Oral administration of insulin

Abstract

Described herein are nanoparticle dispersions comprising bioactive insulin (and insulin alternatives and insulin derivatives), methods for preparation of the nanoparticle dispersions, methods for treatment of diabetes via oral administration of nanoparticles comprising bioactive insulin (and insulin alternatives and insulin derivatives), and formulations comprising the nanoparticle dispersions. This disclosure is believed to represent the first physiologically effective oral administration of insulin.

Description

ORAL ADMINISTRATION OF INSULINREFERENCE TO RELATED APPLICATION

[0001] This International Application filed on May 22, 2025, claims benefit of and priority to U.S. Provisional Application No. 63 / 650,823, filed May 22, 2024, which is hereby incorporated herein in its entirety.TECHNICAL FIELD

[0002] The present disclosure generally relates to the treatment of diabetes and management of its symptoms by oral administration of bioactive insulin, and related compositions, formulations, and methods of administering bioactive insulin orally.BACKGROUND

[0003] Insulin and its alternatives and derivatives have been well known for decades for their use in treatment of diabetes mellitus I and II (diabetes mellitus type 1 and type 2). Administration of insulin (and / or its derivatives and alternatives) has previously been limited to subcutaneous injections, due to its being a relatively large and polar peptide. Treatment of diabetes through oral administration has been a long sought goal, however, none have been successful to date.

[0004] This goal is among those to which the present disclosure is generally directed. The preparation of nanoparticle dispersions comprising bioactive insulin, pharmaceutical formulations comprising the nanoparticle dispersions, and treatment of diabetes via oral administration is described herein.SUMMARY

[0005] This summary is provided to introduce a selection of concepts in a simplified form that are further described herein. This summary is not intended to identify required or essential features of the claimed subject matter. Nor is this summary intended to be used to limit the scope of the claimed subject matter.Methods for treating diabetes comprising orally administering to a patient in need thereof a therapeutically effective dose of a nanoparticle dispersion comprising insulin are disclosed herein. Nanoparticle dispersions comprising insulin are also disclosed. In certain aspects, nanoparticle dispersions can comprise an overall oral bioavailability in mice of at least1% relative to the amount of insulin incorporated within the nanoparticle dispersion. Formulations comprising the nanoparticle dispersion are also disclosed herein.Methods for preparing a nanoparticle dispersion comprising insulin also are disclosed herein, and can comprise preparing an oil phase mixture, preparing an aqueous phase mixture comprising insulin, combining the oil phase mixture and the aqueous phase mixture to form an emulsion, flowing the emulsion through a heat exchanger at a first temperature, and applying a shearing force to the emulsion at a second temperature to form a nanoparticle dispersion. In certain aspects, an amount of bioactive insulin remaining in the nanoparticle dispersion can be at least 25% relative to the of the amount of insulin in the emulsion.

[0006] Both the foregoing summary and the following detailed description provide examples and are explanatory only. Accordingly, the foregoing summary and the following detailed description should not be considered to be restrictive. Further, features or variations may be provided in addition to those set forth herein. For example, certain aspects may be directed to various feature combinations and sub-combinations described in the detailed description.BRIEF DESCRIPTION OF THE FIGURES

[0007] The following figures form part of the present specification and is included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to these figures in combination with the detailed description.

[0008] Figure 1 depicts a graph comparing the blood glucose concentration as a percentage of original blood glucose versus time for both subcutaneously administered native insulin and subcutaneously administered insulin nanoparticle dispersion.

[0009] Figure 2 is a schematic representation of the apparatus employed to produce nanoparticle dispersions of the Examples.

[0010] Figure 3 depicts a graph comparing blood glucose concentration as percentage of original blood glucose versus time for both subcutaneously administered native insulin and an orally administered insulin nanoparticle dispersion.

[0011] While the inventions disclosed herein are susceptible to various modifications and alternative forms, only a few specific aspects have been shown by way of example in the drawing and described in detail below. The figures and detailed description of specific aspects are not intended to limit the breadth or scope of the inventive concepts or the appended claims in any manner. Rather, the figures and detailed description are provided to illustrate theinventive concepts to a person of ordinary skill in the art and to enable such person to make and use the inventive concepts.DEFINITIONS

[0012] To define more clearly the terms used herein, the following definitions are provided. Unless otherwise indicated, the following definitions are applicable to this disclosure. If a term is used in this disclosure but is not specifically defined herein, the definition from the IUPAC Compendium of Chemical Terminology, 2nd Ed (1997), can be applied, as long as that definition does not conflict with any other disclosure or definition applied herein, or render indefinite or non-enabled any claim to which that definition is applied. To the extent that any definition or usage provided by any document incorporated herein by reference conflicts with the definition or usage provided herein, the definition or usage provided herein controls.

[0013] Herein, features of the subject matter are described such that, within particular aspects, a combination of different features can be envisioned. For each and every aspect and each and every feature disclosed herein, all combinations that do not detrimentally affect the compounds, compositions, processes, or methods described herein are contemplated with or without explicit description of the particular combination. Additionally, unless explicitly recited otherwise, any aspect or feature disclosed herein can be combined to describe inventive compounds, compositions, processes, or methods consistent with the present disclosure.

[0014] In this disclosure, while compositions and processes / methods are described in terms of “comprising” various materials or components and steps, the compositions and processes / methods also can “consist essentially of’ or “consist of’ the various materials or components and steps, unless stated otherwise.

[0015] The terms “contacting” and “combining” are used herein to describe compositions and processes / methods in which the materials are contacted or combined together in any order, in any manner, and for any length of time, unless otherwise specified. For example, the materials can be blended, mixed, slurried, dissolved, reacted, treated, impregnated, compounded, or otherwise contacted or combined in some other manner or by any suitable method or technique.

[0016] Several types of ranges are disclosed in the present disclosure. When a range of any type is disclosed or claimed, the intent is to disclose or claim individually each possible number that such a range could reasonably encompass, including end points of the range as well as any sub-ranges and combinations of sub-ranges encompassed therein. For example,nanoparticle dispersions can have various concentrations of insulin in aspects of this disclosure. By a disclosure that a concentration is in a range from 100 ppm to 1000 ppm, the intent is to recite that the concentration can be any ratio in the range and, for example, can include any range or combination of ranges from 100 ppm to 1000 ppm, such as from 200 ppm to 800 ppm, from 500 ppm to 650 ppm, from 450 ppm to 750 ppm, from 525 ppm to 575 ppm, or from 250 ppm to 850 ppm, and so forth. Likewise, all other ranges disclosed herein should be interpreted in a manner similar to this example.

[0017] As used herein, the term "insulin" is used to refer generally to insulin, insulin analogs and derivatives. As used herein insulin can refer to any peptide able to illicit a biological response similar to that of natural human insulin within a patient in need thereof. Insulin therefore can refer to human insulin as the 51 -amino acid peptide hormone composed of two chains: an A-chain with 21 amino acids and a B-chain with 30 amino acids. Insulin as used herein also may refer to insulin lispro, insulin aspart, insulin glulisine, NPH insulin, insulin glargine, insulin detemir, insulin degludec, and combination and mixtures thereof. Additional insulin derivatives also are contemplated herein, and the scope of "insulin" as used herein is not limited to those analogs and derivatives listed above.

[0018] Methods for treating diabetes are described herein. It will be understood that by methods for treating for diabetes disclosed herein will refer to any type of diabetes, in any stage or severity. The oral administration of insulin-containing nanoparticle dispersions generally will be understood as effective for treating diabetes by ultimately managing the glucose concentration within a patient's blood, as a mechanism for the treatment of diabetes. In certain aspects, methods for treating diabetes can comprise treating a patient with prediabetes or diabetes mellitus. Methods for treating for diabetes disclosed herein also will refer to the treatment of type 1 or type 2 diabetes and managing the related symptoms thereof including hyperglycemia.

[0019] In general, an amount, size, formulation, parameter, range, or other quantity or characteristic is “about” or “approximate” whether or not expressly stated to be such. Whether or not modified by the term “about” or “approximately,” the claims include equivalents to the quantities or characteristics.

[0020] As used herein, the term "nanolipid dispersion" can refer to a dispersion of lipid particles in water with a volume average particle size less than 150 nm. Nanolipid dispersions as used herein also can be described more generally as nanoparticle dispersions. Nanoparticle dispersions described herein can include those as described and produced within U.S. Patent 11,504,327, entitled Method of preparing nanoparticles by hot-melt extrusion, andInternational Application No. PCT / US2023 / 080774, filed November 21, 2023, each of which is hereby incorporated in its entirety herein by reference. Accordingly, in certain aspects, nanolipid dispersions can comprise an amount of insulin, an insulin derivative, an insulin alternative, one or more high hydrophile-lipophile-balance (HLB) surfactants, one or more low hydrophile-lipophile-balance (HLB) surfactants; one or more water immiscible oils, and an amount of water, as described in the ‘774 application. Similarly, the terms “capsule” can refer to the capsules as described in the ‘774 application.

[0021] As used herein, the term "emulsion" is generally applied to any mixture of an oil phase and aqueous phase. Emulsions as described herein do not imply a stability or longevity of the mixture in a given state. As used herein "emulsion" is not limited to a particular evenness of particle size or consistency throughout the emulsion, or vesicle size or consistency throughout the emulsion, or homogeneity of the emulsion. It will be understood by those of skill in the art that the term "emulsion" as used herein is liberally applied to the mixture of an oil phase and an aqueous phase in all but mixtures of oils and aqueous phases more fittingly characterized as separated mixtures of oil and aqueous phases.

[0022] Oral administration of nanoparticle dispersions disclosed can, in certain aspects, exhibit the same properties as those described in the ‘774 application. For instance, it is contemplated that the oral administration of nanolipid dispersions disclosed herein can exhibit a Tmax and Cmax may be within any range disclosed within the ‘774 application. Alternatively, the Tmax and / or Cmax may differ according to any particular formulation, as demonstrated by Examples below.

[0023] Features within this disclosure that are provided as minimum values can be alternatively stated as “at least” or “greater than or equal to” any recited minimum value for the feature disclosed herein. Features within this disclosure that are provided as maximum values can be alternatively stated as “less than or equal to” or “below” any recited maximum value for the feature disclosed herein.

[0024] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the typical methods and materials are herein described.

[0025] All publications and patents mentioned herein are incorporated herein by reference in their entirety for the purpose of describing and disclosing, for example, the constructs and methodologies that are described in the publications and patents, which might be used in connection with the presently described disclosure.DETAILED DESCRIPTION

[0026] Disclosed herein are methods for treating diabetes via oral administration of nanoparticle dispersions comprising bioactive insulin. Methods for forming nanoparticle dispersions retaining bioactivity of insulin, and nanoparticle dispersions are also disclosed herein.Methods of treating diabetes by the oral administration of insulin

[0027] Generally, as disclosed herein treating diabetes can include managing blood sugar (e.g., a glucose concentration) levels of a patient suffering from diabetes. In certain aspects methods disclosed herein can comprise monitoring a sugar (glucose) concentration in the blood to determine whether the glucose concentration is within a normal range (e.g., 70-99 mg / dL) or in a hyperglycemic state. Methods disclosed herein can further comprise orally administering a nanoparticle dispersion comprising insulin to lower the glucose concentration from a hyperglycemic state to within the normal range.

[0028] Broadly, methods for the treatment of various forms and stages of diabetes and its related symptoms in patients in need thereof are well-documented throughout the literature, and are not repeated beyond the extent necessary in this disclosure. In the context of this disclosure, methods for treatment can comprise attaining a desirable blood concentration of insulin (and its analogs, derivatives, and variants) for the purpose of managing blood glucose levels in a patient. Again, details surrounding treatment regimens targeting specific blood concentrations are not explicitly recited within this disclosure in the sake of brevity, but will be understood by those of skill in the art as applicable to any treatment regimen that includes administering insulin to a patient and typically in order to effect a desirable blood concentration of insulin and resulting management of blood glucose.

[0029] Unexpectedly, methods disclosed herein benefit from an exceptionally high bioavailability of insulin owing to its inclusion within a nanoparticle dispersion. In certain aspects, the bioavailability of insulin can be at least 3%, at least 5%, at least 8%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% of the total amount of insulin present within the nanoparticle dispersion; alternatively in a range from 15% to 55%, from 20% to 45%, or from 20% to 35%. Methods described herein also can be characterized by the bioavailability of the insulin within the nanoparticle dispersion relative to bioavailability of the insulin administered in a conventional oral formulation, i.e., oral formulations without lipid nanoparticles. In certain aspects, methods contemplated herein can have a bioavailability that is at least 1% greater, at least 3% greater,at least 5% greater, at least 10% greater, at least 25% greater, at least 50% greater, at least 100% greater, or at least 200% greater, than that of an otherwise identical method comprising oral administration of a formulation that does not comprise lipid nanoparticles containing insulin.

[0030] In addition to improved bioavailability, methods comprising oral administration of insulin as disclosed herein have demonstrated a longer time to peak concentration of bioactive insulin within the blood (characterized as Tmax) relative to a subcutaneous injection. Without being bound by theory, methods disclosed herein demonstrating an extended Tmax can allow the delivered dose of insulin to be effective throughout a longer period of time, and reduce the risk and occurrence of sudden drops in blood-glucose concentration (e.g., hypoglycemic spikes) resulting from the concomitant spike of insulin concentration within the blood. In certain aspects, the Tmax of treatment methods disclosed herein can be in a range of 2 hours to 12 hours, from 3 hours to 10 hours, or from 5 hours to 8 hours. Alternatively, the Tmax can be characterized relative to Tmax resulting from subcutaneous injection of insulin, and in certain aspects Tmax of methods disclosed herein can be at least 30 min, at least 1 hour, at least 2 hours, at least 4 hours or at least 8 hours greater than that of an otherwise identical method comprising subcutaneous injection of insulin.

[0031] As a result of the unexpectedly high Tmax achieved, methods for treating diabetes disclosed herein can have uniquely advantageous dosing regimens. The longer Tmax demonstrated by oral administration of nanoparticles containing insulin generally can results in a smoother biodistribution profile compared to subcutaneous inj ections, as depicted in Figure 3. In certain aspects, a larger dose of insulin can be administered at once, without concern of spiking insulin levels due to the steady absorption and release of insulin from nanoparticles within the digestive system. In this manner, a relatively constant concentration of insulin can be maintained in the blood throughout the day by a single daily dose (e.g., every 24 hours, every 18 to 30 hours, or 12 to 36 hours). Alternatively, methods disclosed herein can comprise a dosing regimen of two times a day, three times a day, or four times a day. Still further aspects can comprise dosing every two days, alternatively every three days.

[0032] In certain aspects, a first series of doses may be administered to establish a baseline concentration of insulin within the bloodstream, which can then be supplemented by additional doses spaced at larger times. For instance, methods disclosed herein can comprise oral administration of a nanoparticle dispersion comprising insulin every 6 hours for the first three doses, and then additional doses on a daily dose regimen.

[0033] It is further contemplated that methods of treating diabetes as disclosed herein may be applied in conjunction with conventional treatment methods, for instance subcutaneous administration of insulin. Methods disclosed herein also can comprise additional administration methods for insulin, as a supplement the extended Tmax formulations of the oral administration. For instance, methods disclosed herein can comprise a first oral administration of insulin and a supplemental subcutaneous administration of insulin. In this manner, the prevalence of hyperglycemic events can be reduced by providing a steady baseline of insulin within the bloodstream via oral administration of insulin (higher Tmax) and any hyperglycemic events may be quickly counteracted with a supplemental subcutaneous administration of insulin and having a relatively low T max.

[0034] As defined above, it will be understood within this disclosure that insulin may refer to native insulin, or any number of insulin analogs, derivatives, and substitutes, including short-acting insulin derivatives, long-acting insulin derivatives, fast-acting insulin derivatives and the like. It will further be understood by those of skill in the art that insulin derivatives may have varied oral bioavailability and Tmax. However, it is contemplated within this disclosure that the bioavailability, Tmax, or both for any particular insulin derivative, analog, or substitute may be improved by administration as the nanoparticle dispersions described below. Thus, the Tmax of short-acting insulin derivatives may be extended by formulation as nanoparticle dispersions disclosed herein.Nanoparticle dispersions comprising bioactive insulin

[0035] Nanoparticle dispersions disclosed herein can be formulated for the oral administration of insulin. In this manner, the nanoparticle dispersions disclosed herein can comprise insulin and any number of unique characteristics and components to deliver insulin in its bioactive form through the digestive tract of a patient in need thereof and in suitable concentration for efficacy in treating diabetes.

[0036] Nanoparticle dispersions disclosed herein generally can comprise an exceptional concentration of bioactive insulin. When coupled with the exceptional and unexpected oral bioavailability of the nanoparticle dispersions, the high concentration of insulin within the nanoparticle dispersions disclosed herein allows a pharmaceutically useful amount of insulin to be administered to a patient in need thereof, as described elsewhere. In certain aspects, the nanoparticle dispersions can comprise a concentration of bioactive insulin of at least 50 ppm, at least 100 ppm, at least 200 ppm, at least 300 ppm, at least 400 ppm, at least 500 ppm, or at least 1000 ppm; alternatively, in a range from 100 ppm to 1000 ppm, from200 ppm to 800 ppm, from 250 ppm to 750 ppm, or from 300 ppm to 600 ppm. It will be understood that the nanoparticle dispersions may be diluted (as in Example 2 below) to any appropriate dose concentration, e.g., to a concentration in a range from 5 ppm to 50 ppm.

[0037] In certain aspects, the amount of insulin incorporated within the nanoparticle dispersion can be aided by the presence of an ionic species to pair with the amino acid species present on the exterior of insulin when in the aqueous phase. Without being bound by theory, it is believed that by forming ionic bonds with zinc, insulin may adopt a conformation more suitable for inclusion within the nonpolar interior of a nanolipid. In certain aspects, nanoparticle dispersions can comprise an amount of zinc (e.g., zinc chloride) of at least 0.001 wt. %, at least 0.01 wt. %, at least 0.05 wt. %, at least 0.1 wt. %, at least 0.5 wt. %, at least 1 wt. %, at least 3 wt. %, or at least 5 wt. %, relative to the total weight of the nanoparticle dispersion. In other aspects, nanoparticle dispersions can comprise an amount of zinc in a range from 0.001 wt. % to 10 wt. %, alternatively from 0.001 wt. % to 1 wt. %; alternatively from 0.01 wt. % to 0.5 wt. %. It is further contemplated that this interaction between zinc and insulin may be pH dependent, such that in alkaline environments, the insulin is returned to its native form.

[0038] Nanoparticle dispersions comprising insulin can have any characteristics and components suitable to incorporate insulin within the nanoparticles as are generally disclosed within International Application No. PCT / US2023 / 080774, which is hereby incorporated herein in its entirety. For instance, in certain aspects, nanoparticle dispersions disclosed herein can comprise water, a water immiscible oil, a surfactant, and insulin. In certain aspects, nanoparticle dispersions can further comprise a structure promoting additive that may impart necessary stability in formation of mesophases between the crude mixture of components and formation of the stable nanoparticle dispersions, e.g., during mechanical shearing and kneading conducted within a twin screw extruder. In certain aspects, dispersions contemplated herein can further comprise additives useful for preservation and stability of the dispersions.

[0039] In certain aspects, nanoparticle dispersions can comprise any suitable amount of water that results in a stable nanoparticle dispersion with favorable characteristics (e.g., a particle size less than 150 nm, non-vesicular and latent lamellar structure, adequate incorporation of insulin and additives, acceptable ratio of water immiscible oils to insulin, or any combination thereof). In certain aspects, nanoparticle dispersions can comprise an amount of water in a range from 10 wt. % to 90 wt. %, from 20 wt. % to 80 wt. %, from 25 wt. % to 75 wt. %, from 35 wt. % to 65 wt. %, from 40 wt. % to 60 wt. %, from 40 wt. % to 55 wt. %, from 45 wt. % to 60 wt. %, or from 50 wt. % to 60 wt. %. In other aspects, nanoparticledispersions can comprise an amount of water less than 75 wt. %, less than 65 wt. %, less than 55 wt. %, less than 50 wt. %, less than 45 wt. %, less than 40 wt. %, or less than 35 wt. %.

[0040] Dispersions disclosed herein can further contain water immiscible oils selected from cocoyl caprate / caprylate, alkyl alcohols such as Isofol 12 (2-butyl-l -octanol), benzyl alcohol, diisopropyl adipate, capric / caprylic triglyceride oil, isopropyl myristate, limonene, medium chain triglyceride oil, mineral oil, omega 3 fatty acid, oleyl alcohol, isohexadecane, isododecane, a C13 to Cis alkane, or combinations thereof. In other aspects, suitable water immiscible oils useful in the practice of the invention disclosed herein can include medium chain triglyceride oil, coconut oil, isopropyl palmitate, isopropyl myristate, methyl decanoate, ethyl myristate, ethyl oleate, mineral oil, orange essential oil, cyclopentasiloxane, poly(dimethyl siloxane), hexadecane, propylene glycol dicaprylate, isododecane, isoeicosane, isohexadecane, soy biodiesel, jojoba oil, cocoyl caprylocaprate, Cio to C13 alkanes, squalane, sunflower seed oil, diacetylated monoglycerides, clove essential oil, and limonene.

[0041] Without being bound by theory, water immiscible oils may be provided for the purpose of carrying bioactive ingredients within the dispersion and across biological membranes as may be necessary to achieve efficient incorporation of insulin within the nanoparticle dispersion and delivery of the insulin to or within a biological entity for appropriate treatment, respectively. Thus, it may be advantageous to increase the relative amount of water immiscible oil and surfactants in dispersions to maximize the capacity for insulin as discussed above. It may also be advantageous to maximize the amount of lipid phase in dispersions to maximize protection of peptides that are degraded in aqueous settings, as for example, proteolytic enzymatic degradation of insulin in the gastrointestinal tract. In certain aspects, dispersions can comprise an amount of water immiscible oil in a range from 5 wt. % to 65 wt. %, from 10 wt. % to 45 wt. %, from 15 wt. % to 40 wt. %, or from 20 wt. % to 35 wt. %. In certain aspects, the amount of water immiscible oil can be greater than 10 wt. %, greater than 20 wt. %, greater than 30 wt. %, greater than 35 wt. %, greater than 40 wt. %, greater than 45 wt. %, greater than 50 wt. %, or greater than 55 wt. %.

[0042] Dispersions contemplated herein can further comprise surfactants to stabilize a dispersion comprising the water and water immiscible oils as a dense nanolipid fluid dispersion and prevent phase separation of the water and water immiscible oil(s). Surfactants as disclosed herein may be characterized by their structure and also their general lipophilicity.

[0043] In certain aspects, dispersions disclosed herein can comprise high hydrophile- lipophile-balance (HLB) surfactant, a low hydrophile-lipophile-balance (HLB) surfactant, or both. Generally, the high hydrophile-lipophile-balance (HLB) surfactant can comprise apolyethoxylated high hydrophile-lipophile-balance (HLB) surfactant, an ether type polyethoxylated high hydrophile-lipophile-balance (HLB) surfactant, an ester type polyethoxylated high hydrophile-lipophile-balance (HLB) surfactant, or a non-polyethoxylated high hydrophile-lipophile-balance (HLB) surfactant.

[0044] Suitable polyethoxylated high hydrophile-lipophile-balance (HLB) surfactants, suitable one or more ether type polyethoxylated high hydrophile-lipophile-balance (HLB) surfactants, suitable one or more ester type polyethoxylated high hydrophile-lipophile-balance (HLB) surfactants, and suitable one or more non-polyethoxylated high hydrophile-lipophile- balance (HLB) surfactants each may be as described in PCT / US2023 / 080774, and are not repeated here in the interest of brevity. Suitable one or more low hydrophile-lipophile-balance (HLB) surfactants and suitable one or more phospholipid low hydrophile-lipophile-balance (HLB) surfactants also are described in PCT / US2023 / 080774.

[0045] Nanoparticle dispersions disclosed herein can comprise any amount of the high hydrophile-lipophile-balance (HLB) surfactants suitable to maintain the structural characteristics of the dispersion. In certain aspects, the amount of high HLB surfactant can be in a range from about 0.1 wt. % to about 20 wt. %, from about 1 wt. % to about 15 wt. %, from about 1.5 wt. % to about 15 wt. %, or from about 2 wt. % to about 10 wt. %. In certain aspects, the amount of high HLB surfactant can be in a range from 1.6 wt. % to 11.9 wt. %. Similarly, nanoparticle dispersions can comprise an amount of low HLB surfactant in a range from about 0.1 wt. % to about 20 wt. %, from about 1 wt. % to about 15 wt. %, from about 1.5 wt. % to about 15 wt. %, or from about 2 wt. % to about 10 wt. %. In certain aspects, the amount of high HLB surfactant can be in a range from 2.6 wt. % to 12.0 wt. %.

[0046] In certain aspects a total amount of surfactants (e.g., high, and low HLB surfactants) within the dispersions disclosed herein can be in a range from 2 wt. % to 30 wt. %, from 5 wt. % to 28 wt. %, or from 15 wt. % to 25 wt. %. In other aspects, the total amount of surfactants can be less than about 40 wt. %, less than about 35 wt. %, less than about 30 wt. %, less than about 28 wt. %, less than about 25 wt. %, less than about 22 wt. %, less than about 20 wt. %, less than about 15 wt. %, or less than about 10 wt. %.

[0047] Certain combinations of high and low HLB surfactants can lead to stable nanolipid dispersions with exceptional and advantageous characteristics, which may be extended to the dispersions disclosed herein comprising insulin. In certain aspects, the dispersion can comprise an ester-type poly ethoxylated high HLB surfactant (e.g., PEG100 stearate, PEG20 stearate, PEG30 glyceryl cocoate, PEG32 stearate, polysorbate 20, polysorbate 80) and a phospholipid low HLB surfactant (e.g., phosphatidylcholine and lecithin). In otheraspects, dispersions contemplated herein can comprise an ether type polyethoxylated high HLB surfactant (e.g., laureth-23, laureth-30, steareth-100, steareth-20, steareth-40, ceteareth-20, and ceteareth-30) and an ester type polyethoxylated high HLB surfactant (e.g., PEG100 stearate, PEG20 stearate, PEG30 glyceryl cocoate, PEG32 stearate, polysorbate 20, polysorbate 80). In such aspects the weight ratio of the ether type poly ethoxylated high HLB surfactant to the ester- type poly ethoxylated high HLB surfactant can be greater than 1 : 1, greater than 2: 1, or greater than 4: 1, for example, in a range from 1 : 1 to 4: 1, or from 1 : 1 to 2: 1. Alternatively, dispersions can comprise an ether type polyethoxylated high HLB surfactant can further comprise a nonpolyethoxylated high HLB surfactant (e.g., sodium laurel sulfate).

[0048] Certain aspects disclosed herein can comprise an amount of insulin that may drastically exceed otherwise identical nanoparticulate compositions comprising otherwise identical components but adopting another nanoparticulate form. In certain aspects, it is contemplated that the dispersions can comprise as much as 2 wt. %, 5 wt. %, 10 wt. %, 20 wt. %, 30 wt. % or 40 wt. % of insulin relative to the total weight of the dispersion. Alternatively, dispersions can comprise an amount of the insulin in a range from about 0.01 wt. % to about 40 wt. % of the insulin, from about 0.01 wt. % to about 20 wt. %, or from about 0.1 wt. % to about 10 wt. %, from about 1 wt. % to about 10 wt. % or from about 3 wt. % to about 8 wt. %.

[0049] The ability of nanoparticles to permeate skin is known to be related to particle size, with generally better permeation for smaller particles. The appropriate quantity for particle size in a distribution of particles as it relates to dermal permeation is the weight average particle diameter because it defines the average on the basis of mass fractions of nanoparticles with a particular diameter and what matters is the mass fraction of nanoparticles that have sufficiently small diameters so as to effectively permeate skin. By comparison, an unspecified maj ority by mass fraction of particles can have diameters much greater than the number average particle diameter, making number average particle diameter a poor metric for permeability. In certain aspects, nanoparticle dispersions disclosed herein can have volume average particle diameter less than 150 nm, less than 100 nm, less than 60 nm, or less than 50 nm; alternatively, in a range from about 10 to about 150 nm, from about 20 nm to about 120 nm, from about 30 nm to about 100 nm, or from about 30 nm to about 80 nm.

[0050] Dispersions disclosed herein may also be characterized by their lipid content. Without being bound by theory, it is believed that an increased lipid content can provide additional capacity to load and protect insulin as disclosed herein. In certain aspects, the dispersions disclosed herein can comprise greater than 25 wt. %, greater than wt. 30%, greater than 35 wt. %, greater than 40 wt. %, greater than 45 wt. %, greater than 50 wt. %, greater than55 wt. %, or greater than about 60 wt. %. In other aspects, dispersions can comprise a lipid content in a range from 25 wt. % to 55 wt. %, or from 35 wt. % to 50 wt. %. In certain aspects, dispersions can comprise a lipid content greater than 25 wt. % and a particle size less than 100 nm. In alternative aspects, dispersions can comprise a lipid content in a range from 35 wt. % to 50 wt. %, and a particle size less than 60 nm.

[0051] Separately, nanoparticle dispersions disclosed herein can demonstrate stability with respect to retaining insulin within (or associated with) the nanoparticle dispersion. In certain aspects, insulin does not form crystals within the nanoparticle dispersion at a temperature from about 18 °C to about 22 °C for greater than about 1 month, greater than about 2 months, greater than about 3 months, 8 months, greater than about 12 months, greater than about 18 months, or greater than about 24 months; alternatively, in a range from 1 month to 24 months, in a range from 3 months to 12 months. The nanoparticle dispersions disclosed herein may remain free from crystals even where the concentration of the insulin is greater than the solubility of insulin in the aqueous phase of the dispersion, even outside the context of the nanoparticle.Formulations of nanoparticle dispersions comprising insulin

[0052] Nanoparticle dispersions as described above can be combined with additional components in formulations beneficial for oral administration. Certain exemplary and nonlimiting formulations that may retain the benefits of oral bioavailability, stability, and other beneficial properties of nanoparticle dispersions disclosed above are described.Lipase resistant nanoparticle dispersions

[0053] Nanoparticle dispersions disclosed herein can comprise lipids that lack ester bonds and are therefore not subject to enzymatic decomposition from lipase, or they may comprise compounds that are inhibitors for lipases, or they may comprise surfactants that block access of lipases across oil water interfaces. Lipid nanoemulsions of the present disclosure can have volume average particle diameters sufficiently small to allow direct absorption via endocytosis, particularly caveolin and clathrin mediated endocytosis. In caveolin and clathrin mediated endocytosis, about 60 nm to 100 nm diameter pits or invaginations form in cell walls which yield endocytic vesicles that become increasingly effective vehicles for particle absorption as absorbate particle diameters decrease below about 80 nm.

[0054] The disclosure also provides a nanoparticle dispersion comprising a hydrophobic therapeutic agent encapsulated in a nanoparticle composed of lipophilic materialswherein the nanoparticles have volume average particle size less than 100 nm wherein lipophilic materials are lipolysis resistant meaning that less than 25% of ester bonds in the lipophilic materials hydrolyze within one hour in the presence of a concentration 50 units per milliliter of porcine pancreatic lipase wherein “lipophilic materials” include all surfactants, water immiscible oils, hydrophobic drugs, and hydrophobic therapeutic agents.

[0055] In one embodiment, nanoparticle dispersions of the present disclosure can comprise a high HLB polyethoxylated surfactant that is inert to lipolysis. In one embodiment the nanoparticle dispersion includes a high HLB polyethoxylated surfactant that lacks an ester group. In one embodiment the nanoparticle dispersion includes an ether type high HLB polyethoxylated surfactant.

[0056] In one embodiment the nanoparticle dispersion includes a lipase inhibitor. In one embodiment the lipase inhibitor can be selected from the group consisting of anandamide, diacylglycerol lipase, orlistat, 2-arachidonoylglycerol, cannabinoid, endocannabinoid, fatty acid amidase, acylglycerol lipase, and combinations thereof. In certain aspects, the lipase inhibitor is orlistat. In one embodiment the nanoparticle dispersion comprises greater than 25 weight percent lipophilic material content.

[0057] Nanoparticle dispersions disclosed herein also may comprises lipid nanoparticles with a positive charge. In one embodiment the nanoparticle dispersion comprises lipid nanoparticles with a zeta potential greater than 1.0 millivolt; alternatively, in a range from 0.5 to 5 mV, or from 1 mV to 3 mv). Thus, in certain aspects, nanoparticle dispersions disclosed herein can comprise zwitterionic surfactants. Zwitterionic surfactants comprise separated positively and negatively charged ions which are not conformationally allowed to associate in such a way as to become hydrophobic. The presence of charged ions on lipid nanoparticle surfaces may be effective to reduce bending forces in cell membranes and allow more facile puckering so as to promote endocytosis; alternatively, zwitterionic groups on lipid nanoparticle surfaces may mimic improved attachment, cellular membrane penetration and absorption observed for capsid viruses.

[0058] Accordingly, in certain aspects, nanoparticle dispersions comprise amphipathic compounds with a betaine group. In one aspect, nanoparticle dispersions comprise cocamidopropyl betaine. In one aspect, nanoparticle dispersions comprise amphipathic compounds with a choline phosphate group. In one aspect, DNLF dispersions comprise a phosphatidyl choline compound.

[0059] Lipolysis resistance may be characterized by changes in the particle size, as ester bonds are hydrolyzed within the dispersions. In certain aspects, the nanoparticledispersion can have greater than 80% (or greater than 60%, or greater than 70%, or greater than 90%) of particles in the dispersion on a volume basis have a diameter less than 100 nm after 60 minutes in a lipolysis solution with 2.6% DNLF dispersion, pH about 6.8 containing calcium, one or more bile salts, and 0.4% lipase. In still further aspects, less than 10% (e.g., less than 5%, less than 2%; alternatively, in a range from 1 to 10 %) of the lipid ester bonds are lipolyzed after 60 minutes in a lipolysis solution with pH about 6.8 containing calcium, one or more bile salts, and 0.4% lipase.

[0060] Still, even where lipolysis occurs, it is observed that nanoparticle dispersions as disclosed herein may retain a reduced particle size. For instance, it is seen that greater than 80% of particles on a volume basis can have a diameter less than 100 nm after 30% or more of lipid nanoparticle ester bonds have been hydrolyzed by lipase.Encapsulated nanolipid dispersions

[0061] The high water activity of nanolipid dispersions can cause deterioration of gelatin capsules. Left overnight at temperatures between 4 °C and 50 °C, gelatin capsules filled with DNLFs will become too soft to easily swallow. One solution is to fill the gelatin capsule with DNLF shortly before swallowing, but such a method is inconvenient and creates risk of incorrectly measuring the required dosage.

[0062] A solution to poor systemic absorption is provided by providing drugs and therapeutic agents in dense nanolipid fluid (DNLF) dispersions which are highly concentrated lipid nanoemulsions in a continuous aqueous phase.

[0063] It has been discovered that DNLF dispersions can be encapsulated in so called enteric polymers that have carboxylic acid functional groups if the pH of the DNLF dispersion is below the pKa value of the polymer carboxylic acid groups, even in the case that such polymers may be plasticized by absorption of some of the water in the DNLF dispersion. Furthermore, DNLF dispersions are stable to encapsulation in carboxylic acid functional enteric polymers, even in the case that some of the water in the DNLF dispersion is lost to absorption in the capsule wall.

[0064] The present invention generally provides capsules comprising DNLF dispersions. In certain aspects, the DNLF dispersion can comprise hydrophobic active compounds, hydrophilic active compounds, and or peptides encapsulated in capsules comprising carboxylic acid functional polymers. In certain aspects, the capsules can comprise a carboxylic acid functional polymer and an encapsulated DNLF dispersion.

[0065] In another aspect, the DNLF dispersion can be encapsulated in a carboxylic acid functional polymer. In one embodiment the pH of the DNLF dispersion is less than the pKa value of the encapsulating polymer carboxylic acid groups. In one embodiment the pH of the DNLF dispersion is more than one pH unit less than the pKa value of the encapsulating polymer carboxylic acid groups. In one embodiment, the encapsulating polymer is the product of vinyl polymerization.

[0066] In one embodiment, the encapsulating polymer comprises acrylate monomer units. In one embodiment, the encapsulating polymer comprises methacrylate monomer units. In one embodiment, the encapsulating polymer comprises vinyl acetate monomer units. In one embodiment, the encapsulating polymer comprises acrylic acid monomer units. In one embodiment, the encapsulating polymer comprises methacrylic acid monomer units. In one embodiment, the encapsulating polymer comprises vinyl 4-hydroxyl phthalate monomer units. In one embodiment, the encapsulating polymer comprises modified cellulose. In one embodiment, the encapsulating polymer comprises cellulose esterified with acetic acid and phthalic acid. In one embodiment, the encapsulating polymer comprises cellulose esterified with acetic acid and succinic acid. In one embodiment, the encapsulating polymer is methyl acrylate - methacrylic acid copolymer. In one embodiment, the encapsulating polymer is methyl methacrylate - methacrylic acid copolymer. In one embodiment, the encapsulating polymer is ethyl acrylate - methacrylic acid copolymer. In one embodiment, the encapsulating polymer is vinyl acetate - vinyl 4-hydroxyl phthalate copolymer.

[0067] In one embodiment, the encapsulating polymer is hydroxypropyl methyl cellulose acetate succinate. In one embodiment, the encapsulating polymer is cellulose acetate phthalate. In one embodiment, the encapsulating polymer is Eudragit L 30 D-55.

[0068] In one embodiment, the capsule comprises a surface layer comprising carnauba wax, dimethicone and organo-modified silicone. In one embodiment, the capsule comprises a surface layer comprising carnauba wax.

[0069] In one embodiment, the capsule comprises an innermost layer consisting of a carboxylic acid functional polymer, an intermediate layer of gelatin, and a surface layer comprising carnauba wax.Lyophilized nanoparticle dispersions

[0070] Nanoparticle dispersions disclosed herein also may be formulated for oral administration by lyophilization. Generally, the methods to form nanoparticle dispersions can further comprise a lyophilization step to provide a stable shelf-storage product that may beactivated by dissolution. In certain aspects, lyophilized nanoparticle dispersions can maintain physical characteristics upon being reconstituted from a lyophilized state.

[0071] In certain aspects, nanoparticle dispersions disclosed herein can further comprise one or more cryoprotectants within the formulation to preserve physical characteristics of the dispersions throughout the freezing process. In certain aspects, cryoprotectants suitable for inclusion within nanoparticle dispersions of the present disclosure can include one or more of diethylene glycol, dimethyl sulfoxide, ethylene glycol, glycerin, propylene glycol, sorbitan, trehalose, sucrose, mannitol, and the like.

[0072] Alternately, or additionally, lyophilization processes to dry the nanoparticle dispersions can be promoted under rapid freezing-drying cycles.

[0073] Formulations of nanoparticle dispersions disclosed herein generally can further comprise any amount of a component that assists in the stability, shelf-storage, bioavailability, taste, texture, or any combination thereof, for oral administration of the dispersions.Methods for forming nanoparticle dispersions comprising insulin

[0074] Nanoparticle dispersions disclosed herein can be prepared by any suitable process that provides the dispersions in a stable form, with acceptable characteristics (e.g., particle size, bioactive insulin concentration) as disclosed above. Such methods include those disclosed within PCT / US2023 / 080774, hereby incorporated by reference herein.

[0075] Broadly, methods for preparing a nanoparticle dispersion comprising insulin can comprise preparing an oil phase mixture, preparing an aqueous phase mixture comprising insulin, combining the oil phase mixture and the aqueous phase mixture to form an emulsion, flowing the emulsion through a heat exchanger at a first temperature; then applying a shearing force to the emulsion at a second temperature to form the nanoparticle dispersion. In certain aspects, the first temperature can be in a range from 35 °C to 98 °C, from 50 °C to 98 °C, from 55 °C to 95 °C from 60 °C to 90 °C, or from 65 °C to 85 °C. In these and other aspects, the second temperature can be in a range from 5 °C to 55 °C.

[0076] In certain aspects, applying a shearing force can comprise flowing the emulsion into a stirring aqueous solution. In this manner, the emulsion receives a shearing force while being quickly cooled by the water. Alternatively, applying a shearing force can comprise extruding the emulsion through a twin screw extruder. In such aspects, extruding the emulsion can be conducted at an extrusion temperature in a range from 5 °C to 95°C; alternatively a gradient from a first extrusion temperature to a second extrusion temperature, or from a first extrusion temperature to a plurality of additional extrusion temperatures. In certain aspects, thefirst extrusion temperature is in a range from 60 °C to 95 °C, and the second extrusion temperature is in a range from 5°C to 40 °C.

[0077] Surprisingly, it is found that under these conditions, the insulin loaded into the aqueous phase of the initial mixture remains largely intact in its bioactive form. In certain aspects, the amount of bioactive insulin in the nanoparticle dispersion can be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45%, of an amount of insulin in the emulsion prior to extrusion; alternatively, in a range from 20% to 75%, from 25% to 65%, or from 30% to 55%.EXAMPLES

[0078] The disclosure is further illustrated by the following examples, which are not to be construed in any way as imposing limitations to the scope of this disclosure. Various other aspects, modifications, and equivalents thereof which, after reading the description herein, can suggest themselves to one of ordinary skill in the art without departing from the spirit of the present disclosure or the scope of the appended claims.Examples 1-3. Preparation of lipid nanoparticle comprising bioactive insulin and oral administration in mice.Example 1. Preparation of a lipid nanoparticle comprising bioactive insulin.

[0079] A coarse emulsion was prepared by combining an oil phase mixture and an aqueous phase mixture comprising insulin. The oil phase mixture was made by heating a mixture of 6.04 g of ceteareth-20, 8.13 g of oleic acid, 0.63 g of cetyl pyridinium chloride, 8.4 g of medium chain triglyceride oil, and 8.4 g of food grade mineral oil (70 cps) to give a clear solution. The aqueous phase mixture comprising insulin was prepared by dissolving insulin (35.4 mg) in 27.0 g of an 0.1 molal sodium bicarbonate solution by stirring for 30 minutes to give a clear solution. Polysorbate 80 (0.89 g) was added to the clear solution, and stirring continued to give a clear, homogeneous solution. Zinc chloride solution in deionized water (2.49 g of a 0.5 weight percent solution) was added to the stirring solution of insulin, polysorbate 80 and sodium bicarbonate at a rate of 0.45 g minute using a syringe pump to give a slightly hazy, transparent solution. Citric acid (0.23 g) was added to the aqueous phase mixture which caused it to foam.

[0080] The oil phase mixture (31.7 g) was warmed to about 60 °C and added to the aqueous phase mixture with stirring to give an opaque white dispersion with the viscosity of syrup. The coarse emulsion contained 0.057% insulin, 1.43% polysorbate 80, 9.7% ceteareth-20, 13.0% oleic acid, 13.5% medium chain triglyceride oil, 13.5% food grade mineral oil, 1.0% cetyl pyridinium chloride, 0.36% sodium bicarbonate, 0.37% citric acid, 0.020% zinc chloride, and 47.0% water.

[0081] The coarse emulsion was then extruded to give a concentrated nanoparticle dispersion. An apparatus was made consisting of a heat exchanger, hot plate, modified twin screw extruder, and syringe pump. The heat exchanger consisted of coiled copper tubing (5 mm OD x 4 mm ID) located in a one inch diameter hole in a 3 in x 6 in x 12 in aluminum block in which the one inch diameter hole traversed the 6 inch width of the block. The volume of the copper coil was approximately 11.5 mL. The modified twin screw extruder was a Thermo Fisher Process 11 extruder onto which a water cooled copper block heat exchanger had been added. The coarse emulsion was loaded into a 50 mL syringe and pumped through the heat exchanger heated to 89 °C at a rate of 12.5 mL / min and into the extruder, and during the extrusion processing, the temperatures of the zones were: zone 1 (inlet) = not recorded, zone 2 = 95 °C, zone 3 = 95 °C, zone 4 = 60 °C, zone 5 = 24 °C, zone 6 = 14 °C, zone 7 = 14 °C, zone 8 = 11 °C, and die = 24 °C. The screw rotation rate for extrusion was 400 rpm. The extruded nanoemulsion had the appearance of a translucent soft gel and when measured using a Microtrac NanoFlex DLS, the volume average particle size was 53.5 nm and number average particle size was 35.2 nm. When diluted with 1 / 8 X phosphate buffered saline, at pH 7.00, the zeta potential was negative 9.7 mV. The nominal insulin concentration calculated on the basis of formulation was 568 ppm by weight.Example 2. Subcutaneous injection of nanoparticle dispersions containing insulin

[0082] Three weeks after extrusion, after having been stored at 4 °C, 297 mg of the nanoparticle dispersion was diluted to 5.50 g with 4% bovine serum albumin / IX PBS to give a dilute nanoparticle dispersion with nominal insulin concentration 30.7 ppm, equivalent to 0.83 lU / g. Approximately 150 pL of the nanoparticle dispersion was administered by subcutaneous injection to each of four healthy, non-diabetic female C57BL / 6J mice weighing approximately 25 g which had been fasted for 4 hours to give an administered insulin dose of 5 lU / kg. Separately, approximately 150 pL of insulin dissolved in 4% bovine serum albumin / IX phosphate buffered saline solution at pH 7.4 with 0.82 IU insulin / g was administered by subcutaneous injection to each of four healthy, non-diabetic female C57BL / 6J mice weighing approximately 25 g which had been fasted for 4 hours to give an administered insulin dose of 5 lU / kg. After administration, blood samples were withdrawn at one hour intervals for 5.5 hours, and the blood glucose level determined using Tru Metrix Self Monitoring Blood Glucose Meter (product of Trividia Health). The glucose concentration as a percentage of the bloodglucose concentration just before administration that is, 100 * (blood glucose at time = t) / (blood glucose at t = 0) was calculated at each time interval and the results averaged over four mice. The % glucose concentration vs time for subcutaneously administered native insulin and subcutaneously administered insulin nanoparticle dispersion is shown in Figure 1. The area above the curve (AAC) of % blood glucose (average of 4 mice) was calculated as S (100 - % blood glucose at t = n) * (tn - tn-i) and the % bioavailability of the nanoparticle dispersion calculated as bioavailability = 100 * (AAC5.5 hr, nanoparticle insulin) / (AAC5.5 hr, subcutaneous insulin) was 43%.

[0083] The course emulsion prepared as above contained only ingredients on the US Food and Drug Administration Inert Ingredients Guide for oral administration.

[0084] Surprisingly, it is shown that 43% of the insulin retained its physiological functionality through the extrusion process, and induced a strong physiological response after subcutaneous injection that matches the physiological response to subcutaneously administered native insulin.Example 3. Oral administration of the nanoparticle dispersion comprising bioactive insulin.

[0085] A nanoparticle dispersion comprising bioactive insulin was prepared generally as above for Example 1. An oil phase mixture was made by heating a mixture of 19.7 g of ceteareth-20, 21.6 g of sorbitan stearate, 1.30 g of cetyl pyridinium chloride, 32.5 g of medium chain triglyceride oil, 32.7 g of food grade mineral oil (70 cps), and 10.3 g of linalool to 75 °C to give a clear solution. Separately, an aqueous phase mixture was prepared by dissolving insulin (50.6 mg) in 28.6 g of 0.1 molal sodium bicarbonate solution by stirring for 30 minutes to give a clear solution. Polysorbate 80 (1.47 g) was added and stirring continued to give a clear, homogeneous solution. Zinc chloride solution in deionized water (10.17 g of 0.2 weight percent solution) was added to the stirring solution of insulin, polysorbate 80 and sodium bicarbonate at a rate of 0.30 g minute using a syringe pump to give a slightly hazy, transparent solution. The particle size of the zinc - insulin - polysorbate 80 micelles in the aqueous phase mixture was measured using a Microtrac NanoFlex DLS and found to have a volume average particle size of 7.18 nm and number average particle size of 5.98 nm. A coarse emulsion was then prepared by heating the oil phase mixture (15.4 g) to 57 °C and adding 19.9 g of the aqueous phase zinc-insulin-polysorbate mixture with stirring to give an opaque white dispersion with viscosity of light syrup.

[0086] The coarse emulsion was extruded to produce a nanoparticle dispersion comprising bioactive insulin. As shown in Figure 2, an apparatus 100 consisting of a heatexchanger 102, hot plate 104, magnetic stirrer 106, and syringe 108 was assembled. The heat exchanger contained a coiled copper tubing 112 (4 mm OD x 3 mm ID) located in a one inch diameter hole in a 1 in x 1 in aluminum beam 114 which was filled with tin metal. The volume of the copper coil 112 was approximately 5.7 mL. Underneath the exit of the heat exchanger 102, a 150 mL beaker 116 was filled with 40.1 g of water at 0 °C and stirred using a magnetic stirrer 106. The coarse emulsion was loaded into a 30 mL syringe 108, and with the heat exchanger 102 heated to 94.7 °C, 26.1 g of the coarse emulsion was pumped through the coiled copper tubing 112 and into the stirring water in about 90 seconds, whereupon the temperature of the heat exchanger 102 dropped to 85.6 °C. The product of the addition of heated coarse emulsion to cold water was a nanoparticle dispersion with the appearance of a translucent to opaque liquid. The coarse emulsion nanoparticle dispersion contained 0.028% insulin, 0.82% polysorbate 80, 2.85% ceteareth-20, 3.12% sorbitan stearate, 4.7% medium chain triglyceride oil, 4.7% food grade mineral oil, 0.19% cetyl pyridinium chloride, 1.49% linalool, 0.13% sodium bicarbonate, 0.011% zinc chloride, and 81.9% water.

[0087] When measured using a Microtrac NanoFlex DLS, the volume average particle size was 47.1 nm and number average particle size was 35.7 nm. When the nanoparticle dispersion was diluted (2% by weight) in 0.6 X phosphate buffered saline with pH 7.02, the zeta potential as measured using a Malvern Zetasizer Nano ZS was determined to be positive 3.7 mV. The insulin concentration of the nanoparticle dispersion was 7.6 lU / g. The insulin nanoparticle dispersion contained only ingredients considered as safe for use in oral drugs and food and beverages by the US Food and Drug Administration (FDA).

[0088] Approximately 30 minutes after preparation, approximately 150 pL of the nanoparticle dispersion was administered by oral gavage to each of four healthy, non-diabetic female C57BL / 6J mice weighing approximately 25 g which had been fasted for 4 hours to give an administered insulin dose of 46 lU / kg. Separately, approximately 150 pL of insulin dissolved in 4% bovine serum albumin / IX phosphate buffered saline solution at pH 7.4 with 0.82 IU insulin / g was administered by subcutaneous injection to each of four healthy, nondiabetic female C57BL / 6J mice weighing approximately 25 g which had been fasted for 4 hours to give an administered insulin dose of 5 lU / kg. After administration, blood samples were withdrawn at half hour intervals for one hour and then at one hour intervals for nine more hours, and the blood glucose level determined using Tru Metrix Self Monitoring Blood Glucose Meter (product of Trividia Health). The glucose concentration as a percentage of the blood glucose concentration just before administration, that is, 100 * (blood glucose at time = t) / (blood glucose at t = 0) was calculated at each time interval and the results averaged over four mice.The % glucose concentration vs time for subcutaneously administered native insulin and orally administered insulin nanoparticle dispersion is shown in Figure 3. From this graph, the Tmax (time to maximum % glucose reduction) was determined to be 1 hour for subcutaneously administered native insulin and 7 hours for orally administered insulin nanoparticle dispersion. The area above the curve (AAC) of % blood glucose (average of 4 mice) was calculated as S (100 - % blood glucose at t = n) * (tn - tn-i) and the % bioavailability of the nanoparticle dispersion was calculated as [(AACio hr, nanoparticle insulin) / (dose, lU / kg nanoparticle insulin] / [(AACiohr, subcutaneous insulin) / (dose, lU / kg subcutaneous insulin)].Discussion

[0089] Incredibly, the overall oral bioavailability from formulation and processing to administration was 24%. This experiment is believed to be the first that demonstrates the delivery of a physiologically significant dose of bioactive insulin to each of the mice examined by oral administration of the insulin-containing nanoparticles. Even more surprisingly, the % bioavailability calculated as above includes losses from the destruction of insulin during processing, destruction in the gastrointestinal tract, and incomplete absorption of insulin by the animal.

[0090] Further still, the physiological response to the orally administered insulin nanoparticles resulted in a Tmax (approximately 7 hours) that was greatly increased relative to the subcutaneous insulin injection (approximately 1 hour). Thus, the resulting blood glucose concentration as shown in Figure 3 was shown to be much more consistent across the 10 hour examination than for the subcutaneous insulin injection.

[0091] The extended Tmax further demonstrates that the nanoparticle dispersions containing insulin as disclosed herein provide an effective and convenient oral administration pathway for insulin. Particularly, in view of the results of Figure 3, it is contemplated that nanoparticle dispersions disclosed herein may support treatment methods that rely on dosing regimens of once daily, twice daily, or three times daily, to achieve a consistently steady blood glucose level.

[0092] Without being bound by theory, the presence of Zn within the nanoparticles may play a role in the extended T max.

[0093] It follows that formulations comprising the nanoparticle dispersions are also contemplated herein. For instance, it is contemplated that adding a lyophilization step to the methods of preparing nanoparticle dispersions disclosed herein may retain the insulin nanoparticles in their initial state until ready for use, providing a shelf-storage product that maybe activated by dissolution. Nanoparticle dispersions should maintain physical characteristics upon being reconstituted from a lyophilized state.

[0094] In certain aspects, nanoparticle dispersions disclosed herein can further comprise one or more cryoprotectants within the formulation to preserve physical characteristics of the dispersions throughout the freezing process. In certain aspects, cryoprotectants suitable for inclusion within nanoparticle dispersions of the present disclosure can include one or more of diethylene glycol, dimethyl sulfoxide, ethylene glycol, glycerin, propylene glycol, sorbitan, trehalose, sucrose, mannitol, and the like.

[0095] Alternately, or additionally, lyophilization processes to dry the nanoparticle dispersions can be promoted under rapid freezing-drying cycles.

[0096] It is therefore further contemplated that such dissolution may be accomplished by oral administration, and therefore in certain aspects it is contemplated that the lyophilized nanoparticle dispersions containing insulin may be incorporated into any number of orally acceptable forms (e.g., pills, tablets, capsules, lozenges, gummies).

[0097] It is also contemplated that higher pH formulations may preferentially retain the insulin within the nanoparticles. Thus, nanoparticle dispersions may be prepared (or reconstituted) in a higher pH aqueous solution to retain the insulin within the nanoparticles, particularly prior to oral administration to a patient.

[0098] It is also contemplated herein that alternatives to insulin, (e.g., GLP-1 receptor agonists such as semaglutide, liraglutide, exenatide, dulaglutide) and / or insulin derivatives (e.g., insulin glargine) may be incorporated into the nanoparticle dispersions herein for oral administration, either additionally or alternatively to native insulin. As will be understood by those of skill in the art, the oral administration of insulin via nanoparticles as described herein may also be applied in combination with other diabetes treatments, for instance, pramlintide.

Claims

CLAIMSWhat is claimed is:

1. A method for treating diabetes comprising orally administering to a patient in need thereof a therapeutically effective dose of a nanoparticle dispersion comprising insulin.

2. The method of claim 1, wherein the oral bioavailability of insulin is in a range from 20% to 75%.

3. The method of claim 1, wherein the oral bioavailability of insulin is in a range from 5% to 200% greater than that of an otherwise identical method comprising oral administration of a formulation that does not comprise lipid nanoparticles containing insulin.

4. The method of claim 1, having a Tmax in a range from 5 hours to 8 hours.

5. The method of claim 1, having a Tmax in a range from 1 hour to 8 hours greater than that of an otherwise identical method comprising subcutaneous injection of insulin.

6. A nanoparticle dispersion comprising insulin, wherein the nanoparticle dispersion comprises an overall oral bioavailability in mice of at least 10% relative to the amount of insulin incorporated within the nanoparticle dispersion.

7. The nanoparticle dispersion of claim 6, having a concentration of insulin of in a range from 100 ppm to 1000 ppm.

8. The nanoparticle dispersion of claim 6, further comprising zinc.

9. The nanoparticle dispersion of claim 3, wherein an amount of zinc in the nanoparticle dispersion is in a range from 0.001 wt. % to 1 wt. %.

10. The nanoparticle dispersion of claim 6, having a volume average particle diameter in a range from 20 nm to 120 nm.

11. The nanoparticle dispersion of claim 6, comprising: from 0.01 wt. % to 20 wt. % of one or more bioactive agents; from 0.1 wt.% to 20 wt.% of one or more high hydrophile-lipophile-balance (HLB) surfactants; from 0.1 wt.% to 20wt.% of one or more low hydrophile-lipophile-balance (HLB) surfactants; from 10 wt.% to 45 wt.% of one or more water immiscible oils; and from 25 wt.% to 75 wt.% water.

12. A formulation comprising the nanoparticle dispersion of any one of claims 6- 11.

13. The formulation of claim 12, further comprising one or more pharmaceutically acceptable adjuvants.

14. The formulation of claim 12, wherein the nanoparticle dispersion is a lyophilized nanoparticle dispersion.

15. The formulation of claim 12, wherein the nanoparticle dispersion comprises stabilizer (e.g., a saccharide).

16. A method for preparing a nanoparticle dispersion comprising insulin, the method comprising: preparing an oil phase mixture; preparing an aqueous phase mixture, the aqueous phase mixture comprising insulin; combining the oil phase mixture and the aqueous phase mixture to form an emulsion; flowing the emulsion through a heat exchanger at a first temperature; and applying a shearing force to the emulsion at a second temperature to form a nanoparticle dispersion; wherein an amount of bioactive insulin remaining in the nanoparticle dispersion is at least 25% relative to the of the amount of insulin in the emulsion.

17. The method of claim 16, wherein applying a shearing force to the emulsion comprises stirring the emulsion with a magnetic stirrer.

18. The method of claim 16, wherein the first temperature is in a range from 50 °C to 98 °C.

19. The method of claim 16, wherein the second temperature is in a range from 5°C to 55 °C.

20. The method of claim 16, wherein an amount of bioactive insulin in the nanoparticle dispersion is in a range from 20% to 85% of an amount of insulin in the emulsion prior to forming the nanoparticle dispersion.

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