inorganic acids include hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric (including sulfate and hydrogen sulfate), and phosphoric acids (including hydrogen phosphate and dihydrogen phosphate).

[0110] Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, malonic, saccharin, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, trifluoromethanesulfonic, 2-hydroxyethanesulfonic, p-toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, alginic, P-hydroxybutyric, salicylic, galactaric and galacturonic acid.

[0111] Suitable pharmaceutically acceptable base addition salts of compounds of the disclosure include, for example, metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, calcium, magnesium, potassium, sodium, lithium and copper, iron and zinc salts. Pharmaceutically acceptable base addition salts also include organic salts made from basic amines such as, for example, N,N’-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (A-methylglucamine) and procaine. All of these salts may be prepared from the corresponding compound by reacting, for example, the appropriate acid or base with the compound.

[0112] Disclosed is a kit comprising any composition of the disclosure and an instructional material which describes administering the composition to a tissue of a subject, such as a mammal. This kit may comprise a (preferably sterile) solvent suitable for dissolving or suspending the composition of the disclosure prior to administering the composition to the subject, such as a mammal.

[0113] MethodsAtorney Docket No. 047589-5026WO1 (00244)

[0114] The disclosure provides methods of preparing the lipid-based nanoparticle of the disclosure. In certain embodiments, the method comprises contacting in an aqueous system cholesterol, dicetyl phosphate, amphipathic lipid, and hepatocyte receptor binding molecule. In other embodiments, the method comprises contacting in an aqueous system cholesterol, dicetyl phosphate, amphipathic lipid, hepatocyte receptor binding molecule, and at least one compound selected from the group consisting of a stabilizer and stearoyl lysophosphatidylcholine. In yet other embodiments, the method comprises contacting in an aqueous system cholesterol, dicetyl phosphate, DSPC, and biotin-DHPE. In yet other embodiments, the method comprises contacting in an aqueous system cholesterol, dicetyl phosphate, DSPC, stearoyl lysophosphatidylcholine, m-cresol, and biotin-DHPE.

[0115] In certain embodiments, the nanoparticle is formed in the absence of the therapeutic agent (such as but not limited to insulin), wherein optionally the nanoparticle is at least partially concentrated, purified or isolated, and wherein the therapeutic agent is contacted with the nanoparticle, whereby at least a portion of the therapeutic agent is dispersed within the nanoparticle.

[0116] In certain embodiments, the composition is treated with cellulose acetate phthalate, which can bind non-covalently to at least a portion of the therapeutic agent (such as but not limited to insulin) dispersed within the nanoparticle and protect the therapeutic agent from metabolic degradation. In other embodiments, the cellulose acetate phthalate is covalently bound to the therapeutic agent and / or any of the lipids that constitute the nanoparticle.

[0117] In certain embodiments, the stabilizer is contacted with the membrane, and / or the lipid components that assemble to form the membrane (such as, but not limited to, cholesterol, dicetyl phosphate, DSPC, C12-C24 lysophosphatidylcholine if present, and biotin DHPE), at a (w / w) ratio of the membrane to the stabilizer ranging from about 1 : 1 to about 1 :30. In other embodiments, the stabilizer is contacted with the membrane, and / or the lipid components that assemble to form the membrane, at a (w / w) ratio of the membrane to the stabilizer of about 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1;22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29 or 1:30.

[0118] In certain embodiments, the m-cresol is contacted with the membrane, and / or the lipid components that assemble to form the membrane (such as, but not limited to, cholesterol, dicetyl phosphate, DSPC, C12-C24 lysophosphatidylcholine if present, and biotin DHPE), at a (w / w) ratio of the membrane to the stabilizer ranging from about 1 : 1 to about 1 :30. In other embodiments, the m-cresol is contacted with the membrane, and / or the lipid components thatAtorney Docket No. 047589-5026WO1 (00244)

[0119] assemble to form the membrane, at a (w / w) ratio of the membrane to the stabilizer of about 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1;22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29 or 1:30.

[0120] Further embodiments relating to certain methods for preparing and / or processing and / or purifying a nanoparticle can be found, for example, in U.S. Patent Application Nos. US20110135725 and US20090087479 and PCT Patent Application Publication No. WO 2018 / 169954, all of which are incorporated herein in their entireties by reference.

[0121] The disclosure further provides a method of treating, ameliorating, and / or preventing a disease in a mammal. In certain embodiments, the method comprises administering to the mammal in need thereof a therapeutically effective amount of a nanoparticle and / or a composition of the disclosure.

[0122] In certain embodiments, the disease is diabetes mellitus and the therapeutic agent comprises insulin.

[0123] The disclosure provides a method of treating or ameliorating diabetes in a subject suffering therefrom.

[0124] The disclosure further provides a method of modulating peripheral lipid metabolism and hepatic lipid metabolism in a subject suffering from diabetes.

[0125] The disclosure further provides a method of preventing, ameliorating, minimizing, and / or reversing hepatic accumulation of lipids (fatty liver) in a subject suffering from diabetes.

[0126] In certain embodiments, the method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition of the disclosure.

[0127] In certain embodiments, the administering allows for the subject to achieve liver and / or plasma triglyceride levels that are closer to normal physiological levels than before the subject was subjected to the administering.

[0128] In certain embodiments, the administering allows for the subject to achieve liver and / or plasma free fatty acid levels that are closer to normal physiological levels than before the subject was subjected to the administering.

[0129] In certain embodiments, the administering allows for the subject to achieve liver and / or plasma branched-chain amino acid (BCAA) levels that are closer to normal physiological levels than before the subject was subjected to the administering.Atorney Docket No. 047589-5026WO1 (00244)

[0130] In certain embodiments, the administering allows for the subject to achieve hepatic and / or peripheral glucose storage levels that are closer to normal physiological levels than before the subject was subjected to the administering.

[0131] In certain embodiments, the subject has about 6.5-8.5% A1C. In certain embodiments, the subject has greater than about 8.5% A1C.

[0132] In certain embodiments, the subject has a HbAlc level equal to or greater than about 10%. In certain embodiments, the subject has a HbAlc level equal to or greater than about 9.9%. In certain embodiments, the subject has a HbAlc level equal to or greater than about 9.8%. In certain embodiments, the subject has a HbAlc level equal to or greater than about 9.7%. In certain embodiments, the subject has a HbAlc level equal to or greater than about 9.6%. In certain embodiments, the subject has a HbAlc level equal to or greater than about 9.5%. In certain embodiments, the subject has a HbAlc level equal to or greater than about 9.4%. In certain embodiments, the subject has a HbAlc level equal to or greater than about 9.3%. In certain embodiments, the subject has a HbAlc level equal to or greater than about 9.2%. In certain embodiments, the subject has a HbAlc level equal to or greater than about 9.1%. In certain embodiments, the subject has a HbAlc level equal to or greater than about 9.0%. In certain embodiments, the subject has a HbAlc level equal to or greater than about 8.9%. In certain embodiments, the subject has a HbAlc level equal to or greater than about 8.8%. In certain embodiments, the subject has a HbAlc level equal to or greater than about 8.7%. In certain embodiments, the subject has a HbAlc level equal to or greater than about 8.6%. In certain embodiments, the subject has a HbAlc level equal to or greater than about 8.5%. In certain embodiments, the subject has a HbAlc level equal to or greater than about 8.4%. In certain embodiments, the subject has a HbAlc level equal to or greater than about 8.3%. In certain embodiments, the subject has a HbAlc level equal to or greater than about 8.2%. In certain embodiments, the subject has a HbAlc level equal to or greater than about 8.1%. In certain embodiments, the subject has a HbAlc level equal to or greater than about 8.0%. In certain embodiments, the subject has a HbAlc level equal to or greater than about 7.9%. In certain embodiments, the subject has a HbAlc level equal to or greater than about 7.8%. In certain embodiments, the subject has a HbAlc level equal to or greater than about 7.7%. In certain embodiments, the subject has a HbAlc level equal to or greater than about 7.6%. In certain embodiments, the subject has a HbAlc level equal to or greater than about 7.5%. In certain embodiments, the subject has a HbAlc level equal to or greater than about 7.4%. In certain embodiments, the subject has a HbAlc level equal to or greater than about 7.3%. In certain embodiments, the subject has a HbAlc level equal to or greater than aboutAtorney Docket No. 047589-5026WO1 (00244)

[0133] 7.2%. In certain embodiments, the subject has a HbAlc level equal to or greater than about 7.1%. In certain embodiments, the subject has a HbAlc level equal to or greater than about 7.0%. In certain embodiments, the subject has a HbAlc level equal to or greater than about 6.9%. In certain embodiments, the subject has a HbAlc level equal to or greater than about 6.8%. In certain embodiments, the subject has a HbAlc level equal to or greater than about 6.7%. In certain embodiments, the subject has a HbAlc level equal to or greater than about 6.6%. In certain embodiments, the subject has a HbAlc level equal to or greater than about 6.5%.

[0134] In certain embodiments, the subject has a HbAlc level equal to or lower than about 10%. In certain embodiments, the subject has a HbAlc level equal to or lower than about 9.9%. In certain embodiments, the subject has a HbAlc level equal to or lower than about 9.8%. In certain embodiments, the subject has a HbAlc level equal to or lower than about 9.7%. In certain embodiments, the subject has a HbAlc level equal to or lower than about 9.6%. In certain embodiments, the subject has a HbAlc level equal to or lower than about 9.5%. In certain embodiments, the subject has a HbAlc level equal to or lower than about 9.4%. In certain embodiments, the subject has a HbAlc level equal to or lower than about 9.3%. In certain embodiments, the subject has a HbAlc level equal to or lower than about 9.2%. In certain embodiments, the subject has a HbAlc level equal to or lower than about 9.1%. In certain embodiments, the subject has a HbAlc level equal to or lower than about 9.0%. In certain embodiments, the subject has a HbAlc level equal to or lower than about 8.9%. In certain embodiments, the subject has a HbAlc level equal to or lower than about 8.8%. In certain embodiments, the subject has a HbAlc level equal to or lower than about 8.7%. In certain embodiments, the subject has a HbAlc level equal to or lower than about 8.6%. In certain embodiments, the subject has a HbAlc level equal to or lower than about 8.5%. In certain embodiments, the subject has a HbAlc level equal to or lower than about 8.4%. In certain embodiments, the subject has a HbAlc level equal to or lower than about 8.3%. In certain embodiments, the subject has a HbAlc level equal to or lower than about 8.2%. In certain embodiments, the subject has a HbAlc level equal to or lower than about 8.1%. In certain embodiments, the subject has a HbAlc level equal to or lower than about 8.0%. In certain embodiments, the subject has a HbAlc level equal to or lower than about 7.9%. In certain embodiments, the subject has a HbAlc level equal to or lower than about 7.8%. In certain embodiments, the subject has a HbAlc level equal to or lower than about 7.7%. In certain embodiments, the subject has a HbAlc level equal to or lower than about 7.6%. In certain embodiments, the subject has a HbAlc level equal to or lower than aboutAtorney Docket No. 047589-5026WO1 (00244)

[0135] 7.5%. In certain embodiments, the subject has a HbAlc level equal to or lower than about 7.4%. In certain embodiments, the subject has a HbAlc level equal to or lower than about 7.3%. In certain embodiments, the subject has a HbAlc level equal to or lower than about 7.2%. In certain embodiments, the subject has a HbAlc level equal to or lower than about 7.1%. In certain embodiments, the subject has a HbAlc level equal to or lower than about 7.0%. In certain embodiments, the subject has a HbAlc level equal to or lower than about 6.9%. In certain embodiments, the subject has a HbAlc level equal to or lower than about 6.8%. In certain embodiments, the subject has a HbAlc level equal to or lower than about 6.7%. In certain embodiments, the subject has a HbAlc level equal to or lower than about 6.6%. In certain embodiments, the subject has a HbAlc level equal to or greater than about 6.5%.

[0136] In certain embodiments, the subject experiences fewer hypoglycemia as compared to the treatment without HDV.

[0137] In certain embodiments, the subject experiences weight loss as compared to the treatment without HDV.

[0138] In certain embodiments, the subject does not experience significant iatrogenic hyperinsulinemia.

[0139] In certain embodiments, the pharmaceutical composition comprises basal insulin and is administered continuously to the subject over a period of at least 24 hours.

[0140] In certain embodiments, the pharmaceutical composition is administered continuously to the subject using a pump.

[0141] In certain embodiments, the subject has Type 1 diabetes, Type 2 diabetes, and / or a metabolic derangement. In certain embodiments, the subject has Type 1 diabetes. In certain embodiments, the subject has Type 2 diabetes. In certain embodiments, the subject has a metabolic derangement.

[0142] Administration / Dosage / Formulations

[0143] The disclosure also encompasses pharmaceutical compositions and methods of their use. These pharmaceutical compositions may comprise an active ingredient (which can be one or more compositions of the disclosure, or pharmaceutically acceptable salts thereof) optionally in combination with one or more pharmaceutically acceptable agents. The compositions set forth herein can be used alone or in combination with additional compounds to produce additive, complementary, or synergistic effects.Atorney Docket No. 047589-5026WO1 (00244)

[0144] The regimen of administration may affect what constitutes an effective amount. The therapeutic formulations may be administered to the subject either prior to or after the onset of a disease or disorder contemplated herein. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection, or may be administered inhalationally, buccally and / or orally. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

[0145] Administration of the compositions of the present disclosure to a patient, preferably a mammal, more preferably a human, may be carried out using known procedures, at dosages and for periods of time effective to treat a disease or disorder contemplated herein. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the state of the disease or disorder in the patient; the age, sex, and weight of the patient; and the ability of the therapeutic compound to treat a disease or disorder contemplated herein. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dose range for a therapeutic compound of the disclosure is from about 1 and 5,000 mg / kg of body weight / per day. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.

[0146] Actual dosage levels of the active ingredients in the pharmaceutical compositions of this disclosure may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0147] In particular, the selected dosage level depends upon a variety of factors including the activity of the particular compound employed, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds or materials used in combination with the compound, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well, known in the medical arts.

[0148] A medical doctor, e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds ofAtorney Docket No. 047589-5026WO1 (00244)

[0149] the disclosure employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

[0150] In particular embodiments, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms of the disclosure are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding / formulating such a therapeutic compound for the treatment of a disease or disorder contemplated herein.

[0151] In certain embodiments, the compositions of the disclosure are formulated using one or more pharmaceutically acceptable excipients or carriers. In certain embodiments, the pharmaceutical compositions of the disclosure comprise a therapeutically effective amount of a compound of the disclosure and a pharmaceutically acceptable carrier.

[0152] The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils, as long as the solvent or dispersion medium does not disrupt the nanoparticle significantly. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin.

[0153] In certain embodiments, the compositions of the disclosure are administered to the patient in dosages that range from one to five times per day or more. In other embodiments, the compositions of the disclosure are administered to the patient in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks. It is readily apparent to one skilled in the art that the frequency of administration of the various combination compositions of the disclosure varies from individual to individual depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, theAtorney Docket No. 047589-5026WO1 (00244)

[0154] disclosure should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient is determined by the attending physical taking all other factors about the patient into account.

[0155] Compounds of the disclosure for administration may be in the range of from about 1 pg to about 10,000 mg, about 20 pg to about 9,500 mg, about 40 pg to about 9,000 mg, about 75 pg to about 8,500 mg, about 150 pg to about 7,500 mg, about 200 pg to about 7,000 mg, about 350 pg to about 6,000 mg, about 500 pg to about 5,000 mg, about 750 pg to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg to about 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800 mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80 mg to about 500 mg, and any and all whole or partial increments there between.

[0156] In certain embodiments, the dose of a compound and / or composition of the disclosure is from about 1 mg and about 2,500 mg. In other embodiments, a dose of a compound of the disclosure used in compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in other embodiments, a dose of a second compound as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.

[0157] In certain embodiments, the dose of insulin is per day (daily).

[0158] In certain embodiments, the dose of insulin is about 0.01 units / kg. In certain embodiments, the dose of insulin is about 0.02 units / kg. In certain embodiments, the dose of insulin is about 0.03 units / kg. In certain embodiments, the dose of insulin is about 0.04 units / kg. In certain embodiments, the dose of insulin is about 0.05 units / kg. In certain embodiments, the dose of insulin is about 0.06 units / kg. In certain embodiments, the dose of insulin is about 0.07 units / kg. In certain embodiments, the dose of insulin is about 0.08 units / kg. In certain embodiments, the dose of insulin is about 0.09 units / kg. In certain embodiments, the dose of insulin is about 0.1 units / kg. In certain embodiments, the dose ofAtorney Docket No. 047589-5026WO1 (00244)

[0159] insulin is about 0.15 units / kg. In certain embodiments, the dose of insulin is about 0.2 units / kg. In certain embodiments, the dose of insulin is about 0.25 units / kg. In certain embodiments, the dose of insulin is about 0.3 units / kg. In certain embodiments, the dose of insulin is about 0.35 units / kg. In certain embodiments, the dose of insulin is about 0.4 units / kg. In certain embodiments, the dose of insulin is about 0.45 units / kg. In certain embodiments, the dose of insulin is about 0.5 units / kg. In certain embodiments, the dose of insulin is about 0.55 units / kg. In certain embodiments, the dose of insulin is about 0.6 units / kg. In certain embodiments, the dose of insulin is about 0.65 units / kg. In certain embodiments, the dose of insulin is about 0.7 units / kg. In certain embodiments, the dose of insulin is about 0.75 units / kg. In certain embodiments, the dose of insulin is about 0.8 units / kg. In certain embodiments, the dose of insulin is about 0.85 units / kg. In certain embodiments, the dose of insulin is about 0.9 units / kg. In certain embodiments, the dose of insulin is about 0.95 units / kg. In certain embodiments, the dose of insulin is about 1 unit / kg. In certain embodiments, the dose of insulin is about 1.1 units / kg. In certain embodiments, the dose of insulin is about 1.2 units / kg. In certain embodiments, the dose of insulin is about 1.3 units / kg. In certain embodiments, the dose of insulin is about 1.4 units / kg. In certain embodiments, the dose of insulin is about 1.5 units / kg. In certain embodiments, the dose of insulin is about 1.6 units / kg. In certain embodiments, the dose of insulin is about 1.7 units / kg. In certain embodiments, the dose of insulin is about 1.8 units / kg. In certain embodiments, the dose of insulin is about 1.9 units / kg. In certain embodiments, the dose of insulin is about 2 units / kg. In certain embodiments, the dose of insulin is about 2.1 units / kg. In certain embodiments, the dose of insulin is about 2.2 units / kg. In certain embodiments, the dose of insulin is about 2.3 units / kg. In certain embodiments, the dose of insulin is about 2.4 units / kg. In certain embodiments, the dose of insulin is about 2.5 units / kg. In certain embodiments, the dose of insulin is about 2.6 units / kg. In certain embodiments, the dose of insulin is about 2.7 units / kg. In certain embodiments, the dose of insulin is about 2.8 units / kg. In certain embodiments, the dose of insulin is about 2.9 units / kg. In certain embodiments, the dose of insulin is about 3.0 units / kg. In certain embodiments, the dose of insulin is about 3.2 units / kg. In certain embodiments, the dose of insulin is about 3.4 units / kg. In certain embodiments, the dose of insulin is about 3.5 units / kg. In certain embodiments, the dose of insulin is about 3.6 units / kg. In certain embodiments, the dose of insulin is about 3.8 units / kg. In certain embodiments, the dose of insulin is about 4 units / kg. In certain embodiments, the dose of insulin is about 4.5 units / kg. In certain embodiments, the dose of insulin is about 5 units / kg. In certain embodiments, the dose of insulin is about 5.5Atorney Docket No. 047589-5026WO1 (00244)

[0160] units / kg. In certain embodiments, the dose of insulin is about 6 units / kg. In certain embodiments, the dose of insulin is about 6.5 units / kg. In certain embodiments, the dose of insulin is about 7 units / kg. In certain embodiments, the dose of insulin is about 7.5 units / kg. In certain embodiments, the dose of insulin is about 8 units / kg. In certain embodiments, the dose of insulin is about 8.5 units / kg. In certain embodiments, the dose of insulin is about 9 units / kg. In certain embodiments, the dose of insulin is about 9.5 units / kg. In certain embodiments, the dose of insulin is about 10 units / kg. In certain embodiments, the dose of insulin is about 11 units / kg. In certain embodiments, the dose of insulin is about 12 units / kg. In certain embodiments, the dose of insulin is about 13 units / kg. In certain embodiments, the dose of insulin is about 14 units / kg. In certain embodiments, the dose of insulin is about 15 units / kg. In certain embodiments, the dose of insulin is about 16 units / kg. In certain embodiments, the dose of insulin is about 17 units / kg. In certain embodiments, the dose of insulin is about 18 units / kg. In certain embodiments, the dose of insulin is about 19 units / kg. In certain embodiments, the dose of insulin is about 20 units / kg.

[0161] In certain embodiments, the dose of insulin is greater than about 0.01 units / kg, about 0.02 units / kg, about 0.03 units / kg, about 0.04 units / kg, about 0.05 units / kg, about 0.06 units / kg, about 0.07 units / kg, about 0.08 units / kg, about 0.09 units / kg, about 0.1 units / kg, about 0.15 units / kg, about 0.2 units / kg, about 0.25 units / kg, about 0.3 units / kg, about 0.35 units / kg, about 0.4 units / kg, about 0.45 units / kg, about 0.5 units / kg, about 0.55 units / kg, about 0.6 units / kg, about 0.65 units / kg, about 0.7 units / kg, about 0.75 units / kg, about 0.8 units / kg, about 0.85 units / kg, about 0.9 units / kg, about 0.95 units / kg, about 1 unit / kg, about 1.1 units / kg, about 1.2 units / kg, about 1.3 units / kg, about 1.4 units / kg, about 1.5 units / kg, about 1.6 units / kg, about 1.7 units / kg, about 1.8 units / kg, about 1.9 units / kg, about 2 units / kg, about 2.1 units / kg, about 2.2 units / kg, about 2.3 units / kg, about 2.4 units / kg, about 2.5 units / kg, about 2.6 units / kg, about 2.7 units / kg, about 2.8 units / kg, about 2.9 units / kg, about 3.0 units / kg, about 3.2 units / kg, about 3.4 units / kg, about 3.5 units / kg, about 3.6 units / kg, about 3.8 units / kg, about 4 units / kg, about 4.5 units / kg, about 5 units / kg, about 5.5 units / kg, about 6 units / kg, about 6.5 units / kg, about 7 units / kg, about 7.5 units / kg, about 8 units / kg, about 8.5 units / kg, about 9 units / kg, about 9.5 units / kg, about 10 units / kg, about 11 units / kg, about 12 units / kg, about 13 units / kg, about 14 units / kg, about 15 units / kg, about 16 units / kg, about 17 units / kg, about 18 units / kg, about 19 units / kg, or about 20 units / kg.

[0162] In certain embodiments, the dose of insulin is lower than about 0.01 units / kg, about 0.02 units / kg, about 0.03 units / kg, about 0.04 units / kg, about 0.05 units / kg, about 0.06 units / kg, about 0.07 units / kg, about 0.08 units / kg, about 0.09 units / kg, about 0.1 units / kg,Atorney Docket No. 047589-5026WO1 (00244)

[0163] about 0.15 units / kg, about 0.2 units / kg, about 0.25 units / kg, about 0.3 units / kg, about 0.35 units / kg, about 0.4 units / kg, about 0.45 units / kg, about 0.5 units / kg, about 0.55 units / kg, about 0.6 units / kg, about 0.65 units / kg, about 0.7 units / kg, about 0.75 units / kg, about 0.8 units / kg, about 0.85 units / kg, about 0.9 units / kg, about 0.95 units / kg, about 1 unit / kg, about 1.1 units / kg, about 1.2 units / kg, about 1.3 units / kg, about 1.4 units / kg, about 1.5 units / kg, about 1.6 units / kg, about 1.7 units / kg, about 1.8 units / kg, about 1.9 units / kg, about 2 units / kg, about 2.1 units / kg, about 2.2 units / kg, about 2.3 units / kg, about 2.4 units / kg, about 2.5 units / kg, about 2.6 units / kg, about 2.7 units / kg, about 2.8 units / kg, about 2.9 units / kg, about 3.0 units / kg, about 3.2 units / kg, about 3.4 units / kg, about 3.5 units / kg, about 3.6 units / kg, about 3.8 units / kg, about 4 units / kg, about 4.5 units / kg, about 5 units / kg, about 5.5 units / kg, about 6 units / kg, about 6.5 units / kg, about 7 units / kg, about 7.5 units / kg, about 8 units / kg, about 8.5 units / kg, about 9 units / kg, about 9.5 units / kg, about 10 units / kg, about 11 units / kg, about 12 units / kg, about 13 units / kg, about 14 units / kg, about 15 units / kg, about 16 units / kg, about 17 units / kg, about 18 units / kg, about 19 units / kg, or about 20 units / kg.

[0164] In certain embodiments, the present disclosure is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound and / or composition of the disclosure, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of a disease or disorder contemplated herein.

[0165] In certain embodiments, the container holds a lipid-based nanoparticle, which does not comprise a therapeutic agent of interest, such as but not limited to an insulin or a derivative or analog thereof. In other embodiments, the container holds a lipid-based nanoparticle, which comprises a therapeutic agent of interest, such as but not limited to an insulin or a derivative or analog thereof. In yet other embodiments, the container further holds a therapeutic agent of interest, such as but not limited to an insulin or a derivative or analog thereof.

[0166] Illustrative Non-Limiting Methods of Treating, Ameliorating, and / or Preventing Patients with Type 1 or Type 2 diabetes mellitus, as well as patients with metabolic derangements, such as but not limited to metabolic syndrome with elevated insulin levels, steatosis, and / or steatohepatitis, can be administered an effective amount of a nanoparticle of the disclosure comprising an insulin. When this composition is administered subcutaneously, a portion of the composition enters the circulatory system where the composition is transported to the liver and other areas. The extended amphipathic lipid binds the lipidAtorney Docket No. 047589-5026WO1 (00244)

[0167] construct to receptors of hepatocytes. A portion of the administered composition is exposed to an external gradient in vivo, where insulin can be solubilized and then move from the lipid construct thereby supplying insulin to the muscle and adipose tissue. Insulin that remains with the lipid construct maintains the capability of being directed to the hepatocyte binding receptor on the hepatocytes in the liver. Therefore, two forms of insulin are produced from this particular lipid construct. In an in vivo setting, free and lipid associated insulin are generated in a time-dependent manner.

[0168] Administration of the nanoparticles and compositions comprising same can be through any of the accepted modes of administration for insulin that are desired to be administered. These methods include oral, parenteral, nasal and other systemic or aerosol forms. These methods further include pump delivery systems.

[0169] Oral administration of a nanoparticle of the disclosure is followed by intestinal absorption of insulin associated with the nanoparticle of the disclosure into the circulatory system of the body, where it is also exposed to the physiological pH of the blood. The nanoparticle is targeted for delivery to the liver and may be shielded by the presence of cellulose acetate phthalate within the nanoparticle of the disclosure. In the case of oral administration, the shielded nanoparticle transverses the oral cavity, migrates through the stomach and moves into the small intestine, where the alkaline pH of the small intestine degrades the cellulose acetate phthalate shield. The deshielded nanoparticle is absorbed into the circulatory system. This enables the nanoparticle to be delivered to the sinusoids of the liver. A receptor binding molecule, such as l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(Cap Biotinyl) or any other hepatocyte specific molecule, provides a means for lipid construct to bind to the receptor and then be engulfed or endocytosed by the hepatocytes. Insulin is then released from the nanoparticle where, upon gaining access to the cellular environment, it performs its designated function with regard to acting as an agent to control diabetes mellitus.

[0170] Patients with Type 1 or Type 2 diabetes mellitus, as well as patients with metabolic derangements, such as but not limited to metabolic syndrome with elevated insulin levels, steatosis, and / or steatohepatitis, may be administered an effective amount of a nanoparticle comprising a mixture of free glargine insulin and glargine insulin associated with the nanoparticle. Glargine insulin can be combined with other forms of insulin, such as insulin lispro, insulin aspart (including FIASP®, Novo Nordisk), regular insulin, insulin glargine, insulin zinc, extended human insulin zinc suspension, isophane insulin, human buffered regular insulin, insulin glulisine, recombinant human regular insulin, recombinant humanAtorney Docket No. 047589-5026WO1 (00244)

[0171] insulin isophane, insulin detemir, biphasic human insulin, and insulin degludec (including TRESIBA®, Novo Nordisk) or premixed combinations of any of the aforementioned insulins, a derivative thereof, and a combination of any of the aforementioned insulins. The composition can be administered by a subcutaneous or oral route.

[0172] After a composition is administered to a patient by subcutaneous injection, the in situ physiological environment in the injection area, the morphology and chemical structures of free insulin and the insulin associated with the nanoparticle begin to change. For example, as the pH of the environment around the free glargine insulin and the glargine insulin associated with the nanoparticle increases after being diluted with physiological media, the pH reaches the isoelectric point of glargine insulin, where flocculation, aggregation and precipitation reactions occur for both free glargine insulin and glargine insulin associated with the nanoparticle. In certain embodiments, free glargine insulin changes from a soluble form at injection, to a insoluble form at a pH near its isoelectric point of pH 5.8-6.2, and then to a soluble form at physiological pH. The rates at which these processes occur differ between free glargine insulin and glargine insulin associated with the nanoparticle. The free glargine insulin is directly exposed to changes in pH and dilution. Exposure of glargine insulin associated with the nanoparticle to small changes in pH and dilution at physiological pH is delayed due to the time required for diffusion of physiological fluids or media through the lipid bilayer in the nanoparticle. The delay in the release of insulin from the lipid construct as well as the delay of the release of the insulin associated with the nanoparticle is a feature of the disclosure since it affects and augments the biological and pharmacological response in vivo.

[0173] Oral administration of a pharmaceutical composition that combines free glargine insulin and glargine insulin associated with a nanoparticle is followed by intestinal absorption of glargine insulin associated with the nanoparticle into the circulatory system of the body, where it is also exposed to the physiological pH of the blood. In certain embodiments, the composition comprises a delayed release matrix which releases HDV glargine over a prolonged period of time, in order to achieve a 24-hour dose regimen. All or a portion of the nanoparticle is delivered to the liver.

[0174] Patients with Type 1 or Type 2 diabetes mellitus, as well as patients with metabolic derangements, such as but not limited to metabolic syndrome with elevated insulin levels, steatosis, and / or steatohepatitis, can be administered an effective amount of a hepatocyte targeted composition comprising a mixture of free recombinant human insulin isophane (NPH) plus free recombinant human regular insulin along with recombinant human insulinAtorney Docket No. 047589-5026WO1 (00244)

[0175] isophane and recombinant human regular insulin which are both associated with a nanoparticle. Recombinant human insulin isophane can be combined with other forms of insulin, such insulin lispro, insulin aspart (including FIASP®, Novo Nordisk), regular insulin, insulin glargine, insulin zinc, extended human insulin zinc suspension, isophane insulin, human buffered regular insulin, insulin glulisine, recombinant human regular insulin, recombinant human insulin isophane, insulin detemir, biphasic human insulin, and insulin degludec (including TRESIBA®, Novo Nordisk, ultralong-acting basal insulin analogue; has one single amino acid deleted in comparison to human insulin, and is conjugated to hexadecanedioic acid via gamma-L-glutamyl spacer at the amino acid lysine at position B29), or any (premixed) combinations thereof.

[0176] In certain embodiments, the composition comprises a delayed release matrix which releases HDV NPH over a prolonged period of time, in order to achieve a 24-hour dose regimen.

[0177] Oral administration of a pharmaceutical composition that combines free recombinant human insulin isophane and recombinant human insulin isophane associated with a nanoparticle is followed by intestinal absorption of recombinant human insulin isophane associated with the nanoparticle into the circulatory system of the body where it is also exposed to the physiological pH of the blood. All or a portion of the nanoparticle is delivered to the liver, while the non-HDV isophane is slowly absorbed from a slow release matrix for release into the general circulation.

[0178] As the physiological dilution is increased in situ in the subcutaneous space or upon entering into the circulatory system, free recombinant human insulin isophane and recombinant human insulin isophane associated with the nanoparticle encounter a normal physiological pH environment of pH 7.4. As a result of dilution free recombinant human insulin isophane changes from an insoluble form at injection, to a soluble form at physiological pH. In the soluble form, recombinant human insulin isophane migrates through the body to sites where it is capable of eliciting a pharmacological response. Recombinant human insulin isophane associated with the nanoparticle becomes solubilized and released from the nanoparticle at a different rate that is slower than that of free recombinant human insulin isophane. This is because recombinant human insulin isophane associated with the nanoparticle has to traverse the core volume and lipid domains of the nanoparticle before it contacts the bulk phase media.

[0179] The amount of insulin administered will be dependent on the subject being treated, the type and severity of the affliction, the manner of administration and the judgment of theAtorney Docket No. 047589-5026WO1 (00244)

[0180] prescribing physician. Although effective dosage ranges for specific biologically active substances of interest are dependent upon a variety of factors and are generally known to one of ordinary skill in the art, some dosage guidelines can be generally defined. For most forms of administration, the nanoparticle will be suspended in an aqueous solution and generally not exceed 4.0% (w / v) of the total formulation. The drug component of the formulation will in certain embodiments be less than 20% (w / v) of the formulation and generally greater than 0.01% (w / v).

[0181] In certain embodiments, the pharmaceutical composition comprises HDV insulin, and no free insulin. In such cases, all of the insulin within the composition is targeted to the liver. In other embodiments, the pharmaceutical composition comprises HDV insulin and free insulin (non-HDV insulin). The ratio between HDV insulin and free insulin can be, in nonlimiting example, about 0.1:99.9, 0.2:99.8, 0.3:99.7, 0.4:99.6, 0.5:99.5, 0.6:99.4, 0.7:99.3, 0.8:99.2, 0.9:99.1, 1:99, 2:98, 3:97, 4:96, 5:95, 6:94, 7:93, 8:92, 9:91, 10:90, 12:88, 14:86, 16:84, 18:82, 20:80, 22:78, 24:76, 25:75, 26:74, 28:72, 30:70, 32:68, 34:66, 36:64, 38:62, 40:60, 42:58, 44:56, 46:54, 48:52, and / or 50:50.

[0182] Dosage forms or compositions containing active ingredient in the range of 0.005% to 5% with the balance made up from non-toxic carriers can be prepared.

[0183] The exact composition of these formulations may vary widely depending on the particular properties of the drug in question. In certain embodiments, they comprise from 0.01% to 5%, and preferably from 0.05% to 1% active ingredient for highly potent drugs, and from 2%-4% for moderately active drugs.

[0184] The percentage of active ingredient contained in such parenteral compositions is highly dependent on the specific nature thereof, as well as the activity of the active ingredient and the needs of the subject. However, percentages of active ingredient of 0.01% to 5% in solution are employable, and will be higher if the composition is a solid which will be subsequently diluted to the above percentages. In certain embodiments, the composition comprises 0.2%-2.0% of the active agent in solution.

[0185] Administration

[0186] Formulations may be employed in admixtures with conventional excipients, / .< ., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents,Atorney Docket No. 047589-5026WO1 (00244)

[0187] emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and / or aromatic substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic agents.

[0188] Routes of administration of any of the compositions of the disclosure include oral, nasal, rectal, intravaginal, parenteral, buccal, sublingual or topical. The compounds and / or compositions for use in the disclosure may be formulated for administration by any suitable route, such as for oral or parenteral, for example, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g, trans- and perivaginally), (intra)nasal and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.

[0189] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present disclosure are not limited to the particular formulations and compositions that are described herein.

[0190] Oral Administration

[0191] For oral application, particularly suitable are tablets, dragees, liquids, drops, suppositories, or capsules, caplets and gelcaps. The compositions intended for oral use may be prepared according to any method known in the art and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutically excipients that are suitable for the manufacture of tablets. Such excipients include, for example an inert diluent such as lactose; granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agents such as magnesium stearate. The tablets may be uncoated or they may be coated by known techniques for elegance or to delay the release of the active ingredients. Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert diluent.

[0192] For oral administration, the compounds and / or compositions of the disclosure may be in the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g, polyvinylpyrrolidone, hydroxypropylcellulose or hydroxypropyl methylcellulose); fillers (e.g., cornstarch, lactose,Atorney Docket No. 047589-5026WO1 (00244)

[0193] microcrystalline cellulose or calcium phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrates (e.g., sodium starch glycollate); or wetting agents e.g., sodium lauryl sulphate). If desired, the tablets may be coated using suitable methods and coating materials such as OPADRY™ film coating systems available from Colorcon, West Point, Pa. (e.g., OPADRY™ OY Type, OYC Type, Organic Enteric OY-P Type, Aqueous Enteric OY-A Type, OY-PM Type and OPADRY™ White, 32K18400). Liquid preparation for oral administration may be in the form of solutions, syrups or suspensions. The liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agent (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters or ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxy benzoates or sorbic acid).

[0194] Parenteral Administration

[0195] For parenteral administration, the compounds and / or compositions of the disclosure may be formulated for injection or infusion, for example, intravenous, intramuscular or subcutaneous injection or infusion, or for administration in a bolus dose and / or continuous infusion. Suspensions, solutions or emulsions in an oily or aqueous vehicle, optionally containing other formulatory agents such as suspending, stabilizing and / or dispersing agents may be used.

[0196] Pulmonary administration

[0197] A pharmaceutical composition of the disclosure may be prepared, packaged, or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 microns, and preferably from about 1 to about 6 microns. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder or using a self-propelling solvent / powder-dispensing container such as a device comprising the active ingredient dissolved or suspended in a low-boiling propellant in a sealed container. Preferably, such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 microns and at least 95% of the particles by number have a diameter less than 7 microns. More preferably, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6Atorney Docket No. 047589-5026WO1 (00244)

[0198] microns. Dry powder compositions preferably include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.

[0199] Low boiling propellants generally include liquid propellants having a boiling point of below 65°F at atmospheric pressure. Generally, the propellant may constitute 50 to 99.9% (w / w) of the composition, and the active ingredient may constitute 0.1 to 20% (w / w) of the composition. The propellant may further comprise additional ingredients such as a liquid non-ionic or solid anionic surfactant or a solid diluent (preferably having a particle size of the same order as particles comprising the active ingredient).

[0200] Pharmaceutical compositions of the disclosure formulated for pulmonary delivery may also provide the active ingredient in the form of droplets of a solution or suspension. Such formulations may be prepared, packaged, or sold as aqueous or dilute alcoholic solutions or suspensions, optionally sterile for administration by injection, comprising the active ingredient, and may conveniently be administered using any nebulization or atomization device. In certain embodiments, the compounds and / or compositions of the disclosure are sterile filtered before administration to the subject. Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, or a preservative such as methylhydroxybenzoate. The droplets provided by this route of administration preferably have an average diameter in the range from about 0.1 to about 200 microns.

[0201] Intranasal Delivery

[0202] The formulations described herein as being useful for pulmonary delivery are also useful for intranasal delivery of a pharmaceutical composition of the disclosure.

[0203] Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 to 500 microns. Such a formulation is administered in the manner in which snuff is taken i.e. by rapid inhalation through the nasal passage from a container of the powder held close to the nares.

[0204] Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% (w / w) and as much as 75% (w / w) of the active ingredient, and may further comprise one or more of the additional ingredients described herein.

[0205] Additional Administration FormsAtorney Docket No. 047589-5026WO1 (00244)

[0206] Additional dosage forms of this disclosure include dosage forms as described in U.S. Patents Nos. 6,340,475; 6,488,962; 6,451,808; 5,972,389; 5,582,837; and 5,007,790.

[0207] Additional dosage forms of this disclosure also include dosage forms as described in U.S. Patent Applications Nos. 20030147952; 20030104062; 20030104053; 20030044466;

[0208] 20030039688; and 20020051820. Additional dosage forms of this disclosure also include dosage forms as described in PCT Applications Nos. WO 03 / 35041; WO 03 / 35040; WO 03 / 35029; WO 03 / 35177; WO 03 / 35039; WO 02 / 96404; WO 02 / 32416; WO 01 / 97783; WO 01 / 56544; WO 01 / 32217; WO 98 / 55107; WO 98 / 11879; WO 97 / 47285; WO 93 / 18755; and WO 90 / 11757.

[0209] Controlled Release Formulations and Drug Delivery Systems

[0210] In certain embodiments, the formulations of the present disclosure may be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations.

[0211] The term sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may, although not necessarily, result in substantially constant blood levels of a drug over an extended time period. The period of time may be as long as a month or more and should be a release that is longer that the same amount of agent administered in bolus form.

[0212] For sustained release, the compositions may be formulated with a suitable polymer or hydrophobic material that provides sustained release properties to the compounds and / or compositions. As such, the compositions and / or compositions for use the method of the disclosure may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation.

[0213] In certain embodiments, the compounds and / or compositions of the disclosure are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation.

[0214] The term delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that mat, although not necessarily, includes a delay of from about 10 minutes up to about 12 hours.

[0215] The term pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration.Atorney Docket No. 047589-5026WO1 (00244)

[0216] The term immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration.

[0217] As used herein, short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug administration.

[0218] As used herein, rapid-offset refers to any period of time up to and including about 16 hours, about 15 hours, about 14 hours, about 13 hours, about 12 hours, about 11 hours, about 10 hours, about 9 hours, about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration.

[0219] Dosing

[0220] The therapeutically effective amount or dose of a compound and / or composition of the present disclosure depends on the age, sex and weight of the patient, the current medical condition of the patient and the progression of a disease or disorder contemplated herein in the patient being treated. The skilled artisan is able to determine appropriate dosages depending on these and other factors.

[0221] A suitable dose of a compound and / or composition of the present disclosure may be in the range of from about 0.01 mg to about 5,000 mg per day, such as from about 0.1 mg to about 1,000 mg, for example, from about 1 mg to about 500 mg, such as about 5 mg to about 250 mg per day. The dose may be administered in a single dosage or in multiple dosages, for example from 1 to 4 or more times per day. When multiple dosages are used, the amount of each dosage may be the same or different. For example, a dose of 1 mg per day may be administered as two 0.5 mg doses, with about a 12-hour interval between doses.

[0222] It is understood that the amount of compound and / or composition dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on.

[0223] In the case wherein the patient’s status does improve, upon the doctor’s discretion the administration of the inhibitor of the disclosure is optionally given continuously;Atorney Docket No. 047589-5026WO1 (00244)

[0224] alternatively, the dose of drug being administered is temporarily reduced or temporarily suspended for a certain length of time (i.e., a “drug holiday”). The length of the drug holiday optionally varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. The dose reduction during a drug holiday includes from 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0225] Once improvement of the patient’s conditions has occurred, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, is reduced, as a function of the viral load, to a level at which the improved disease is retained. In certain embodiments, patients require intermittent treatment on a long-term basis upon any recurrence of symptoms and / or infection.

[0226] The compounds and / or compositions for use in the method of the disclosure may be formulated in unit dosage form. The term “unit dosage form” refers to physically discrete units suitable as unitary dosage for patients undergoing treatment, with each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier. The unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.

[0227] Toxicity and therapeutic efficacy of such therapeutic regimens are optionally determined in cell cultures or experimental animals, including, but not limited to, the determination of the LDso (the dose lethal to 50% of the population) and the EDso (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LDso and EDso. The data obtained from cell culture assays and animal studies are optionally used in formulating a range of dosage for use in human. The dosage of such compounds and / or compositions lies preferably within a range of circulating concentrations that include the ED50 with minimal toxicity. The dosage optionally varies within this range depending upon the dosage form employed and the route of administration utilized.Atorney Docket No. 047589-5026WO1 (00244)

[0228] Definitions

[0229] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure belongs. Generally, the nomenclature used herein and the laboratory procedures in organic chemistry and protein chemistry are those well known and commonly employed in the art.

[0230] The articles “a” and “an” are used herein to refer to one or to more than one ( / .< ., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0231] The term “Ale” or “A1C” or “HbAlC” or “hemoglobin Ale” or “HBA1C” or “HgbAlc” or “haemoglobin Ale” or “HbAlc” or “Hblc” refers to a form of hemoglobin that is covalently bound to glucose. Ale is formed in a non-enzymatic glycation pathway by hemoglobin’s exposure to plasma glucose. Ale is measured primarily to identify the three-month average plasma glucose concentration, and thus can be used as a diagnostic test for diabetes and as assessment test for glycemic control in people with diabetes. The ratio of Ale to total hemoglobin (% Ale) (generally measured as mass / mass) is used to diagnose diabetes (according to 1993 Diabetes Control and Complications Trial or DCCT): normal individuals have less than 5.7% Al, pre-diabetic individuals have 5-7-6.4% Ale, and diabetic individuals have greater than 6.5% Ale. The DCCT % Ale value can be converted to the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) units using the formula:

[0232] IFCC HbAlc (mmol / mol) = [DCCT HbAlc (%) - 2.14] x 10.929 As used herein, the term “about” is understood by persons of ordinary skill in the art and varies to some extent on the context in which it is used. As used herein when referring to a measurable value such as an amount, a temporal duration, and the like, the term “about” is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0233] As used herein, the term “active ingredient” refers to a therapeutic agent that is to be delivered to a subject to produce a therapeutic effect in the subject. Non-limiting examples of active ingredients contemplated within the disclosure are insulin, interferon, parathyroid hormone, calcitonin, serotonin, serotonin agonist, serotonin reuptake inhibitor, human growth hormone, GIP, anti-GIP monoclonal antibody, metformin, bromocriptine, dopamine, glucagon and / or GLP-1.Atorney Docket No. 047589-5026WO1 (00244)

[0234] The term “amphipathic lipid” means a lipid molecule having a polar and non-polar end.

[0235] By “aqueous media” is meant water or water containing buffer or salt.

[0236] As used herein, the term “basal insulin” or “background insulin” is insulin that is taken to keep blood glucose levels at consistent levels during periods of fasting. Basal insulin is thus needed to keep blood glucose levels under control, and to allow the cells to take in glucose for energy. Basal insulin is usually taken once or twice a day depending on the insulin. Basal insulin needs to act over a relatively long period of time, and thus is either long acting insulin or intermediate insulin.

[0237] As used herein, the term “basal glucose control” refers to the glucose control that is afforded by use of basal insulin, or an equivalent thereof.

[0238] The term “bioavailability” refers to a measurement of the rate and extent that insulin reaches the systemic circulation and is available at the sites of action.

[0239] As used herein, the term “bolus insulin” refers to insulin that is specifically taken just before, at, or just after meal times to keep blood glucose levels under control following a meal. Bolus insulin needs to act quickly and is generally short acting insulin or rapid acting insulin.

[0240] As used herein, the term “bolus glucose control” refers to the glucose control that is afforded by use of bolus insulin, or an equivalent thereof.

[0241] As used herein, the term “CGM” refers to continuous glucose monitoring.

[0242] In one aspect, the terms “co-administered” and “co-administration” as relating to a subject refer to administering to the subject a compound of the disclosure or salt thereof along with a compound that may also treat any disease or disorder contemplated herein and / or with a compound that is useful in treating other medical conditions but which in themselves may cause or facilitate any disease or disorder contemplated herein. In certain embodiments, the co-administered compounds are administered separately, or in any kind of combination as part of a single therapeutic approach. The co-administered compound may be formulated in any kind of combinations as mixtures of solids and liquids under a variety of solid, gel, and liquid formulations, and as a solution.

[0243] As used herein, a “disease” is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated then the subject’s health continues to deteriorate.

[0244] As used herein, a “disorder” in a subject is a state of health in which the subject is able to maintain homeostasis, but in which the subject’s state of health is less favorable thanAtorney Docket No. 047589-5026WO1 (00244)

[0245] it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the subject’s state of health.

[0246] As used herein, the term “EDso” refers to the effective dose of a formulation that produces 50% of the maximal effect in subjects that are administered that formulation.

[0247] As used herein, an “effective amount,” “therapeutically effective amount” or “pharmaceutically effective amount” of a compound is that amount of compound that is sufficient to provide a beneficial effect to the subject to which the compound is administered.

[0248] The term “free active ingredient” or “free therapeutic agent” refers to an active ingredient or therapeutic agent that is not dispersed within the lipid particle (i.e., located within, adsorbed on and / or bound to the lipid particle membrane).

[0249] The terms “glargine” and “glargine insulin” both refer to a recombinant human insulin analog which differs from human insulin in that the amino acid asparagine at position A21 is replaced by glycine and two arginines are added to the C-terminus of the B-chain.

[0250] Chemically, it is 21A- Gly-30Ba-L- Arg-30Bb-L-Arg-human insulin and has the empirical formula C267H404N72O78S6 and a molecular weight of 6063.

[0251] As used herein, the term “hyperinsulinemia” refers to a condition in which there are excess levels of insulin circulating in the blood relative to the level of glucose.

[0252] Hyperinsulinemia can be an unwanted side effect of administration of exogenous insulin to a diabetic patient (thus being a form of iatrogenic hyperinsulinemia; see Cryer, 2008, Diabetes 57(12):3169-76, McCrinson & Sherwin, 2010, Diabetes 59(10):2333-9; Wang, et a!., 2013, J. Diab. & Its Compl. 27(l):70-74; all of which are incorporated herein in their entireties by reference). That condition can trigger complications such as metabolic disease, hypoglycemia, increased risk of polycystic ovary syndrome (PCOS), increased synthesis of VLDL (hypertriglyceridemia), hypertension (insulin increases sodium retention by the renal tubules), coronary artery disease (CAD; increased insulin damages endothelial cells), increased risk of cardiovascular disease, and / or weight gain and lethargy.

[0253] As used herein, the term “hypoglycemic event” or “hypoglycemia event” refers to an event wherein the subject’s blood sugar is lower than 70 mg / dL for a significant amount of time, such as but not limited to 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes. In certain embodiments, a hypoglycemic event is defined as a series of CGM values less than about 54 mg / dL, separated by 20 min or more, with no intervening values of 54 mg / dL or more. In certain embodiments, a hypoglycemic event is defined as over 15 min of CGM values less than about 54 mg / dL.Atorney Docket No. 047589-5026WO1 (00244)

[0254] “Instructional material,” as that term is used herein, includes a publication, a recording, a diagram, or any other medium of expression that can be used to communicate the usefulness of the composition and / or compound of the disclosure in a kit. The instructional material of the kit may, for example, be affixed to a container that contains the compound and / or composition of the disclosure or be shipped together with a container that contains the compound and / or composition. Alternatively, the instructional material may be shipped separately from the container with the intention that the recipient uses the instructional material and the compound cooperatively. Delivery of the instructional material may be, for example, by physical delivery of the publication or other medium of expression communicating the usefulness of the kit, or may alternatively be achieved by electronic transmission, for example by means of a computer, such as by electronic mail, or download from a website.

[0255] The term “insulin” refers to natural or recombinant forms of insulin, and derivatives of the aforementioned insulins. Examples of insulin include, but are not limited to insulin lispro (such as, for example, ADMELOG®, Sanofi), insulin aspart (such as, for example, FIASP®, Novo Nordisk), regular insulin, insulin glargine (such as, for example, BASAGLAR®, Lilly), insulin zinc, human insulin zinc extended, isophane insulin, human buffered regular insulin, insulin glulisine, recombinant human regular insulin, recombinant human insulin isophane, insulin detemir, biphasic human insulin, and insulin degludec (including TRESIBA®, Novo Nordisk, ultralong-acting basal insulin analogue; has one single amino acid deleted in comparison to human insulin, and is conjugated to hexadecanedioic acid via gamma-L-glutamyl spacer at the amino acid lysine at position B29). Also included are animal insulins, such as bovine or porcine insulin.

[0256] As used herein, the term “iotrogenic” refers to any illness caused by a medical examination or treatment.

[0257] The term “isoelectric point” refers to the pH at which the concentrations of positive and negative charges on the protein are equal and, as a result, the protein will express a net zero charge. At the isoelectric point, a protein will exist almost entirely in the form of a zwitterion, or hybrid between forms of the protein. Proteins are least stable at their isoelectric points, and are more easily coagulated or precipitated at this pH. However, proteins are not denatured upon isoelectric precipitation since this process is essentially reversible.Atorney Docket No. 047589-5026WO1 (00244)

[0258] The term “lipid construct” refers to a lipid and / or phospholipid particle in which individual lipid molecules interact to create a bipolar lipid membrane that defines the boundaries of the lipid construct.

[0259] The term “lipid metabolism” refers to synthesis and degradation of lipids in cells, involving breakdown and storage of fats for energy and synthesis of structural and functional lipids. Physiological lipids include membrane lipids (for example, phospholipids, sphingolipids, glycolipids, and glycerophospholipids), cholesterols, steroids, triacylglycerols, fatty acids, bile salts, eicosanoids, and ketone bodies. In animals, some lipids are obtained from diet and some are synthesized in the liver. The overall lipid metabolism process includes lipid digestion, lipid absorption, lipid transportation, lipid storage, lipid biosynthesis, and lipid catabolism. Lipid metabolism takes place in the liver (hepatic lipid metabolism) and ex-liver, especially in fat tissue and muscle (peripheral lipid metabolism). Proper lipid metabolism ensures that the liver and plasma lipid concentrations are properly regulated. Altered lipid metabolism leads to unhealthy changes in liver and plasma levels of certain molecules, such as but not limited to triglycerides, free fatty acids, and branched-chain amino acids. Proper lipid metabolism ensures that there is no pathological accumulation of lipids in the liver (fatty liver) and that glucose is properly stored in the liver and in the periphery.

[0260] As the term is used herein, “to modulate” or “modulation of’ a biological or chemical process or state refers to the alteration of the normal course of the biological or chemical process, or changing the state of the biological or chemical process to a new state that is different than the present state. For example, modulation of the isoelectric point of a polypeptide may involve a change that increases the isoelectric point of the polypeptide. Alternatively, modulation of the isoelectric point of a polypeptide may involve a change that decreases the isoelectric point of a polypeptide.

[0261] As used herein, a “metabolic derangement” refers to a metabolic disorder or disease relating to uncontrolled, elevated, or fluctuating insulin levels, such as but not limited to metabolic syndrome with elevated insulin levels, steatosis, and / or steatohepatitis.

[0262] The term “non-glargine insulin” refers at all insulins, either natural or recombinant that are not glargine insulin. The term includes insulin-like moieties, including fragments of insulin molecules, that have biological activity of insulins.

[0263] As used herein, the term “pharmaceutical composition” or “composition” refers to a mixture of at least one compound useful within the disclosure with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a subject.Atorney Docket No. 047589-5026WO1 (00244)

[0264] As used herein, the term “pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound useful within the disclosure, and is relatively non-toxic, / .< ., the material may be administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0265] As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the disclosure within or to the subject such that it may perform its intended function. Typically, such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, including the compound useful within the disclosure, and not injurious to the subject. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. As used herein, “pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful within the disclosure, and are physiologically acceptable to the subject. Supplementary active compounds may also be incorporated into the compositions. The “pharmaceutically acceptable carrier” may further include a pharmaceutically acceptable salt of the compound useful within the disclosure. Other additional ingredients that may be included in the pharmaceutical compositions used in the practice of the disclosure are known in the art and described, for example in Remington’s Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.Atorney Docket No. 047589-5026WO1 (00244)

[0266] As used herein, the language “pharmaceutically acceptable salt” refers to a salt of the administered compound prepared from pharmaceutically acceptable non-toxic acids and bases, including inorganic acids, inorganic bases, organic acids, inorganic bases, solvates, hydrates, and clathrates thereof.

[0267] The term “prevent,” “preventing” or “prevention,” as used herein, means avoiding or delaying the onset of symptoms associated with a disease or condition in a subject that has not developed such symptoms at the time the administering of an agent or compound commences. Disease, condition and disorder are used interchangeably herein.

[0268] By the term “specifically bind” or “specifically binds,” as used herein, is meant that a first molecule preferentially binds to a second molecule (e.g., a particular receptor or enzyme), but does not necessarily bind only to that second molecule.

[0269] As used herein, a “subject” may be a human or non-human mammal or a bird. Nonhuman mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals. In certain embodiments, the subject is human.

[0270] The term “treat,” “treating” or “treatment,” as used herein, means reducing the frequency or severity with which symptoms of a disease or condition are experienced by a subject by virtue of administering an agent or compound to the subject.

[0271] The term “well controlled diabetes” refers to a diabetic or pre-diabetic subject that receives treatment that allows for keeping fasting blood sugars below 140 mg / dL. In certain embodiments, the fasting blood sugars threshold is below 140 mg / dL, below 130 mg / dL, below 120 mg / dL, below 110 mg / dL, or below 100 mg / dL. In certain embodiments, the fasting blood sugars range is 70-120 mg / dL. In certain embodiments, the fasting blood sugars range is 80-100 mg / dL. In certain embodiments, the fasting blood sugars range is 70-120 mg / dL. In certain embodiments, the fasting blood sugars range is 70-100 mg / dL.

[0272] Throughout this disclosure, various aspects of the disclosure may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range and, when appropriate, partial integers of the numerical values within ranges. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.Atorney Docket No. 047589-5026WO1 (00244)

[0273] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents are considered to be within the scope of this disclosure and covered by the claims appended hereto. For example, it should be understood, that modifications in reaction conditions, including but not limited to reaction times, reaction size / volume, and experimental reagents, such as solvents, catalysts, pressures, atmospheric conditions, e.g., nitrogen atmosphere, and reducing / oxi dizing agents, with art-recognized alternatives and using no more than routine experimentation, are within the scope of the present application.

[0274] It is to be understood that wherever values and ranges are provided herein, all values and ranges encompassed by these values and ranges, are meant to be encompassed within the scope of the present disclosure. Moreover, all values that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the present application.

[0275] The following examples further illustrate aspects of the present disclosure. However, they are in no way a limitation of the teachings or disclosure of the present disclosure as set forth herein.

[0276] EXPERIMENTAL EXAMPLES

[0277] The disclosure is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only and the disclosure should in no way be construed as being limited to these Examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0278] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present disclosure and practice the claimed methods. The following working examples therefore, point out specific embodiments of the present disclosure, and are not to be construed as limiting in any way the remainder of the disclosure.

[0279] The materials and methods used in the experiments presented in this Experimental Example are now described.

[0280] Example 1: Suppression of endogenous glucose production by injectable HDV-Insulin Lispro: A dose response study in human subjects with type 1 diabetesAtorney Docket No. 047589-5026WO1 (00244)

[0281] A Single-center, double-blind, random-sequence study assessing the HDV doseresponse relationship to Endogenous Glucose Production (EGP), Free Fatty Acids (FFA) and Glucose Disposal Rate (GDR) during a euglycemic clamp procedure following overnight stabilization of blood glucose with intravenous insulin (and, if needed intravenous glucose). EGP and GDR will be determined using established radioisotope methodology. The concentration of Hepatic Directed Vesicles (HDV) in the insulin lispro (LIS) infused during the clamp procedure will be varied such that the percentage of HDV-bound LIS will range from 0%, 1%, 10%, and 100%.

[0282] Each participant will thus undergo four clamp procedures at the four different HDV levels. LIS will be infused at a constant dose (6 mU / m2 / min) for each of the four procedures.

[0283] Non-limiting objectives:

[0284] • To establish the dose-response relationship between HDV and endogenous glucose production at a constant dose of insulin lispro (LIS) during euglycemic clamp procedure.

[0285] • To establish the dose-response relationship between HDV and peripheral free faty acid (FFA) concentration at a constant dose of LIS during euglycemic clamp procedure.

[0286] • To establish the dose-response relationship between HDV and glucose disposal rate (GDR) at a constant dose of LIS during euglycemic clamp procedure.

[0287] • To confirm safety and tolerability of HDV-L across a range of HDV concentrations.

[0288] Non-limiting endpoints:

[0289] Endpoint EGP, %EGP, endpoint FFA, %FFA change, GDR and %GDR change will be calculated from baseline (start of infusion at t = 0) using the average of the last two measurements of the infusion as endpoint. EGP, FFA and GDR will be compared from baseline to endpoint by pairwise t-test. Dose response for HDV will be plotted using endpoint data from each of the four clamp procedures.

[0290] Study population:

[0291] Total of up to 18 subjects with Type 1 diabetes may be recruited. Male or female of age 18 to 65 years, inclusive, who:

[0292] • Has at Screening been diagnosed as T1DM for at least 12 months;

[0293] • Has at Screening C-peptide <0.8 ng / mL (single retest allowed);

[0294] • Has at Screening a BMI >18.0 kg / m2 and <33.0 kg / m2;Atorney Docket No. 047589-5026WO1 (00244)

[0295] • Has at Screening HbAlc >6.5% and <8.5 %.

[0296] Recruited participants will refrain from using basal insulin injections for at least 24 hours before the clamp procedures; preference will be given to potential participants who use insulin pump therapy since this eliminates the need for long-acting insulin injections. During the clamp procedure, participants will receive a fixed, constant dose of intravenous insulin either alone or in combination with varying concentrations of HDV. Other study medications will include intravenous glucose with radiotracer labeling, intravenous unlabeled glucose, and lispro. Including the screening period, each participant will be enrolled for a maximum of 10 weeks

[0297] In certain embodiments, the observed reductions in hypoglycemia risk observed in ISLE-1 and OPTI-1 result from the hepato-preferential effects of HDV-L as compared to LIS alone. By providing adequate insulin to hepatocytes, the liver is able to take up and store mealtime carbohydrate intake as glycogen, which is then more readily available for the prevention of hypoglycemia that may occur in the post-absorptive state. In certain nonlimiting embodiments, the present study is designed to support this hypothesis by demonstrating a hepato-preferential metabolic effect (i.e. suppression of endogenous glucose production) of HDV-L as compared to LIS alone during a eugly cemic clamp procedure in persons with T1D.

[0298] In certain non-limiting embodiments, the primary endpoint for this study is change in EGP. From previous literature, doses of 4 and 8 mU / m2 / min of insulin infusion suppressed EGP by approximately 12.5% and 40% respectively. In certain non-limiting embodiments, the goal for initial EGP suppression (without HDV added) is -20%. Therefore, an insulin infusion rate of 6 mU / m2 / min was chosen for all subjects as this dose is halfway between 4-8 and should achieve a roughly 20% suppression in EGP. The concentration of HDV in the LIS infused during the clamp procedure will be varied such that the percentage of HDV-bound LIS will range from 0%, 1%, 10%, and 100%. Each participant will thus undergo four clamp procedures at the four different HDV levels for this dose finding study.

[0299] In certain non-limiting embodiments, a participant is considered to have completed the study if he or she has completed all phases of the study including the last visit or the last scheduled procedure shown in the Schedule of Activities (SoA). In certain non-limiting embodiments, the end of the study is defined as completion of the last visit or procedure shown in the SoA in the trial at our site.

[0300] Study PopulationAtorney Docket No. 047589-5026WO1 (00244)

[0301] Total of up to 18 subjects may be recruited, with a goal for 14 subjects completing all four clamp procedures.

[0302] Inclusion criteria

[0303] Male or female of age 18 to 65 years, inclusive, who:

[0304] • Has at Screening been diagnosed as T1D for at least 12 months; preference will be given to potential

[0305] • participants who currently use continuous subcutaneous insulin infusion (CSII) therapy;

[0306] • Has at Screening C-peptide <0.8 ng / mL (single retest allowed);

[0307] • Has at Screening a BMI >18.0 kg / m2and <33.0 kg / m2;

[0308] • Has at Screening HbAlc >6.5% and <8.5 %.

[0309] Exclusion criteria:

[0310] Potential participant who:

[0311] • Has known or suspected allergy to any component of any of the study drugs in this trial;

[0312] • Is, at Screening, pregnant or breast-feeding, or intends to become pregnant at any time during the duration of the study;

[0313] • Has, at Screening, as judged by the Site Investigator, a history or current evidence of any of advance complications of diabetes;

[0314] • Is, at Screening, judged by the Site Investigator to have a current addiction to alcohol or substances of abuse;

[0315] • Is, at Screening, using one or more drugs that may interfere with the interpretation of trial results or are known to cause clinically relevant interference with insulin action, glucose utilization, or recovery from hypoglycemia (e.g., beta blockers, systemic corticosteroids at pharmacologic doses, cancer chemotherapies);

[0316] • Has, within one (1) month prior to Screening, used either oral anti-diabetic medication or noninsulin anti-diabetic injection therapies (e.g. SGLT-2 inhibitors, pramlintide, GLP-1 agonists, etc.);

[0317] • Has, within one (1) month prior to Screening, received any investigational drug;

[0318] • Has, within three (3) months prior to Screening, smoked tobacco or used any smokeless tobacco or nicotine delivery system (inhaled, oral or buccal);

[0319] • Has at Screening, as judged by the Site Investigator, any condition (intrinsic or extrinsic) that could reasonably be expected to interfere with trial participation,Atorney Docket No. 047589-5026WO1 (00244)

[0320] confound evaluation of the data, or pose additional risk to adhering to the study protocol. Examples of such conditions include but are not limited to:

[0321] • Clinically significant active disease of the gastrointestinal, cardiovascular, hepatic, neurological, renal, genitourinary, or hematological systems;

[0322] • History of such an illness or disease;

[0323] • Diminished mental capacity, psychological or behavioral dysfunction, unwilling or resistant to protocol requirements, language barriers. Lifestyle Considerations

[0324] Excluded concomitant medication and activities:

[0325] • Insulin and insulin analogs (except as specified under this protocol)

[0326] • Corticosteroids and anabolic steroids, unless used for topical or intraarticular use • Nicotine-containing products, including e-cigarette products

[0327] • Initiation of weight loss medications or therapies during study.

[0328] Dosing and Administration

[0329] The concentration of HDV in the LIS infused during the clamp procedure will be varied such that the percentage of HDV-bound LIS will range from 0%, 1%, 10%, and 100%. Each participant will thus undergo four clamp procedures at the four different HDV levels. LIS will be infused at a constant dose (6 mU / m2 / min) for each of the four procedures. Route of Administration: IV infusion

[0330] As an additive to commercial insulin vials, 0.8ml of liquid (equivalent to lOmg) HDV is provided in 3ml glass vials and is aseptically added by pharmacists or other trained healthcare professional at clinical sites on a once-per-vial basis.

[0331] After the HDV is added to the Insulin, it is to be stored in refrigerator or room temperature (2-25°C).

[0332] A properly qualified study site staff member or pharmacist will prepare the study drug admixture by adding HDV additive (0.8 mL) to commercially obtained vials of insulin lispro (U100, lOmL). LIS will be similarly diluted with sterile water to equalize the insulin concentration (approximately 93 U / mL) in both study drugs.

[0333] Double-blind randomization block size of 4 will be used. The concentration of HDV in the LIS infused during the clamp procedure will be varied such that the percentage of HDV-bound LIS will range from 0%, 1%, 10%, and 100%. Each participant will be randomly assigned during the clamp procedures at the four different HDV levels (0%, 1%,Atorney Docket No. 047589-5026WO1 (00244)

[0334] 10%, and 100%). LIS will be infused at a constant dose (6 mU / m2 / min) for each of the four procedures.

[0335] For this protocol, a prescription medication is defined as a medication that can be prescribed only by a properly authorized / licensed clinician. Medications to be reported in the Case Report Form (CRF) are concomitant prescription medications, over-the-counter medications and supplements. Excluded medications include:

[0336] • Insulin and insulin analogs (except as specified under this protocol)

[0337] • Corticosteroids and anabolic steroids, unless used for topical or intraarticular use • Nicotine-containing products, including e-cigarette products

[0338] • Initiation of weight loss medications or therapies during study.

[0339] Study Assessments and Procedures: Efficacy Assessments

[0340] Randomization

[0341] Double-blind randomization block size of 4 will be used. The concentration of HDV in the LIS infused during the clamp procedure will be varied such that the percentage of HDV-bound LIS will range from 0%, 1%, 10%, and 100%. Each participant will be randomly assigned during the clamp procedures at the four different HDV levels (0%, 1%, 10%, and 100%).

[0342] Duration of Participation

[0343] 2 weeks screening period followed by 4-8 weeks for completion of four clamp procedures with 7-14 days washout between clamps. This means the total participation is up to 10 weeks.

[0344] Procedures

[0345] The study has a screening period followed by four euglycemic glucose clamp procedures (including isotope-labeled glucose tracers to allow calculation of EGP and GDR. Each clamp will be preceded by an overnight in-clinic stabilization period and will be followed by a 7-14 day washout period until the next clamp.

[0346] • Glucose Clamp Procedure

[0347] Following successful screening, participants who use injectable basal insulin will be advised to discontinue basal injections for 24 hours prior to each clamp procedure, using only rapid-acting analog insulin to control diabetes prior to clinic presentation.

[0348] • Overnight Glucose StabilizationAtorney Docket No. 047589-5026WO1 (00244)

[0349] Prior to visits 1-4, participants will be admitted to the research clinic to complete an overnight glucose stabilization. Participants will arrive at approximately 5:00 pm for a single overnight stay to monitor blood glucose levels until the start of the euglycemic clamp procedure the following morning.

[0350] Upon arrival, the following assessments will be completed: vital signs, weight, urine pregnancy test (for female participants of child-bearing potential), CGM download and assessment of adverse events. At approximately 6:00 pm a standardized dinner will be provided and the participant will initiate a typical bolus for the carbohydrate / protein / fat content of the meal.

[0351] A research nurse will place two intravenous (IV) lines. One IV line will be for the overnight infusion of insulin and glucose (20% dextrose), as needed. The other IV will be for drawing small blood samples, in order to monitor subject’s blood sugar levels every hour, with the goal of maintaining overnight blood glucose in the range of 120-140 mg / dL.

[0352] • Participants Using Continuous Subcutaneous Insulin Infusion Therapy (Insulin Pump)

[0353] At approximately 7:00 pm the participants will suspend and disconnect their insulin pump and a standard insulin drip will begin. A catheter placed into antecubital or forearm area vein with a 3 -tailed Y-connector will be used. FIG. X contains the algorithm that will be followed to maintain overnight euglycemia (90-120 mg / dL). As noted on the algorithm, pump participants will start the initial rate of insulin at U standard (column 4) based on their blood glucose level at that time.

[0354] • Participants Using Multiple Daily Injection Therapy (MDI)

[0355] At approximately 7:00 pm a standard insulin drip will begin based on the participant’s usual injection of basal insulin. For participants who inject basal insulin in the evening, the initial rate of insulin will be % standard (column 3) based on their blood glucose level at that time. For participants who inject in the morning, the initial rate of insulin will be % standard (column 2) based on their blood glucose level at that time.

[0356] For all participants, plasma glucose will be measured hourly via Yellow Springs Instrument (YSI) biochemistry analyzer and the results will dictate the rate of insulin infusion based on the algorithm. In addition to the algorithm information, the present disclosure also contains protocols for hypoglycemia and hyperglycemia should they occur during the overnight glucose stabilization.

[0357] • Euglycemic Glucose ClampAtorney Docket No. 047589-5026WO1 (00244)

[0358] Following the overnight glucose stabilization, a euglycemic glucose clamp will be initiated.

[0359] At -05:30 hr, subject will be asked to empty bladder and discard urine. Attach INF infusate to Y-connector port. Take blood samples for measurement of background tracer glucose, serum creatinine, urea and uric acid. A primed continuous infusion of 6,6-2H2-glucose (tracer) in normal saline will be started and maintained throughout the procedure to target a 2% enrichment. The insulin infusion will be continued and set at a constant rate of 6 mU / m2 / min for 3 hours.

[0360] At -180 min, -06:00 hr, begin the INF infusion at flow rate determined on clamp sheet. Continue for duration of procedure. At t = -90 min, Draw 1 mL of INF from sample port.

[0361] Attach GINF and insulin (HDV-Lispro) to Y-connector ports. Timing and number of blood samples collected throughout the procedure are detailed in Appendix.

[0362] At t = 0 min, Start priming insulin infusion. At t = 0 min to t= 10 min, adjust insulin infusion rate according to the algorithm detailed in Clamp Flowsheet.

[0363] At t= 10 min (after completion of the priming of the insulin infusion), start GINF infusion when necessary to maintain target BG. Blood will be collected at 10 min intervals for real-time determination of BG. At t = -60 min, draw 1 mL of the GINF from sample port. At t = 180 min, stop the insulin infusion. At approximately 9:00am the single-step euglycemic glucose clamp will begin with a constant insulin (insulin containing HDV at 0, 1, 10 or 100% saturation level) infusion rate of 6 mU / m2 / min and be maintained for 3 hours. A variable infusion of 20% Dextrose (D2O) containing 6,6-2H2-glucose (tracer) will be adjusted to maintain blood glucose values at -100 mg / dL as measured by YSI at 10 minute intervals. Blood samples for stable isotope analyses will be collected immediately before the start of the insulin infusion and then every 30 minutes for the first 2 hours. Beginning at the 150 minute mark, samples will be collected every 10 minutes until the 180 minute mark. After 180 minutes the insulin infusion will be discontinued and the participant will begin a recovery period.

[0364] During the recovery period the D2O GINF infusion will continue while the participant is provided a post-procedure meal. Once the meal is complete and blood glucose is above 100 mg / dL and deemed stable by a study investigator, the IVs will be removed and the participant will be discharged from the research clinic.

[0365] Other AssessmentsAtorney Docket No. 047589-5026WO1 (00244)

[0366] Prospective subjects will be provided with informed consent and then screened for inclusion and exclusion criteria. No more than 14 days later, qualified subjects will be entered into the study. The following safety and eligibility procedures will occur during that time:

[0367] • Informed Consent - Participants will provide written informed consent prior to any other study procedures.

[0368] • Physical Exam, Medical History and Study Eligibility - A study investigator will complete an assessment of general appearance and a review of systems (dermatologic, head, eyes, ears, nose, mouth / throat / neck, thyroid, lymph nodes, respiratory, cardiovascular, gastrointestinal, extremities, musculoskeletal, neurologic, and psychiatric systems). In addition, at study investigator will verbally review all medical history, medications and the inclusion / exclusion criteria with the participants.

[0369] • Vital Signs - These include height, weight, blood pressure, heart rate, temperature and respiratory rate. Vitals signs will be measured to monitor safety and eligibility.

[0370] • Electrocardiogram (ECG) - A 12-lead ECG will be performed for screening eligibility purposes only. A study investigator will read and interpret the ECG.

[0371] • Specimen Collection - Blood will be collected via standard venipuncture by a study nurse for safety evaluations including: Complete Blood Count (CBC), Comprehensive Metabolic Panel (CMP) and Hemagiobin Ale (HbAlc). The laboratory samples will be sent for analysis on the same day as collection and results will be reported through their web portal within one week. A study investigator will review all laboratory reports and record significance of any abnormal value. The volume of blood collected at the screening visit is approximately 12 mL or 2.5 teaspoons.

[0372] Urine will be collected from all participants and a urine dip stick urinalysis will be completed for safety evaluation at screening and at final visit (Visit 4 or ET). The dipstick will be done at the ACTRI clinic laboratory by trained study staff. The ACTRI clinic has an up-to-date CLIA certificate.

[0373] In addition, individuals who are able to have children will have a urine pregnancy test completed, also done at the ACTRI clinic laboratory, at every visit.

[0374] During overnight glucose stabilization period, bedside blood glucose is checked as outlined elsewhere herein in order to stabilize blood glucose between 120-140 mg / dL.Attorney Docket No. 047589-5026WO1 (00244)

[0375] During Clamp procedure, bedside blood glucose is checked by a nurse via YSI every 5-10 minutes as needed, in order to keep subject euglycemic (-100 mg / dL).

[0376] Table 1: Laboratory Assessments

[0377]

[0378] The total amount of blood collected during the course of the entire study will be approximately 612 mL (about 41 tablespoons) over a period of about 10 weeks. Subjects will be advised not to donate any additional blood during the study, or for 60 days after completing the study.

[0379] Study subjects will also be asked to consent to optional long-term storage of additional blood samples collected at visits 1-4. An extra 10 ml of blood will be collected at each of the visits. This will be a total of 40 ml of blood (2.7 tablespoons).

[0380] Statistical Considerations

[0381] Statistical Hypothesis: Endpoints

[0382] • To establish the dose-response relationship between Hepatic-Directed Vesicles (HDV) and endogenous glucose production at a constant dose of insulin lispro (LIS) during euglycemic clamp procedure.Atorney Docket No. 047589-5026WO1 (00244)

[0383] • To establish the dose-response relationship between Hepatic-Directed Vesicles (HDV) and peripheral free fatty acid (FFA) concentration at a constant dose of LIS during euglycemic clamp procedure.

[0384] • To establish the dose-response relationship between HDV and glucose disposal rate (GDR) at a constant dose of LIS during euglycemic clamp procedure.

[0385] Statistical Analyses

[0386] The primary endpoint for this study is change in EGP. Doses of 4 and 8 mU / m2 / min of insulin infusion suppressed EGP by approximately 12.5% and 40% respectively. In certain non-limiting embodiments, the goal for initial EGP suppression (without HDV added) is -20%. Therefore, an insulin infusion rate of 6 mU / m2 / min was chosen for all subjects as this dose is halfway between 4-8 and should achieve a roughly 20% suppression in EGP. As the study is a dose finding study to evaluate the effects of additional doses of HDV, no formal power analysis was done. In certain non-limiting embodiments, 14 subjects are sufficient to demonstrate differential hepatic effects of the various doses of HDV.

[0387] Analysis of secondary endpoint(s)

[0388] To establish the dose-response relationship between HDV and peripheral free faty acid (FFA) concentration at a constant dose of LIS during euglycemic clamp procedure: To evaluate this secondary outcome, we will measure FFA concentrations at bassline (prior to clamp procedure) and during the final minutes of the clamp. FFA suppression will be calculated as % reduction of FFA concentration at the completion of the clamp compared to baseline values.

[0389] To establish the dose-response relationship between HDV and glucose disposal rate (GDR) at a constant dose of LIS during euglycemic clamp procedure: To evalute this outcome, we will calculate GDR for each patient during each clamp procedure. Briefly, this is calculated using the glucose infusion rate during the final 30 minutes of the clamp procedure and concerting to a weight based glucose disposal rate per minute (mg / Kg / min). This value will be averaged for all patients per dose of HDV and then compared to one another. Therefore, we will calculate an average GDR for 0% HDV, 1% HDV, 10% HDV, and 100% HDV bound insulin.Attorney Docket No. 047589-5026WO1 (00244)

[0390] Table 2: Non-Limiting Abbreviations

[0391] &

[0392]

[0393] Enumerated Embodiments:

[0394] The following exemplary embodiments are provided, the numbering of which is not to be construed as designating levels of importance.

[0395] Embodiment 1 : A pharmaceutical composition comprising an insulin and a lipid-based nanoparticle, wherein the lipid-based nanoparticle is a Hepatic Directed Vesicle (HDV), wherein at least a fraction of the insulin is dispersed within the HDV; wherein theAtorney Docket No. 047589-5026WO1 (00244)

[0396] percentage of HDV-dispersed insulin as compared to the total insulin in the pharmaceutical composition ranges from about 0.1% to about 100%; wherein the lipid nanoparticle is enclosed by a bipolar lipid membrane comprising cholesterol, dicetyl phosphate, an amphipathic lipid, and a hepatocyte receptor binding molecule; wherein the amphipathic lipid comprises at least one selected from the group consisting of l,2-distearoyl-sn-glycero-3-phosphocholine, l,2-dipalmitoyl-sn-glycerol-[3-phospho-rac-(l-glycerol)], 1,2-distearoyl-sn-glycero-3 -phosphoethanolamine, l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-( succinyl), 1 ,2-dimyri stoyl-sn-gly cero-3 -phosphate, 1 ,2-dimyri stoyl-sn-gly cero-3 -phosphocholine, l,2-distearoyl-sn-glycero-3-phosphate, l,2-dipalmitoyl-sn-glycero-3-phosphate, and 1,2-dipalmitoyl-sn-gly cero-3 -phosphocholine; wherein the at least one hepatocyte receptor binding molecule extends outward from the nanoparticle; and wherein the size of the nanoparticle ranges from about 10 nm to about 200 nm.

[0397] Embodiment 2: The pharmaceutical composition of embodiment 1, wherein the percentage of HDV-dispersed insulin as compared to the total insulin in the pharmaceutical composition is about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 20.5%, 21%, 21.5%, 22%, 22.5%, 23%, 23.5%, 24%, 24.5%, 25%, 25.5%, 26%, 26.5%, 27%, 27.5%, 28%, 28.5%, 29%, 29.5%, 30%, 30.5%, 31%, 31.5%, 32%, 32.5%, 33%, 33.5%, 34%, 34.5%, 35%, 35.5%, 36%, 36.5%, 37%, 37.5%, 38%, 38.5%, 39%, 39.5%, 40%, 40.5%, 41%, 41.5%, 42%, 42.5%, 43%, 43.5%, 44%, 44.5%, 45%, 45.5%, 46%, 46.5%, 47%, 47.5%, 48%, 48.5%, 49%, 49.5%, 50%, 50.5%, 51%, 51.5%, 52%, 52.5%, 53%, 53.5%, 54%, 54.5%, 55%, 55.5%, 56%, 56.5%, 57%, 57.5%, 58%, 58.5%, 59%, 59.5%, 60%, 60.5%, 61%, 61.5%, 62%, 62.5%, 63%, 63.5%, 64%, 64.5%, 65%, 65.5%, 66%, 66.5%, 67%, 67.5%, 68%, 68.5%, 69%, 69.5%, 70%, 70.5%, 71%, 71.5%, 72%, 72.5%, 73%, 73.5%, 74%, 74.5%, 75%, 75.5%, 76%, 76.5%, 77%, 77.5%, 78%, 78.5%, 79%, 79.5%, 80%, 80.5%, 81%, 81.5%, 82%, 82.5%, 83%, 83.5%, 84%, 84.5%, 85%, 85.5%, 86%, 86.5%, 87%, 87.5%, 88%, 88.5%, 89%, 89.5%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100%.

[0398] Embodiment 3: The pharmaceutical composition of any one of embodiments 1-2, wherein the membrane further comprises at least one agent selected from the group consisting of a stabilizer and stearoyl lysophosphatidylcholine.Atorney Docket No. 047589-5026WO1 (00244)

[0399] Embodiment 4: The pharmaceutical composition of embodiment 3, wherein the stabilizer is selected from the group consisting of m-cresol, benzyl alcohol, methyl 4-hydroxybenzoate, thiomersal, and butylated hydroxytoluene (2,6-di- / c / 7-butyl-4-m ethylphenol).

[0400] Embodiment 5: The pharmaceutical composition of any one of embodiments 3-4, wherein the stabilizer ranges from about 0.5 % to about 25 % (w / w) in the membrane.

[0401] Embodiment 6: The pharmaceutical composition of embodiment 3, wherein the stearoyl lysophosphatidylcholine ranges from about 1% to about 30% (w / w) in the membrane.

[0402] Embodiment 7: The pharmaceutical composition of any one of embodiments 1-6, wherein the insulin is covalently bound to the lipid-based nanoparticle.

[0403] Embodiment 8: The pharmaceutical composition of any one of embodiments 1-6, wherein the insulin is not covalently bound to the lipid-based nanoparticle.

[0404] Embodiment 9: The pharmaceutical composition of any one of embodiments 1-8, wherein the insulin is suspended in an aqueous solution comprising a free dissolved insulin that is not dispersed within the lipid-based nanoparticle.

[0405] Embodiment 10: The pharmaceutical composition of any one of embodiments 1-9, wherein the HDV-dispersed insulin and the free dissolved insulin are independently selected from the group consisting of insulin lispro, insulin aspart, regular insulin, insulin glargine, insulin zinc, extended human insulin zinc suspension, isophane insulin, human buffered regular insulin, insulin glulisine, recombinant human regular insulin, recombinant human insulin isophane, insulin detemir, biphasic human insulin, and insulin deglude, and any combinations thereof.

[0406] Embodiment 11 : The pharmaceutical composition of any one of embodiments 1-10, wherein the amphipathic lipid comprises at least one selected from the group consisting of 1.2-distearoyl-sn-glycero-3-phosphocholine, l,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1.2-dipalmitoyl-sn-glycero-3-[phospho-rac-(l-glycerol)], l,2-distearoyl-sn-glycero-3-phosphoethanolamine, and l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl).

[0407] Embodiment 12: The pharmaceutical composition of any one of embodiments 1-11, wherein the hepatocyte receptor binding molecule comprises biotin.

[0408] Embodiment 13: The pharmaceutical composition of embodiment 12, wherein the biotin-containing hepatocyte receptor binding molecule comprises at least one selected from the group consisting of N-hydroxy succinimide (NHS) biotin; sulfo-NHS-biotin; N-hydroxysuccinimide long chain biotin; sulfo-N-hydroxysuccinimide long chain biotin; D-Atorney Docket No. 047589-5026WO1 (00244)

[0409] biotin; biocytin; sulfo-N-hydroxysuccinimide-S-S-biotin; biotin-BMCC; biotin-HPDP; iodoacetyl-LC-biotin; biotin-hydrazide; biotin-LC-hydrazide; biocytin hydrazide; biotin cadaverine; carboxybiotin; photobiotin; / ?-aminobenzoyl biocytin trifluoroacetate; p-diazobenzoyl biocytin; biotin DHPE (2,3-diacetoxypropyl 2-(5-((3aS,6aR)-2-oxohexahydro-lH-thieno[3,4-d]imidazol-4-yl)pentanamido)ethyl phosphate); biotin-X-DHPE (2,3-diacetoxypropyl 2-(6-(5-((3aS,6aR)-2-oxohexahydro-lH-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido) ethyl phosphate); 12-((biotinyl)amino)dodecanoic acid; 12-((biotinyl)amino)dodecanoic acid succinimidyl ester; S-biotinyl homocysteine; biocytin-X; biocytin x-hydrazide; biotinethylenediamine; biotin-XL; biotin-X-ethylenediamine; biotin-XX hydrazide; biotin-XX-SE; biotin-XX, SSE; biotin-X-cadaverine; a-(t-BOC)biocytin; N-(biotinyl)-N’ -(iodoacetyl) ethylenediamine; DNP-X-biocytin-X-SE; biotin-X-hydrazide; norbiotinamine hydrochloride; 3-(N-maleimidylpropionyl)biocytin; ARP; biotin-l-sulfoxide; biotin methyl ester; biotin-maleimide; biotin-poly(ethyleneglycol) amine; (+) biotin 4-amidobenzoic acid sodium salt; Biotin 2-N-acetylamino-2-deoxy-P-D-glucopyranoside; Biotin-a-D-N-acetylneuraminide; Biotin-a-L-fucoside; Biotin lacto-N-bioside; Biotin-Lewis-A trisaccharide; Biotin-Lewis-Y tetrasaccharide; Biotin-a-D-mannopyranoside; and biotin 6-O-phospho-a-D-mannopyranoside.

[0410] Embodiment 14: The pharmaceutical composition of embodiment 12, wherein the biotin-containing hepatocyte receptor binding molecule comprises at least one selected from the group consisting of biotin DHPE and biotin-X-DHPE.

[0411] Embodiment 15: The pharmaceutical composition of any one of embodiments 1-14, wherein the composition further comprises cellulose acetate phthalate, which is at least partially bound to the insulin dispersed within the lipid-based nanoparticle.

[0412] Embodiment 16: The pharmaceutical composition of any one of embodiments 1-15, wherein the composition further comprises at least one charged organic molecule bound to the insulin dispersed within the lipid-based nanoparticle, wherein the charged organic molecule is at least one selected from the group consisting of protamines, polylysine, poly (arg-pro-thr)« in a mole ratio of 1 : 1 : 1, poly (DL-Ala-poly-L-lys)n in a mole ratio of 6: 1, histones, sugar polymers comprising a primary amino group, polynucleotides with primary amino groups, proteins comprising amino acid residues with carboxyl (COO ) or sulfhydral (S") functional groups, and acidic polymers.

[0413] Embodiment 17: The pharmaceutical composition of any one of embodiments 1-16, wherein the cholesterol ranges from about 5% to about 25% (w / w) in the membrane.Atorney Docket No. 047589-5026WO1 (00244)

[0414] Embodiment 18: The pharmaceutical composition of any one of embodiments 1-17, wherein the dicetyl phosphate ranges from about 10% to about 25% (w / w) in the membrane.

[0415] Embodiment 19: The pharmaceutical composition of any one of embodiments 1-18, wherein the DSPC ranges from about 40% to about 75% (w / w) in the membrane.

[0416] Embodiment 20: The pharmaceutical composition of any one of embodiments 1-19, wherein the hepatocyte receptor binding molecule ranges from about 0.5% to about 10% (w / w) in the membrane.

[0417] Embodiment 21 : The pharmaceutical composition of any one of embodiments 3-20, wherein the amount of the stearoyl lysophosphatidylcholine in the membrane is about 5%-30% (w / w) of the amount of DSPC in the membrane.

[0418] Embodiment 22: The pharmaceutical composition of any one of embodiments 3-21, wherein the membrane comprises one of the following:

[0419] (a) cholesterol, dicetyl phosphate, DSPC, stearoyl lysophosphatidylcholine, m- cresol, and at least one selected from the group consisting of biotin DHPE and biotin-X-DHPE;

[0420] (b) cholesterol, dicetyl phosphate, DSPC, m-cresol, and at least one selected from the group consisting of biotin DHPE and biotin-X-DHPE; and (c) cholesterol, dicetyl phosphate, DSPC, stearoyl lysophosphatidylcholine, and at least one selected from the group consisting of biotin DHPE and biotin-X- DHPE.

[0421] Embodiment 23: The pharmaceutical composition of any one of embodiments 3-22, wherein the membrane comprises cholesterol, dicetyl phosphate, DSPC, stearoyl lysophosphatidylcholine, m-cresol, and biotin DHPE in a % (w / w) ratio selected from the group consisting of:

[0422] (a) about 9.4 : 18.1 : 56.8 : 14.1 : 0.0 : 1.5;

[0423] (b) about 7.7 : 15.0 : 58.6 : 0.0 : 17.4 : 1.3; and

[0424] (c) about 8.4 : 16.2 : 47.5 : 7.6 : 19.0 : 1.3.

[0425] Embodiment 24: A method of treating or ameliorating diabetes in a subject suffering therefrom, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition of any one of embodiments 1-23.

[0426] Embodiment 25: A method of modulating peripheral lipid metabolism and hepatic lipid metabolism in a subject suffering from diabetes, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition of any one of embodiments 1-23.Atorney Docket No. 047589-5026WO1 (00244)

[0427] Embodiment 26: The method of embodiment 25, wherein the administering allows for the subject to achieve liver and / or plasma triglyceride levels that are closer to normal physiological levels than before the subject was subjected to the administering.

[0428] Embodiment 27: The method of embodiment 25, wherein the administering allows for the subject to achieve liver and / or plasma free fatty acid levels that are closer to normal physiological levels than before the subject was subjected to the administering.

[0429] Embodiment 28: The method of embodiment 25, wherein the administering allows for the subject to achieve liver and / or plasma branched-chain amino acid (BCAA) levels that are closer to normal physiological levels than before the subject was subjected to the administering.

[0430] Embodiment 29: The method of embodiment 25, wherein the administering allows for the subject to achieve hepatic and / or peripheral glucose storage levels that are closer to normal physiological levels than before the subject was subjected to the administering.

[0431] Embodiment 30: A method of preventing, ameliorating, minimizing, and / or reversing hepatic accumulation of lipids (fatty liver) in a subject suffering from diabetes, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition of any one of embodiments 1-23.

[0432] Embodiment 31 : The method of any one of embodiments 24-30, wherein the subject has about 6.5-8.5% A1C.

[0433] Embodiment 32: The method of any one of embodiments 24-30, wherein the subject has greater than about 8.5% Al C.

[0434] Embodiment 33: The method of any one of embodiments 24-32, wherein the subject experiences fewer hypoglycemia as compared to the treatment without HDV.

[0435] Embodiment 34: The method of any one of embodiments 24-33, wherein the subject experiences weight loss as compared to the treatment without HDV.

[0436] Embodiment 35: The method of any one of embodiments 24-34, wherein the subject does not experience significant iatrogenic hyperinsulinemia.

[0437] Embodiment 36: The method of any one of embodiments 24-35, wherein the pharmaceutical composition comprises basal insulin and is administered continuously to the subject over a period of at least 24 hours.

[0438] Embodiment 37: The method of any one of embodiments 24-36, wherein the pharmaceutical composition is administered continuously to the subject using a pump.

[0439] Embodiment 38: The method of any one of embodiments 24-37, wherein the subject has Type 1 diabetes, Type 2 diabetes, and / or a metabolic derangement.Attomey Docket No. 047589-5026WO1 (00244)

[0440] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this disclosure has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this disclosure may be devised by others skilled in the art without departing from the true spirit and scope of the disclosure. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

Attorney Docket No. 047589-5026WO1 (00244)CLAIMSWhat is claimed is:

1. A pharmaceutical composition comprising an insulin and a lipid-based nanoparticle, wherein the lipid-based nanoparticle is a Hepatic Directed Vesicle (HDV), wherein at least a fraction of the insulin is dispersed within the HDV;wherein the percentage of HDV-dispersed insulin as compared to the total insulin in the pharmaceutical composition ranges from about 0.1% to about 100%;wherein the lipid nanoparticle is enclosed by a bipolar lipid membrane comprising cholesterol, dicetyl phosphate, an amphipathic lipid, and a hepatocyte receptor binding molecule;wherein the amphipathic lipid comprises at least one selected from the group consisting of l,2-distearoyl-sn-glycero-3-phosphocholine, l,2-dipalmitoyl-sn-glycerol-[3-phospho-rac-(l -glycerol)], l,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl), l,2-dimyristoyl-sn-glycero-3-phosphate, l,2-dimyristoyl-sn-glycero-3-phosphocholine, l,2-distearoyl-sn-glycero-3-phosphate, l,2-dipalmitoyl-sn-glycero-3 -phosphate, and l,2-dipalmitoyl-sn-glycero-3-phosphocholine;wherein the at least one hepatocyte receptor binding molecule extends outward from the nanoparticle; andwherein the size of the nanoparticle ranges from about 10 nm to about 200 nm.

2. The pharmaceutical composition of claim 1, wherein the percentage of HDV-dispersed insulin as compared to the total insulin in the pharmaceutical composition is about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 20.5%, 21%, 21.5%, 22%, 22.5%, 23%, 23.5%, 24%, 24.5%, 25%, 25.5%, 26%, 26.5%, 27%, 27.5%, 28%, 28.5%, 29%, 29.5%, 30%, 30.5%, 31%, 31.5%, 32%, 32.5%, 33%, 33.5%, 34%, 34.5%, 35%, 35.5%, 36%, 36.5%, 37%, 37.5%, 38%, 38.5%, 39%, 39.5%, 40%, 40.5%, 41%, 41.5%, 42%, 42.5%, 43%, 43.5%, 44%, 44.5%, 45%, 45.5%, 46%, 46.5%, 47%, 47.5%, 48%, 48.5%, 49%, 49.5%, 50%, 50.5%, 51%, 51.5%, 52%, 52.5%, 53%, 53.5%, 54%, 54.5%, 55%, 55.5%, 56%, 56.5%, 57%, 57.5%, 58%, 58.5%, 59%, 59.5%, 60%, 60.5%, 61%, 61.5%, 62%, 62.5%, 63%, 63.5%,Attorney Docket No. 047589-5026WO1 (00244)64%, 64.5%, 65%, 65.5%, 66%, 66.5%, 67%, 67.5%, 68%, 68.5%, 69%, 69.5%, 70%, 70.5%, 71%, 71.5%, 72%, 72.5%, 73%, 73.5%, 74%, 74.5%, 75%, 75.5%, 76%, 76.5%, 77%, 77.5%, 78%, 78.5%, 79%, 79.5%, 80%, 80.5%, 81%, 81.5%, 82%, 82.5%, 83%, 83.5%, 84%, 84.5%, 85%, 85.5%, 86%, 86.5%, 87%, 87.5%, 88%, 88.5%, 89%, 89.5%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100%.

3. The pharmaceutical composition of any one of claims 1-2, wherein the membrane further comprises at least one agent selected from the group consisting of a stabilizer and stearoyl lysophosphatidylcholine.

4. The pharmaceutical composition of claim 3, wherein the stabilizer is selected from the group consisting of m-cresol, benzyl alcohol, methyl 4-hydroxybenzoate, thiomersal, and butylated hydroxytoluene (2,6-di- / c77-butyl-4-methylphenol).

5. The pharmaceutical composition of any one of claims 3-4, wherein the stabilizer ranges from about 0.5 % to about 25 % (w / w) in the membrane.

6. The pharmaceutical composition of claim 3, wherein the stearoyl lysophosphatidylcholine ranges from about 1% to about 30% (w / w) in the membrane.

7. The pharmaceutical composition of any one of claims 1-6, wherein the insulin is covalently bound to the lipid-based nanoparticle.

8. The pharmaceutical composition of any one of claims 1-6, wherein the insulin is not covalently bound to the lipid-based nanoparticle.

9. The pharmaceutical composition of any one of claims 1-8, wherein the insulin is suspended in an aqueous solution comprising a free dissolved insulin that is not dispersed within the lipid-based nanoparticle.

10. The pharmaceutical composition of any one of claims 1-9, wherein the HDV-dispersed insulin and the free dissolved insulin are independently selected from the group consisting of insulin lispro, insulin aspart, regular insulin, insulin glargine, insulin zinc,Attomey Docket No. 047589-5026WO1 (00244)extended human insulin zinc suspension, isophane insulin, human buffered regular insulin, insulin glulisine, recombinant human regular insulin, recombinant human insulin isophane, insulin detemir, biphasic human insulin, and insulin deglude, and any combinations thereof.

11. The pharmaceutical composition of any one of claims 1-10, wherein the amphipathic lipid comprises at least one selected from the group consisting of l,2-distearoyl-sn-glycero-3-phosphocholine, l,2-dipalmitoyl-sn-glycero-3 -phosphocholine, l,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(l -glycerol)], l,2-distearoyl-sn-glycero-3-phosphoethanolamine, and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl).

12. The pharmaceutical composition of any one of claims 1-11, wherein the hepatocyte receptor binding molecule comprises biotin.

13. The pharmaceutical composition of claim 12, wherein the biotin-containing hepatocyte receptor binding molecule comprises at least one selected from the group consisting of N-hydroxysuccinimide (NHS) biotin; sulfo-NHS-biotin; N-hydroxysuccinimide long chain biotin; sulfo-N-hydroxysuccinimide long chain biotin; D-biotin; biocytin; sulfo-N-hydroxysuccinimide-S-S-biotin; biotin-BMCC; biotin-HPDP; iodoacetyl-LC-biotin; biotinhydrazide; biotin-LC-hydrazide; biocytin hydrazide; biotin cadaverine; carboxybiotin; photobiotin; / ?-aminobenzoyl biocytin trifluoroacetate; p-diazobenzoyl biocytin; biotin DHPE (2,3 -diacetoxypropyl 2-(5-((3aS,6aR)-2-oxohexahydro-lH-thieno[3,4-d]imidazol-4-yl)pentanamido)ethyl phosphate); biotin-X-DHPE (2,3 -diacetoxypropyl 2-(6-(5-((3aS,6aR)-2-oxohexahydro-lH-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido) ethyl phosphate); 12-((biotinyl)amino)dodecanoic acid; 12-((biotinyl)amino)dodecanoic acid succinimidyl ester; S-biotinyl homocysteine; biocytin-X; biocytin x-hydrazide; biotinethylenediamine; biotin-XL; biotin-X-ethylenediamine; biotin-XX hydrazide; biotin-XX-SE; biotin-XX, SSE; biotin-X-cadaverine; a-(t-BOC)biocytin; N-(biotinyl)-N’ -(iodoacetyl) ethylenediamine; DNP-X-biocytin-X-SE; biotin-X-hydrazide; norbiotinamine hydrochloride; 3-(N-maleimidylpropionyl)biocytin; ARP; biotin-l-sulfoxide; biotin methyl ester; biotin-maleimide; biotin-poly(ethyleneglycol) amine; (+) biotin 4-amidobenzoic acid sodium salt; Biotin 2-N-acetylamino-2-deoxy-P-D-glucopyranoside; Biotin-a-D-N-acetylneuraminide; Biotin-a-L-fucoside; Biotin lacto-N-bioside; Biotin-Lewis-A trisaccharide; Biotin-Lewis-Y tetrasaccharide; Biotin-a-D-mannopyranoside; and biotin 6-O-phospho-a-D-mannopyranoside.Attomey Docket No. 047589-5026WO1 (00244)14. The pharmaceutical composition of claim 12, wherein the biotin-containing hepatocyte receptor binding molecule comprises at least one selected from the group consisting of biotin DHPE and biotin-X-DHPE.

15. The pharmaceutical composition of any one of claims 1-14, wherein the composition further comprises cellulose acetate phthalate, which is at least partially bound to the insulin dispersed within the lipid-based nanoparticle.

16. The pharmaceutical composition of any one of claims 1-15, wherein the composition further comprises at least one charged organic molecule bound to the insulin dispersed within the lipid-based nanoparticle, wherein the charged organic molecule is at least one selected from the group consisting of protamines, polylysine, poly (arg-pro-thr)n in a mole ratio of 1:1:1, poly (DL-Ala-poly-L-lys)n in a mole ratio of 6: 1, histones, sugar polymers comprising a primary amino group, polynucleotides with primary amino groups, proteins comprising amino acid residues with carboxyl (COO ) or sulfhydral (S") functional groups, and acidic polymers.

17. The pharmaceutical composition of any one of claims 1-16, wherein the cholesterol ranges from about 5% to about 25% (w / w) in the membrane.

18. The pharmaceutical composition of any one of claims 1-17, wherein the dicetyl phosphate ranges from about 10% to about 25% (w / w) in the membrane.

19. The pharmaceutical composition of any one of claims 1-18, wherein the DSPC ranges from about 40% to about 75% (w / w) in the membrane.

20. The pharmaceutical composition of any one of claims 1-19, wherein the hepatocyte receptor binding molecule ranges from about 0.5% to about 10% (w / w) in the membrane.

21. The pharmaceutical composition of any one of claims 3-20, wherein the amount of the stearoyl lysophosphatidylcholine in the membrane is about 5%-30% (w / w) of the amount of DSPC in the membrane.Attorney Docket No. 047589-5026WO1 (00244)22. The pharmaceutical composition of any one of claims 3-21, wherein the membrane comprises one of the following:(a) cholesterol, dicetyl phosphate, DSPC, stearoyl lysophosphatidylcholine, m- cresol, and at least one selected from the group consisting of biotin DHPE and biotin-X-DHPE;(b) cholesterol, dicetyl phosphate, DSPC, m-cresol, and at least one selected from the group consisting of biotin DHPE and biotin-X-DHPE; and (c) cholesterol, dicetyl phosphate, DSPC, stearoyl lysophosphatidylcholine, and at least one selected from the group consisting of biotin DHPE and biotin-X- DHPE.

23. The pharmaceutical composition of any one of claims 3-22, wherein the membrane comprises cholesterol, dicetyl phosphate, DSPC, stearoyl lysophosphatidylcholine, m-cresol, and biotin DHPE in a % (w / w) ratio selected from the group consisting of:(a) about 9.4 : 18.1 : 56.8 : 14.1 : 0.0 : 1.5;(b) about 7.7 : 15.0 : 58.6 : 0.0 : 17.4 : 1.3; and(c) about 8.4 : 16.2 : 47.5 : 7.6 : 19.0 : 1.3.

24. A method of treating or ameliorating diabetes in a subject suffering therefrom, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition of any one of claims 1-23.

25. A method of modulating peripheral lipid metabolism and hepatic lipid metabolism in a subject suffering from diabetes, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition of any one of claims 1-23.

26. The method of claim 25, wherein the administering allows for the subject to achieve liver and / or plasma triglyceride levels that are closer to normal physiological levels than before the subject was subjected to the administering.

27. The method of claim 25, wherein the administering allows for the subject to achieve liver and / or plasma free fatty acid levels that are closer to normal physiological levels than before the subject was subjected to the administering.Attomey Docket No. 047589-5026WO1 (00244)28. The method of claim 25, wherein the administering allows for the subject to achieve liver and / or plasma branched-chain amino acid (BCAA) levels that are closer to normal physiological levels than before the subject was subjected to the administering.

29. The method of claim 25, wherein the administering allows for the subject to achieve hepatic and / or peripheral glucose storage levels that are closer to normal physiological levels than before the subject was subjected to the administering.

30. A method of preventing, ameliorating, minimizing, and / or reversing hepatic accumulation of lipids (fatty liver) in a subject suffering from diabetes, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition of any one of claims 1-23.

31. The method of any one of claims 24-30, wherein the subject has about 6.5-8.5% A1C.

32. The method of any one of claims 24-30, wherein the subject has greater than about 8.5% A1C.

33. The method of any one of claims 24-32, wherein the subject experiences fewer hypoglycemia as compared to the treatment without HDV.

34. The method of any one of claims 24-33, wherein the subject experiences weight loss as compared to the treatment without HDV.

35. The method of any one of claims 24-34, wherein the subject does not experience significant iatrogenic hyperinsulinemia.

36. The method of any one of claims 24-35, wherein the pharmaceutical composition comprises basal insulin and is administered continuously to the subject over a period of at least 24 hours.

37. The method of any one of claims 24-36, wherein the pharmaceutical composition is administered continuously to the subject using a pump.Attorney Docket No. 047589-5026WO1 (00244)38. The method of any one of claims 24-37, wherein the subject has Type 1 diabetes, Type 2 diabetes, and / or a metabolic derangement.

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