CN116897038A

CN116897038A - Delivery of oral therapeutic agents

Info

Publication number: CN116897038A
Application number: CN202180092373.4A
Authority: CN
Inventors: C·E·科恩; J·斯特拉坎
Original assignee: RMIT University
Current assignee: RMIT University
Priority date: 2020-11-30
Filing date: 2021-11-30
Publication date: 2023-10-17

Abstract

The present application relates to a dosage form for oral delivery of a therapeutic agent to a subject, the dosage form comprising: (i) A lipid nanocarrier formulation comprising a therapeutic agent and a lipid in the form of an mesophase; and (ii) an enteric coating encapsulating the lipid nanocarrier formulation.

Description

Delivery of oral therapeutic agents

The present application claims priority from australian provisional patent application No. 2020904426, 2021, australian provisional patent application No. 2021900348, filed 11, 30, 2021, 2, 12, the contents of which are incorporated herein by reference.

Technical Field

The present disclosure relates to formulations for oral delivery of therapeutic agents, methods of making the formulations, and uses thereof.

Background

Intravenous (IV), intramuscular (IM) and Subcutaneous (SC) injections are the three injection routes most commonly used in drug delivery.

The mechanism of absorption and the nature of the drug are fundamental factors in determining an appropriate delivery system to achieve maximum bioavailability and effectiveness. For example, insulin may be 1) injected subcutaneously (intradermally) by an insulin syringe, a pre-filled pen device, or an insulin pen, as the case may be; 2) For some type 1 diabetics, delivery may be in the form of insulin infusion through a wearable personal insulin pump; or 3) by intravenous insulin administration.

For the intravenous route, the needle is inserted directly into the vein. The drug-containing solution may be administered in a single dose or as a continuous infusion. For infusion, the solution is moved by gravity (from a collapsible plastic bag) or, more commonly, by an infusion pump through a thin flexible tube to a tube (catheter) inserted into the vein (typically in the forearm). Intravenous administration is the best method for delivering precise doses in a rapid, well controlled manner throughout the body.

When administered intravenously, the drug is immediately delivered into the blood stream and tends to be more rapidly effective than when administered by any other route.

For the subcutaneous route, the needle is inserted into adipose tissue just below the skin. After injection, the drug enters small blood vessels (capillaries) and is carried away by the blood stream. Alternatively, the drug passes through lymphatic vessels to the blood stream. Large-sized protein drugs (e.g., insulin) typically reach the blood stream through lymphatic vessels, as these drugs slowly migrate from the tissue into capillaries. Many protein drugs are administered subcutaneously because oral administration of these drugs can be disrupted in the digestive tract.

When a larger volume of drug is required, the intramuscular route is superior to the subcutaneous route. Because the muscles are located below the skin and adipose tissue, longer needles are used. The drug is typically injected into the muscles of the upper arm, thigh or buttocks. The rate at which the drug is absorbed into the blood stream depends in part on the blood supply to the muscle: the less the blood supply, the longer the time it takes for the drug to be absorbed.

Drugs administered by IV, SC or IM avoid the Gastrointestinal (GI) environment. However, injection can lead to significant problems including needle-related phobia and pain, unsafe and mishandled needles, trained medical personnel, muscle atrophy, and bone and nerve injuries.

Because of these problems, oral drug delivery remains the preferred route of administration. However, not all drugs have desirable physicochemical and pharmacokinetic properties that favor oral administration, mainly due to poor bioavailability. In some cases, this results in the selection of other routes of administration, which may compromise patient convenience and increase the risk of non-compliance. Poor bioavailability necessitates administration of higher than normally required oral doses, which often results in economic waste, risk of toxicity, instability and unpredictable responses. There remains a need to provide drug delivery dosage forms that enhance the oral bioavailability of drugs.

Disclosure of Invention

Surprisingly, as disclosed herein, oral delivery dosage forms have been developed that increase the bioavailability of malabsorptive therapeutic agents; to improve the clinical efficacy of oral administration. The dosage form comprises a formulation of the therapeutic agent in a lipid nanocarrier. The formulation is then encapsulated in an enteric coating. Surprisingly, it has been shown that the dosage form can be used to improve the absorption and bioavailability of insulin following oral administration.

It has also surprisingly been found that the dosage form can also be used to improve the absorption and bioavailability of therapeutic agents such as proteins (e.g. hormones) and small molecules (e.g. antibiotics) following oral administration.

Thus, in one aspect, the present disclosure relates to a dosage form for oral delivery of a therapeutic agent to a subject, the dosage form comprising: a lipid nanocarrier formulation comprising a therapeutic agent; enteric coating encapsulating the lipid nanocarrier formulation.

Another aspect of the present disclosure relates to a dosage form for oral delivery of a therapeutic agent to a subject, the dosage form comprising a lipid nanocarrier formulation comprising a therapeutic agent and a lipid in an mesophase form; enteric coating encapsulating the lipid nanocarrier formulation.

In some embodiments, the lipid nanocarrier formulation comprises a lipid in the mesophase form.

The lipid mesophase may comprise, for example, an inverse bicontinuous cubic phase or a bicontinuous cubic phase.

In other embodiments, the mesophase may comprise an inverse hexagonal phase.

In other embodiments, the mesophase may comprise an inverse bicontinuous cubic phase, an original cubic phase, a double diamond cubic phase, a spiral twenty tetrahedron (gyroid) cubic phase, a hexagonal phase, an inverse hexagonal phase, a cubic structure (cubosomes), or a hexagonal structure (hexasomes).

In some embodiments, the mesophase may comprise a cubic structure or a hexagonal structure. Thus, other embodiments relate to mesophases configured as cubic structures or hexagonal structures.

In some embodiments, the lipid nanocarrier comprises a lipid selected from the group consisting of a mono-, di-, or tri-substituted glycerol, a charged lipid, a branched lipid, and a glycolipid. In one embodiment, the lipid nanocarrier is a long chain lipid.

In some embodiments, the charged lipid is dioleoyl-3-trimethylammoniopropane (DOTAP) which is present in an amount up to and including 10% of the lipid nanocarrier formulation.

Such lipids can advantageously direct the transfer of insulin or derivatives thereof into the lymphatic system, avoiding first pass metabolism and improving bioavailability.

In other embodiments, the lipid nanocarrier comprises a lipid of formula I:

wherein at least one R is of formula II and the remaining R groups are independently selected from hydrogen or formula II:

wherein w, x, y and z are independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12; wherein the dashed line represents the presence or absence of a bond; and wherein the wavy bonds represent E or Z bond geometry in the presence of the bond.

In other embodiments, the lipid nanocarrier comprises a lipid of formula III:

In some embodiments, the lipid nanocarrier comprises a lipid selected from the group consisting of: glycerol monooleate, phytantriol and glycerol monopalmitoleate.

In some embodiments, the glycerol monooleate is present at about 40% to 80% by weight of the formulation.

In some embodiments, the phytantriol is present at about 60% to 75% by weight of the formulation.

In some embodiments, the therapeutic agent is selected from insulin or a derivative thereof, a steroid hormone, an antimicrobial agent such as an antibiotic, a protein such as a hormone, and a peptide such as a neuropeptide.

In some embodiments, the insulin or derivative thereof is selected from insulin glargine (Lantus, basaglar, toujeo), insulin deltoid (Levemir), insulin deltoid (Tresiba), NPH (eurine N, norand reion insulin N), fast acting insulin, and short acting insulin.

In some embodiments, the insulin or derivative thereof is present in the formulation from 0.01% to 1% by weight of the formulation.

In some embodiments, the nanocarriers comprise aqueous channels having a size of 1nm to 17 nm. Therapeutic agents, such as insulin, may be entrapped within the aqueous channels of the nanocarriers.

In some embodiments, the enteric coating is soluble in a range of about pH 4.5 to pH 7.2.

In other embodiments, the enteric coating is soluble in a range of about pH 5.0 to pH 6.0. Advantageously, the solubility of the enteric coating at these pH ranges may allow the coating to dissolve in the small intestine.

In other embodiments, the thickness of the enteric coating is in the range of 0.01nm to 1.00 mm.

In some embodiments, the lipid nanocarrier formulation is encapsulated in an enteric coating having a thickness in the range of 0.07mm to 0.4 mm.

In some embodiments, the lipid nanocarrier formulation is aqueous. In a further embodiment, the water content of the lipid nanocarrier formulation is from 1% to 70% by weight of the lipid nanocarrier formulation.

In some embodiments, the lipid nanocarrier formulation has a water content up to and including 48% by weight of the lipid nanocarrier formulation, and wherein the lipid is glycerol monooleate, or has a water content up to and including 48% by weight of the lipid nanocarrier formulation, and wherein the lipid is phytantriol.

Advantageously, such aqueous lipid nanocarrier formulations may help to improve degradation of therapeutic agents in vivo, for example in the human gastrointestinal tract.

In other embodiments, the dosage form may be a capsule comprising a fill and a shell encapsulating the fill, the fill comprising a lipid nanocarrier formulation, and the shell comprising an enteric coating.

In other embodiments, the shell is coated with an enteric coating on at least one of the shell surface facing the filler and the outer shell surface.

In other embodiments, the enteric coating on the shell surface facing the filling and on the outer shell surface each independently has a thickness in the range of 30 μm to 380 μm.

In a further embodiment, the lipid nanocarrier formulation further comprises a swelling agent. In some embodiments, the water content of the formulation ranges up to and including 70% by weight of the lipid nanocarrier formulation.

Another aspect of the invention relates to a method of preparing a dosage form comprising the steps of providing a lipid nanocarrier formulation and encapsulating the lipid nanocarrier formulation in an enteric coating.

In some embodiments, the method comprises contacting the lipid, the therapeutic agent, and the aqueous solvent under conditions sufficient to, for example, promote self-assembly of the lipid into an mesophase.

In some embodiments, a lipid to aqueous solvent ratio of about 60:40w/w is used.

In some embodiments, high pressure homogenization is used to promote lipid self-assembly into an mesophase.

In some embodiments, an enteric coating is applied to at least one of the shell surface and the outer shell surface facing the filler. In alternative embodiments, the enteric coating may be applied directly to the lipid nanocarrier formulation.

In another aspect, the present disclosure provides a dosage form as described for use in the treatment of diabetes, wherein the therapeutic agent is insulin or a derivative thereof.

In another aspect, the present disclosure provides a dosage form as described for use in the treatment or prevention of diabetes, a condition mediated by a bacterial infection, a condition mediated by human growth hormone, or a condition mediated by blood coagulation, and wherein the therapeutic agent is present in a therapeutically or prophylactically effective amount.

In another aspect, the present disclosure provides a method for treating or preventing diabetes comprising administering to a subject in need thereof the described dosage form, wherein the therapeutic agent is insulin or a derivative thereof.

In another aspect, the present disclosure provides a method for treating or preventing diabetes, a disorder mediated by a bacterial infection, a disorder mediated by human growth hormone, or a disorder mediated by coagulation, comprising administering to a subject in need thereof the described dosage form and wherein the therapeutic agent is present in a therapeutically or prophylactically effective amount.

In another aspect, the present disclosure provides the use of a therapeutic agent in the manufacture of a described dosage form for the treatment or prevention of diabetes, wherein the therapeutic agent is insulin or a derivative thereof.

In another aspect, the present disclosure provides the use of a dosage form as described in the manufacture of a medicament for the treatment or prevention of diabetes, a condition mediated by a bacterial infection, a condition mediated by human growth hormone, or a condition mediated by coagulation, and wherein the therapeutic agent is present in a therapeutically or prophylactically effective amount.

The scope of the present disclosure is not limited by the specific examples described herein, which are intended for purposes of illustration only. Functionally equivalent products, compositions, and methods are clearly within the scope of the disclosure.

Unless specifically stated otherwise, any examples/embodiments of the disclosure herein should be considered as applied to any other examples/embodiments of the disclosure mutatis mutandis.

Drawings

Fig. 1 is an example showing that the lipid nanocarriers promote lymphatic absorption of a therapeutic agent in, for example, the small intestine, without wishing to be bound by theory.

Fig. 2 is a diagram of a specific set of examples showing a dosage form comprising the following formulation: (1) a lipid nanocarrier comprising the therapeutic agent (2); (3) enteric coating. The figure represents a specific embodiment in which the lipid nanocarriers are in a bicontinuous cubic phase.

FIG. 3, panel (A) blood fluorescence of Green Fluorescent Protein (GFP) in plasma as determined by fluorescence measurement (470/515 excitation/emission) within 6 hours after SC injection of 500mL GFP (100 pg/mL) in Sprague Dawley rats. Panel (B) blood fluorescence of GFP in plasma of Sprague Dawley rats, determined by fluorescence measurement (470/515 excitation/emission) within 6 hours after administration of 50pg GFP contained in the lipid cubic phase in an enteric capsule. Panel (C) average blood fluorescence of GFP in Sprague Dawley rats over 6 hours after administration by SC injection or enteric capsule/cubic phase. Panel (D) GFP fluorescence (470/515 excitation/emission) from plasma obtained from Sprague Dawley rats, all samples were collected within 6 hours.

Fig. 4. Blood glucose levels change over time within 24 hours after four (4) individual rats (rats 2, 49 and 12 in subsequent trials) were injected with 15IU of actipid insulin by SC. Data are shown as two different days per rat (triangle and circle symbols). The average increase in blood glucose was determined from data between 450 and 1450 minutes.

Fig. 5 change in blood glucose levels over time after delivery of fast acting (actipid) insulin by SC injection (circles) and perforated lipid cubic phase filled enteric vesicles (triangles). The estimated Blood Glucose (BG) increase in rat 1 over this period of time (dashed line) is based on the data in fig. 4.

Fig. 6 change in blood glucose levels over time after delivery of fast acting (actipid) insulin by SC injection (circles) and lipid cubic phase filled enteric vesicles (triangles). The estimated Blood Glucose (BG) increase in rats 2-4 during this period (dashed line) is based on the data in fig. 4.

Fig. 7 change in blood glucose levels over time after delivery of fast acting (actipid) insulin to four (4) rats (rats 5-8) by SC injection (circles) and filling of the enteric capsule (triangles) by lipid cubic phase. The estimated Blood Glucose (BG) increase in rats 5-8 over this period of time (dashed line) is based on the data in fig. 4.

Fig. 8 change in blood glucose levels over time following delivery of slow acting (Levemir) insulin to six (6) rats (rats 9-14) by SC injection (circles) and lipid cubic phase filled enteric vesicles (triangles). The estimated Blood Glucose (BG) increase in rats 9-14 during this period (dashed line) is based on the data in fig. 4.

Fig. 9. (a) change in average blood glucose levels over time after delivery of fast acting (actipid) insulin (1U) by SC injection (triangle) and lipid cubic phase filled enteric capsule (open circles) (rats 2-4) (stage 1). As a result of capsule perforation, rat 1 (capsule) was not used. (B) Changes in average blood glucose levels over time (phase 1) following slow acting (Levemir) insulin (1U) delivery by SC injection (asterisk) and filling of the enteric capsule (filled circles) by lipid cubic phase (rats 9-10). (C) The average blood glucose level varies with time after delivery of fast acting (actipid) insulin (1U) by SC injection (triangle) and lipid cubic phase filling of the enteric capsule (open circles) (rats 5-8) (phase 2). (D) Changes in average blood glucose levels over time after slow acting (Levemir) insulin (1U) delivery by SC injection (asterisk) and filling of the enteric capsule (filled circles) by lipid cubic phase (rats 11-14) (phase 2). All blood glucose readings were normalized to initial BG level 0.

FIG. 10 shows that Human Growth Hormone (HGH) was found in plasma collected from Sprague Dawley rats within 5 hours. (A) Plasma concentrations measured within 300 minutes (5 hours) after administration by SC injection are shown. For all four rats, the drug concentration in the plasma increased immediately to a maximum in the range of 20-32ng/ml at 60 minutes, then gradually decreased to 0ng/ml over the following 4 hours, reaching the baseline drug concentration by 300 minutes. Panel (B) shows the measured concentration of HGH in plasma over 300 minutes (5 hours) after oral capsule administration.

FIG. 11 shows that human factor (HCFX) was found in plasma collected from Sprague Dawley rats over 5 hours. (A) The HCFO concentration in plasma measured within 300 minutes (5 hours) after administration by SC injection is shown. For all four rats, the drug concentration in the plasma increased immediately to a maximum in the range of 9-10ng/ml at 30 minutes, followed by a sharp decrease to +.1 ng/ml in the following 90 minutes. (B) The HCFO concentration in plasma measured within 300 minutes (5 hours) after oral capsule administration is shown.

Figure 12 shows that vancomycin was found in plasma collected from Sprague Dawley rats over 5 hours. (A) The concentration of vancomycin in plasma measured within 300 minutes (5 hours) after administration by SC injection is shown. For all four rats, the drug concentration in the plasma increased immediately to a maximum of about 1500ng/ml at 30 minutes, followed by a sharp drop to the baseline value within 180 minutes. (B) The concentration of vancomycin in plasma measured within 300 minutes (5 hours) after oral capsule administration is shown.

Fig. 13 shows that meropenem was found in plasma collected from Sprague Dawley rats over 5 hours. (A) The concentration of meropenem in plasma measured within 300 minutes (5 hours) after administration by SC injection is shown. For all four rats, the drug concentration in the plasma increased immediately at 30 minutes to a maximum in the range of 37-40ng/ml, and then gradually decreased to the baseline value over 240 minutes. (B) The concentration of vancomycin in plasma measured within 300 minutes (5 hours) after oral capsule administration is shown.

FIG. 14 shows a 1D SAXS pattern of human growth hormone (1 mg/ml) encapsulated in MO at 38% aqueous phase.

FIG. 15. 1DSAXS pattern of human coagulation factor X (1 mg/ml) encapsulated in MO at 38% aqueous phase content. Calculated lattice parameters of QIID phase are

FIG. 16. 1D SAXS pattern of vancomycin (15 mg/ml) encapsulated in MO at 38% aqueous phase.

Figure 17 (left) double enteric (Eudragit L100) coated capsules remained stable inside and outside after 24 hours in pH 4 with (middle) standard capsules empty, showing t=0. The (right) inner and outer were coated with a single outer coating of pH 4 medium, indicating that the capsule was destroyed after 8 hours.

Fig. 18. Lattice parameters of MO and PT host (bulk) cubic phases taken with this phase after addition of insulin at 25 ℃ at concentrations ranging from 0 to 10 mg/ml. The water content of the main phase of MO was 48% and the water content of the main phase of PT was 28%. Error bars represent standard deviation of three (3) replicates.

FIG. 19 is a 1D SAXS pattern of intensities versus q for MO and PT host cubic phases with encapsulated insulin concentrations ranging from 0 to 10mg/ml.

FIG. 20 (A) percentage of encapsulated insulin (2 mg/ml) released from MO over time. The data are in accordance with the Ritger-Peppas model in (B) (solid line). The percentage of encapsulated insulin (2 mg/ml) released from MO as a function of time. For the first 60% of release of encapsulated insulin, the data were in accordance with Higuchi model (solid line). (C) Percentage of encapsulated insulin released from PT over time (2.5 mg/ml). The data were in accordance with the Ritger-Peppas model (solid line). D. The percentage of encapsulated insulin released from PT (2.5 mg/ml) as a function of time. For the first 60% of release of encapsulated insulin, the data were in accordance with Higuchi model (solid line). Error bars represent standard deviation of three (3) replicates.

FIG. 21 (left) far UV CD spectra of free insulin (0.2 mg/ml) over time within 30 minutes after chymotrypsin (0.02 mg/ml) addition. (right) after chymotrypsin (0.02 mg/ml) addition, insulin encapsulated in phytantriol host cubic phase (0.2 mg/ml) was subjected to far UV CD spectra over time in 132 minutes.

FIG. 22 (above) representative far UV CD spectra of insulin (0.2 mg/ml) at 2 minutes and 30 minutes after chymotrypsin (0.02 mg/ml) addition. Representative far UV CD spectra of insulin (0.2 mg/ml) encapsulated in phytantriol host cubic phase at 2, 30 and 130 minutes after chymotrypsin (0.02 mg/ml) addition (below).

FIG. 23 (left) near UV CD spectra of free insulin (0.5 mg/ml) over time within 30 minutes after chymotrypsin (0.05 mg/ml) addition. Near UV CD spectra of insulin (0.5 mg/ml) in the MO host cubic phase over time within 132 minutes after chymotrypsin (0.05 mg/ml) addition.

FIG. 24 (above) representative near UV CD spectra of insulin (0.5 mg/ml) in solution at 2 minutes and 30 minutes after chymotrypsin (0.05 mg/ml) addition. Representative near UV CD spectra of insulin (0.5 mg/ml) encapsulated in MO host cubic phase at 2, 30 and 130 minutes after chymotrypsin (0.05 mg/ml) addition (below).

Detailed Description

Before describing the present invention in detail, it is to be understood that the terminology used herein is for the purpose of describing embodiments and examples only and is not intended to be limiting.

As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a lipid nanocarrier" includes a combination of two or more such lipid nanocarriers. Similarly, reference to "a therapeutic agent" includes a combination of two or more such therapeutic agents.

Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprises" and "comprising", are not intended to exclude other additives, components, integers or steps. As used herein, "include" means "include". Thus, "comprising" a or B "means" including "A, B, or a and B", without excluding additional elements.

Unless otherwise defined explicitly, all technical and scientific terms used herein should be considered to have the same meaning as commonly understood by one of ordinary skill in the art.

The reference herein to a document or other item given as prior art should not be taken as an admission that the document or item is known or that the information it contains is part of the common general knowledge as at the priority date of any claim.

As used herein, the term "therapeutic agent" refers to a drug, protein, hormone, peptide, compound, or other pharmaceutically or biologically active ingredient.

As used herein, the term "subject" is understood to mean any mammal, preferably a human. The subject may have or be at risk for diabetes.

As used herein, "diabetes" refers to diabetes and related conditions, including type 1 diabetes, type 2 diabetes, gestational diabetes, latent autoimmune diabetes in adulthood, juvenile onset diabetes, neonatal diabetes, and type 3 diabetes.

As used herein, a subject who is "at risk" for developing a disease or recurrence thereof may or may not have a detectable disease or disease symptom, and may or may not have exhibited a detectable disease or disease symptom prior to treatment according to the present disclosure. By "at risk" is meant that the subject has one or more risk factors, which are measurable parameters associated with disease progression, as known in the art and/or as described herein.

As used herein, "disease," "disorder," "condition," and the like, as they relate to the health of a subject, are used interchangeably and have the meaning ascribed to each and all of these terms.

As used herein, the terms "treatment", "treatment" or "treatment" include administration of a therapeutic agent, e.g., to reduce or eliminate at least one symptom of a particular disease or to slow the progression of the disease.

As used herein, the terms "prevention", "prevention" or "prevention" include providing prophylaxis against the occurrence or recurrence of a particular disease. An individual may be susceptible to, or at risk of suffering from, the disease or recurrence, but has not yet been diagnosed with the disease or recurrence.

An "effective amount" refers to an amount effective to achieve the desired result, at least over the necessary dosage and period of time. For example, the desired outcome may be a therapeutic or prophylactic outcome. An effective amount may be provided in one or more administrations. In some examples of the present disclosure, the term "effective amount" refers to an amount necessary to achieve effective treatment or prevention of the diseases described herein. In some examples of the present disclosure, the term "effective amount" refers to the amount required to treat or prevent diabetes. The effective amount may vary depending on the disease to be treated or the factors to be altered, as well as depending on weight, age, ethnic background, sex, health and/or physical condition, and other factors associated with the subject to be treated. In general, an effective amount will fall within a relatively broad range (e.g., a "dose" range), which can be determined by a practitioner through routine experimentation and experimentation. Therefore, the term should not be construed as limiting the disclosure to a particular number. The effective amount may be administered in a single dose or in doses that are repeated one or more times during the course of treatment.

As used herein, the term "prophylactically effective amount" is understood to mean a sufficient amount of a therapeutic agent to prevent or inhibit or delay the onset of one or more detectable symptoms of a disease or its complications, such as inhibiting or delaying the progression of diabetes.

As used herein, "oral delivery" refers to the administration of a therapeutic dosage form by oral cavity for local action or systemic absorption along the gastrointestinal tract (GI).

As used herein, the term "Gastrointestinal (GI) tract" is intended to encompass the mouth, esophagus, stomach, duodenum, small intestine, large intestine (colon), rectum and anus.

As used herein, "fatty acid derivative" or "fatty acid-derived lipid" refers to a product in the form of a condensation reaction with a pendant group of a suitable functional group (e.g., mono-, di-or tri-substituted glycerol), glycolipid, phospholipid, or ethanolamine that is pendant on the head group, resulting in an acyl fatty acid residue. The lipids disclosed herein may be prepared or obtained by any means known in the art, including by biological means and synthetic means.

As used herein, "dosage form" refers to any pharmaceutical formulation suitable for oral delivery of a therapeutic agent, such as insulin or a derivative thereof, including, for example, a pill, tablet, or capsule.

Formulations for oral delivery of therapeutic agents to a subject

The present disclosure relates to dosage forms for oral delivery of therapeutic agents. The therapeutic agent is first formulated into a lipid nanocarrier.

Colloidal drug carriers, such as micelles, nanoemulsions, nanosuspensions, polymeric nanoparticles and liposomes, can overcome many problems in drug delivery, such as solubility and stability. However, these systems are associated with drawbacks such as limited physical stability, aggregation, drug leakage upon storage, lack of suitable low cost mass production methods to produce products of quality approved by regulatory authorities, the presence of organic solvent residues in the final product, cytotoxicity, etc.

The lipids can self-assemble to form 1-D, 2-D, or 3-D symmetrical structured host lipid self-assembled materials. Such host non-dispersed lipid materials have long range order (long range order) in 1D (lamellar phase), 2D (hexagonal phase), or 3D (bicontinuous cubic phase). Lipids can also form self-assembled particles that are either 2D symmetric (hexagonal structures) or 3D symmetric (cubic structures).

The present disclosure relies on structured lipid self-assembled materials as nanocarriers for delivering therapeutic agents. The lipid nanocarriers of the present disclosure may take the form of 2D (hexagonal phase) or 3D (bicontinuous cubic phase) symmetry. The lipid nanocarriers of the present disclosure also include dispersed particles of 2D symmetry (hexagonal structures) or 3D symmetry (cubic structures).

In some examples, the lipids used to prepare the nanocarrier formulation are molecules in which physiological lipids (biocompatible and biodegradable) are present. Preferably, the lipids used have low acute and chronic toxicity. The lipid may be naturally occurring.

In some examples, the lipid has a chain length of C7-C35. In other examples, the lipid is a long chain lipid, such as a C13-C35 chain length. The lipid may be unsaturated, saturated or branched. In other examples, the lipid chain length is C13 to C19. Advantageously, long chain lipids have been demonstrated to enhance lymphatic absorption of therapeutic agents and bypass first pass metabolism by the liver. This effect may be enhanced with increasing lipid chain length, particularly C13 to C19 chain lipids, including glycerol monooleate, phytantriol and glycerol monopalmitoleate. Lymphatic transport may also increase with increasing degree of lipid unsaturation, such as glycerol monooleate. In some embodiments, the therapeutic agent is insulin, e.g., hydrophilic insulin or a derivative thereof.

Examples of long chain lipids that can be used to prepare nanocarrier and therapeutic agent formulations include mono-, di-or tri-substituted glycerol, glycolipids, phospholipids, or ethanolamine derivatives of linear fatty acids.

Examples of such linear fatty acids include: caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachic acid, behenic acid, lignoceric acid, cerotic acid, myristoleic acid, palmitoleic acid, hexadecenoic acid (sapienic acid), oleic acid, elaidic acid, isooleic acid, linoleic acid, trans-linoleic acid, alpha-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, microglial acid, and docosahexaenoic acid, pelargonic acid, undecanoic acid, tridecanoic acid, pentadecanoic acid, heptadecanoic acid, nonadecanoic acid, behenic acid, tricosanoic acid, eicosanoic acid, heptadecanoic acid, nonadecanoic acid, melissic acid, tridecanoic acid, triacontanoic acid, and tetradecanoic acid.

In some examples, the lipid is a mono-, di-, or trisubstituted glycerol of formula I:

wherein at least one R is formula II and the remaining R groups are independently selected from hydrogen or formula II:

wherein w, x, y and z are independently selected from 0, 1, 2, 3, 4, 5, 6 and 7; wherein the dashed line represents the presence or absence of a bond; and wherein the wavy bonds represent E or Z bond geometry in the presence of the bond.

In other examples, w, x, y, and z have formula II and are independently selected from 0, 1, 2, 3, 4, 5, and 6. In other examples, w, x, y, and z have formula II and are independently selected from 0, 1, 2, 3, 4, and 5. In other examples, w, x, y, and z have formula II and are independently selected from 0, 1, 2, 3, and 4. In other examples, w, x, y, and z have formula II and are independently selected from 0, 1, 2, and 3. In other examples, w, x, y, and z have formula II and are independently selected from 0, 1, and 2. In other examples, w, x, y, and z have formula II and are independently selected from 1 and 2.

In a further example, the lipid is a monosubstituted glycerol derived from a fatty acid, comprising a compound of formula III:

wherein w, x, y and z are selected from 0, 1, 2, 3, 4, 5, 6 and 7; wherein the dashed line represents the presence or absence of a bond; and wherein the wavy bonds represent E or Z bond geometry in the presence of the bond.

In other examples, w, x, y, and z have formula III and are independently selected from 0, 1, 2, 3, 4, 5, and 6. In other examples, w, x, y, and z have formula II and are independently selected from 0, 1, 2, 3, 4, and 5. In other examples, w, x, y, and z have formula II and are independently selected from 0, 1, 2, 3, and 4. In other examples, w, x, y, and z have formula II and are independently selected from 0, 1, 2, and 3. In other examples, w, x, y, and z have formula II and are independently selected from 0, 1, and 2. In other examples, w, x, y, and z have formula II and are independently selected from 1 and 2.

In some examples, the lipid is an unsaturated long chain lipid, such as a long chain monoglyceride, such as glycerol monooleate or glycerol monopalmitoleate.

In other examples, the lipid is a branched lipid.

Examples of branched lipids include fatty acid derivatives of mono-, di-, or trisubstituted glycerol, glycolipids, phospholipids, or ethanolamine derivatives. Such fatty acids include mycophenolic acid (mycolipanolic acid), mycocerotic acid, mycolic acid, micolipodienoic acid, trimethylstearic acid, phthioceranic acids, dolichoic acids, phytanic acid, pristanic acid, from branched hydroxy fatty acids (mycolic acid), methoxymycolic acid, ketomycolic acid, 1-monomethyl branched fatty acid, 1-methyl-12-enoic acid and 12-methyl-10-enoic acid, cis-11-methyl-2-dodecenoic acid, tuberculosis stearic acid, lactylic acid, 7-methyl-6-octadecenoic acid and 17-methyl-7-octadecenoic acid, and laetiphoric acid.

Other examples of branched lipids relate to multi-branched lipids, including isoprenoid-like lipids such as phytantriol and those derived from retinoic acid.

In a further example, the lipid is a charged lipid. Examples of charged lipids include phospholipids, such as glycerophospholipids including phosphatidate, phosphatidylserine, phosphoinositide, phosphatidylinositol, phosphophosphatidylinositol, diphosphatidylinositol, triphosphatidylinositol, and phosphosphingolipid (phosphosphingolipid).

In other examples, the phospholipid lipid may be phosphatidylethanolamine or phosphatidylcholine.

Other examples of charged phospholipids include sphingolipids such as ceramide phosphorylcholine, ceramide phosphorylethanolamine, and ceramide phosphoryl lipid.

In some examples, the lipid is a glycolipid lipid. Examples of glycolipids include glyceroglycolipids, galactolipids, gangliosides, globosides, glycosphingolipids and glycophosphatidylinositol.

In other examples, the formulation of the lipid nanocarriers comprises one or more of the described lipids.

Lipid nanocarrier structures

When mixed with an aqueous solution (typically water), lipids can form different structures, such as a lyotropic liquid crystal phase, which can be distinguished by, for example, its characteristic small angle X-ray scattering pattern, particle size, zeta potential, and polydispersity index.

The term "mesophase" is used to denote a unique self-assembled structure between liquid and solid crystalline phases. The liquid phase is a fluid and the ordered crystal structure defines a solid state. The different mesophases include cubic phase, hexagonal phase, lamellar phase, micelles, which are in continuous and dispersed form. Other possible forms of liquid crystal phase include reverse hexagonal phase (H2), bicontinuous reverse cubic phase (V2), including primary phase (Im 3 m), double rhombohedral phase (Pn 3 m), spiral icosahedral phase (Ia 3 d), reverse cubic micelle (I2), sponge (L3), reverse micelle (L2), disordered micelle, vesicle (La), lamellar (La) and liposome.

Self-assembly of lipids can be modulated by varying formulation conditions (e.g., temperature, lipid concentration, homogenization) and by adding modulators and stabilizers. This allows the mesophase to assemble lipid nanoparticles of different morphology. Typically, morphology results in a three-dimensional network of lipid and solvent channels that can be tailored to accommodate different therapeutic agents and have different properties, such as dissolution profiles.

Modulation of the dissolution profile may result in delayed release, allowing targeted release of the therapeutic agent in specific regions of the gastrointestinal tract. For example, eudragit L100-55 may be specifically targeted to the duodenum, while Eudragit L100 may be specifically targeted to the upper small intestine. Lipid nanocarriers comprising a mesophase form of a therapeutic agent (e.g., insulin) have been demonstrated to provide protection from enzymatic degradation in systems that mimic the gastrointestinal environment in vivo. Such environments include at least 2 hours after chymotrypsin addition. In contrast, complete degradation of insulin in water was observed in less than 35 minutes without the protective matrix. This work shows that the mesophase form of the lipids is effective in protecting the encapsulated therapeutic agent from degradation by digestive enzymes.

Without wishing to be bound by theory, aqueous lipid nanocarriers with mesophase form comprising therapeutic agents provide further protection against the gastrointestinal environment. Such dosage forms avoid hydration in the intestinal tract, thereby avoiding exposure of the therapeutic agent to various lipases and peptidases, including chymotrypsin, in the gastrointestinal environment. Furthermore, by avoiding intestinal hydration, the mesophase lipid nanocarriers comprising the therapeutic agent are theoretically excreted from the intestinal tract through the lymphatic system, thereby avoiding circulatory system and first pass metabolism, thereby improving bioavailability and biodistribution.

The therapeutic agent and lipid may be formulated into an intermediate phase by any method known in the art, including, for example, mixing in a syringe, cold or hot high pressure homogenization, emulsion-sonication, solvent emulsification-evaporation, solvent diffusion, microemulsion, solvent injection, and/or double emulsion.

Cubic phase

In some examples, the disclosure relates to lipid nanocarriers in the form of cubic phases. The cubic phase is a non-dispersed lipid nanocarrier formed by lipid self-assembly and has long range order in the three (3) dimensions.

Bicontinuous cubic phase

In a further example, the lipid nanocarriers are in a bicontinuous cubic phase, which is arranged in a pattern of Infinite Period Minimum Surfaces (IPMS). This is further divided into an original phase (Im 3 m), a double diamond phase (Pn 3 m) and a spiral icosahedron phase (Ia 3 d).

The bicontinuous cubic mesophase forms a three-dimensional network of lipid bilayers separated by two water channels. The nanocarrier formulations of this stage may incorporate therapeutic agents of different physicochemical properties.

In some examples, the bicontinuous cubic mesophase may be formulated using unsaturated or long chain monoglycerides (e.g., glycerol monooleate or glycerol monopalmitoleate) or long chain branched lipids (e.g., phytantriol); and varying concentrations of water, resulting in adjustable characteristics including stability and dissolution profile in the digestive tract (e.g., small intestine).

In some examples, the lipid concentration of the bicontinuous cubic phase formulation is 10% to 95% by weight of the formulation. In other examples, the nanocarrier has a lipid concentration of 25 wt% to 85 wt%, in other examples, the nanocarrier has a lipid concentration of 35 wt% to 80 wt%, and in other examples, the nanocarrier has a lipid concentration of 55 wt% to 75 wt%.

In some embodiments, the bicontinuous cubic phase formulation is aqueous and has a water content at room temperature as follows:

cubic phase	From the slave	To the point of
			Water (%) weight of formulation	1	70
Water (%) weight of formulation	20	60
			Water (%) weight of formulation	30	50
Water (%) weight of formulation	36	42

In the specific example of a bicontinuous cubic phase, the water content is about 38% by weight of the formulation.

In a more specific example, the inverse (or inverse) form of the bicontinuous cubic phase comprises a lipid concentration of glycerol monooleate of about 40% to 80% by weight of the total formulation. In other examples, glycerol monooleate is present at 50% to 65%, 50% to 64%, 60% to 63% by weight of the total formulation. In particular embodiments, the glycerol monooleate content is greater than or equal to 52% by weight of the total formulation. In these examples, and when the bicontinuous cubic phase formulation is aqueous, the water content of the formulation at room temperature is up to and including 48% by weight of the formulation. In other examples, the water content is 1% to 48%, 20% to 44%, 30% to 42%, or 36% to 42% by weight of the formulation. In one embodiment, the water content is about 38% by weight of the formulation.

In a more specific example, the inverse (or reverse) form of the bicontinuous cubic phase comprises a lipid concentration of phytantriol of about 60% to 75% by weight of the total formulation. In other examples, phytantriol is present at 65% to 75%, 68% to 74%, 70% to 73% by weight of the total formulation. In one embodiment, the phytantriol is present in an amount of 72% by weight of the total formulation. In these examples, and when the bicontinuous cubic phase formulation is aqueous, the water content of the formulation is up to and including 28% at room temperature. In other examples, the water content is 1% to 28%, 20% to 25%, 22% to 36% by weight of the formulation.

In other examples, the inverse (or inverse) form of the bicontinuous cubic phase comprises a combination of the described lipids.

The aqueous channel size may be, for example, between 1nm and 17 nm. For example, the aqueous channel size may be 1nm to 7nm. In a further example, a suitable surfactant may be used to break up the cubic mesophase into a dispersion of cubic particles. Such surfactants include, for example, poloxamer 407 (Poloxamer 407).

Cube structure

In some examples, the lipid nanocarriers take the form of cubic structures. The cubic structure is a dispersed sub-micron nanostructure particle with three (3) dimensional symmetry.

The cubic structure may be formulated to a specific pore size for incorporation into a therapeutic agent using methods known in the art. Their structure provides a high surface area for loading the therapeutic agent.

In some examples, a stabilizer may be added to stabilize the structure of the nanocarrier when in the form of a cubic structure.

Examples of stabilizers include, for example, poloxamer 407, polyethylene glycol, pluronic F108, F68, F38, F127, F87NF, P105, P85, L35, P104, P84, L64, P123, P103, L43, L92, L62, L121, L101, L81, and L61.

The stabilizing agent may be present in a range of 0.01% to 10% by weight of the lipid present, for example, 5% to 10% by weight of the lipid. In some examples, the stabilizing agent is present at 10% by weight of the lipid. In these examples and when the formulation is aqueous, the water content of the formulation at room temperature is up to 48% by weight and includes 48% by weight of the formulation. In other examples, the water content is 1% to 48%, 20% to 40%, 25% to 35%, 38% to 40% by weight of the formulation.

Hexagonal phase

In some examples, the disclosure relates to lipid nanocarriers in the form of hexagonal phases. The hexagonal phase is a non-dispersed lipid nanocarrier formed by lipid self-assembly and has long-range order in the two (2) dimensions.

In some examples, the lipid used to form the hexagonal phase is dioleoyl phosphatidylethanolamine (DOPE).

In a specific example, the disclosure relates to lipid nanocarriers in the form of inverted hexagonal phases (HIIs).

Hexagonal structure

In some examples, the lipid nanocarriers are in the form of hexagonal structures. The hexagonal structures are dispersed sub-micron nanostructured particles with two (2) dimensional symmetry.

In some examples, the lipid nanocarriers are in the form of hexagonal structures. The hexagonal structure is a reverse hexagonal phase consisting of a hexagonal close-packed infinite aqueous layer covered by a monolayer of surfactant.

The hexagonal structures may be formulated with the therapeutic agent by, for example, being contained within an aqueous layer.

Examples of surfactants include amino acid based cationic surfactants such as arginine-N-lauroyl-amide dihydrochloride (ALA) and N-lauroyl-arginine-methyl ester hydrochloride (LAM), which have two and one positive charge per head group, respectively, and hydrogenated sodium tallow glutamate (HS).

Other examples of surfactants include anionic surfactants such as Sodium Octyl Sulfate (SOS) and Sodium Cetyl Sulfate (SCS).

Additional modifiers may be present in the hexagonal structure formulation, including oleic acid, tetradecane, and vitamin E acetate.

In some examples, when the nanocarrier is in the form of a hexagonal structure, the stabilizer may be added to stabilize the structure of the nanocarrier.

Solid lipid nanoparticles

In some examples, the lipid nanocarriers take the form of nanoparticles, including nanospheres and nanocapsules, dendrimers, solid lipid nanoparticles, transfersomes, and nanogels.

Solid Lipid Nanoparticles (SLNs) are typically prepared from lipids that are solid at room temperature and body temperature. Solid lipid nanoparticles can protect photosensitive, moisture sensitive, and chemically unstable therapeutic agents. Typically, SLN dispersions contain a large amount of water.

Examples of lipids that can form solid lipid nanoparticles include tripalmitin, cetyl alcohol, cetyl palmitate, compritol 888ATO, glyceryl monostearate,AT05, trimyristin, tristearin, stearic acid and900。

excipients, diluents, adjuvants, swelling agents and modulators

The lipid nanocarrier formulation may further comprise one or more of an excipient, diluent, adjuvant, swelling agent, modulator, and/or stabilizer.

For example, the formulation may comprise one or more of charged lipids, branched lipids, carboxylic acids, ionic surfactants, polyelectrolytes, water soluble surfactants, water insoluble surfactants, hydrophilic co-solvents, and/or small molecules.

In specific examples, the formulation may comprise one or more of charged lipids, branched lipids, carboxylic acids such as oleic acid.

Specific examples include pyridylmethyl linoleate, 2-hydroxy oleic acid, pluronic F127, phloroglucinol, N-oleoyl-glycine, N- (2-aminoethyl) -oleamide, isooleic acid, oleic acid, giant whale acid (mondioic acid), erucic acid, and nervonic acid.

In some examples, the formulation comprises one or more cationic lipids, such as dioleoyl-3-trimethylammonium propane (DOTAP) and 1, 2-dioleoyl-3-trimethylammonium propane. These cationic lipids may be present in the range of, for example, 1% to 10% by weight.

In some examples, DOTAP is present in an amount up to and including 10% by weight of the lipid nanocarrier formulation. In other examples, DOTAP is present at 0.1% to 15%, 1% to 10%, 4% to 10%, or 6% to 10% by weight of the lipid nanocarrier formulation.

In some examples, the formulation comprises cholesterol, which is typically used as a swelling agent for the cubic phase swelling. The swelling agent, e.g. cholesterol, may be present in an amount of up to, e.g., up to 50% by weight.

In some examples, the lipid nanocarrier formulation is swollen, in particular to accommodate large proteins, with a water content of up to and including 70% by weight of the lipid nanocarrier formulation. In further examples, the water content is 48% to 70%, 50% to 65%, or 60% to 65% by weight of the formulation.

Enteric coating

In some examples, after formulating the therapeutic agent into the lipid nanocarrier, the formulation may be encapsulated in an enteric coating to provide the dosage form.

In another set of examples, an enteric coating may be applied to the capsule shell and capsule fill containing the formulation. In examples where the coating is applied to the shell, the shell may be composed of any suitable soluble material that is soluble in the gastrointestinal environment, particularly the small intestine.

Examples of enteric coatings that may be used include, for example, those shown in table 1:

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In some examples, the enteric coating is soluble in a range of about pH 5.6 to pH 7.2, and in further examples, the enteric coating is soluble in a range of about pH 5.8 to pH 6.5. In a further example, the enteric coating is soluble in a range of about pH 4.5 to pH 7.2. In a further example, the enteric coating is soluble in a range of about pH 5.0 to pH 6.0.

In some examples, the thickness of the enteric coating is in the range of 0.01nm to 1.00 mm. In other examples, the range is 10 μm to 500 μm, and in further examples, the range is 100 μm to 200 μm.

In some examples, when the dosage form is encapsulated in an enteric coating, the thickness of the coating is in the range of 0.05 μm to 1mm, 1 μm to 0.5mm, 10 μm to 0.1mm, or 50 μm to 0.5 mm. In a specific example, the range is 0.07mm to 0.4mm.

In some examples, the dosage form is a capsule comprising a fill comprising a lipid nanocarrier formulation and a shell encapsulating the fill, the shell being coated with an enteric coating.

In some examples, an enteric coating is applied to at least one of the shell surface and the outer shell surface facing the filler. In those examples, the shell surface and the outer shell surface facing the filler each independently have a thickness in the range of 30 μm to 500 μm. In other examples, the range is 200 μm to 400 μm, and in other examples, the range is 80 μm to 180 μm.

In examples where the lipid nanocarrier is aqueous, having a thicker enteric coating, the enteric coating may aid in the stability of the dosage form ex vivo.

Table 2 provides further examples of enteric coating thicknesses:

the left hand side of figure 17 shows that the double enteric (Eudragit L100) coated capsule remained stable inside and outside after 24 hours in pH 4 medium. The middle part of the figure shows that the standard capsule is empty when t=0. The right hand side shows a single outer coating with both the inner and outer pH 4 medium, showing that the capsule is destroyed after 8 hours. In some examples, the inner enteric coating may theoretically prevent contact of the hydrated cubic phase with the capsule and help prevent degradation ex vivo.

Therapeutic agent

The present disclosure relates to formulations for oral delivery of therapeutic agents.

The therapeutic agent may be dispersed, contained, conjugated and/or absorbed within the lipid nanocarrier. In addition, the therapeutic agent may be incorporated into the lipid nanocarrier by any means, such as a homogeneous matrix, enriched on the surface of the lipid nanocarrier, within the membrane of the lipid nanocarrier, and/or within the cavity.

In some examples, the therapeutic agent is a peptide, such as insulin.

Insulin or a derivative thereof may be long acting, ultra long acting or medium acting insulin. Examples of such insulins include insulin glargine (Lantus, basaglar, toujeo), insulin detention (Levemir), insulin deglutide (Tresiba) and NPH (eurine N, norand reion insulin N). Alternatively, the insulin may be a fast acting or short acting insulin. These insulins are well suited for preventing postprandial blood glucose elevation. Examples of such insulins include insulin aspart (NovoLog, fiasp), insulin glulisine (apitra), insulin lispro (Humalog, admelog) and conventional insulins (eurin R, norand R).

In other examples, the peptide is a neuropeptide, such as somatostatin (SST), also known as Growth Hormone Inhibitory Hormone (GHIH) or oxytocin.

Other therapeutic agents include steroid hormones. Examples of steroid hormones include glucocorticoids, mineralocorticoids, androgens, estrogens and progestins.

In a further example, the therapeutic agent is a protein, e.g., a protein up to 170kDa in size.

In some embodiments, the therapeutic agent size is between 5 and 150kDa, between 5 and 100kDa, between 5 and 50kDa, between 5 and 40kDa, between 5 and 30kDa, between 5 and 20kDa, or between 5 and 10 kDa. In one embodiment, the therapeutic agent is about 5kDa in size.

In some examples, the protein is a hormonal protein, such as human growth hormone or human coagulation factor X. In some examples, the therapeutic agent is a small molecule. For example, the small molecule may be an antibiotic or an antimicrobial agent. Such antibiotics include glycopeptides such as vancomycin or beta-lactam antibiotics such as meropenem.

Oral administration of these therapeutic agents is often ineffective due to chemical and/or enzymatic degradation in the gastrointestinal tract. Current modes of administration include intravenous, intramuscular, and/or subcutaneous injection due to lack of bioavailability for oral administration. These modes of administration are sometimes accompanied by side effects associated with injection.

In examples where the nanocarrier comprises bicontinuous cubic-phase lipids, the insulin derivative may be present at 0.01% to 1% by weight of the formulation. In other examples, the insulin derivative may be present at 0.05% to 0.5% by weight of the formulation. In a further example, the insulin derivative may be present at 0.1% to 0.3% by weight of the formulation.

Method for preparing capsules

In some examples, the dosage form may be a capsule and may be prepared by the steps of providing a capsule fill comprising the described formulation and encapsulating the capsule fill in an enteric coated shell or encapsulating the dosage form directly with an enteric coating, respectively.

The formulation may be prepared by contacting the lipid, therapeutic agent, and aqueous solvent under conditions sufficient to induce nanocarrier formation.

In other examples, the formulation may be formed by contacting the lipid, aqueous solvent, and therapeutic agent using the following methods known in the art: such as mixing in a syringe, cold or hot high pressure homogenization, emulsion-sonication, solvent emulsification-evaporation, solvent diffusion, microemulsion, solvent injection, and/or double emulsion.

An example of contacting the lipid in an aqueous solution may be a lipid concentration in the range of 10% to 90% by weight of the solution. In examples where the formulation is aqueous, the water content of the formulation at room temperature is 1% to 70%, 20% to 60%, 30% to 50% or 36% to 42% by weight of the formulation.

In a specific example associated with the cubic phase of swelling, the lipid concentration is 10% to 50%. In these examples, the water content is up to and including 70% by weight of the lipid nanocarrier formulation. In further examples, the water content is 48% to 70%, 50% to 65%, or 60% to 65% by weight of the formulation.

In some examples, the formulation is prepared by including a lipid, a therapeutic agent, and an aqueous solvent to obtain a multiphase mixture. The mixture is then introduced into a first mixing chamber which is connected to a second mixing chamber by a mixing attachment suitable for homogenization. The mixture is passed through a mixing attachment suitable for homogenization until an inverse bicontinuous cubic phase is formed.

In a specific example, the ratio of lipid to aqueous solvent is about 60:40w/w and the first and second mixing chambers are syringes, which can be run up to 50 times through a mixing attachment suitable for homogenization.

The formulation may be carried out at 20℃to 90 ℃. In other examples, the temperature is in the range of 20 ℃ to 50 ℃. In other examples, the temperature is in the range of 20 ℃ to 35 ℃.

In some examples, a capsule fill comprising a formulation of lipid nanocarriers and a therapeutic agent may be inserted into a pre-prepared enteric coated shell. In other examples, the enteric coated capsule shell is applied to a fill comprising a lipid nanocarrier formulation. The application of the enteric coating may be performed by any suitable means, including dipping or spraying.

In a further example, an enteric coating is applied to at least one of the shell surface and the outer shell surface facing the filler.

In a particular example, a capsule, such as a gelatin capsule, may be coated with an enteric coating, such as by using an dip pan, to form an enteric coated capsule shell. For example, the capsules may be impregnated up to 3 times and then dried for about 15 minutes.

The enteric coated capsule shell may then be filled with a formulation comprising a lipid cubic phase and a therapeutic agent. Alternatively, the two halves of the capsule may be joined to encapsulate a capsule fill comprising the lipid nanocarrier and the therapeutic agent formulation.

For industrial scale, when the scale is enlarged to mass production of capsule shells, the enteric capsules may be sprayed.

In specific examples, the therapeutic agent of the dosage form is insulin, an antibiotic or a protein hormone present in a therapeutically or prophylactically effective amount. The mesophase may be a cubic phase, an original cubic phase (Pn 3 m), a rhombohedral cubic phase (Ia 3 d) or a spiral icosahedron cubic phase, or an inverted hexagonal phase. The mesophase is aqueous and has a water content of from 0.1% to 48% by weight of the formulation or mixture of phases. The lipid may be a long chain lipid, such as glycerol monooleate, phytantriol or glycerol monopalmitoleate, present in an amount of from 35% to 62% by weight of the formulation. The enteric coating may be present in a thickness of 160 μm to 500 μm. Without wishing to be bound by theory, the inner enteric coating may prevent the hydrated cubic phase from contacting the capsule and helping to prevent its degradation ex vivo.

Therapeutic method

Oral administration of therapeutic agents, including insulin, is preferred because of its convenience, relatively low cost of manufacture, and high levels of patient safety. However, a significant portion of the therapeutic agents do not exhibit the properties required for oral administration.

A prerequisite for the effectiveness of a therapeutic agent following oral administration is that it largely avoids a series of continuous barriers in the gastrointestinal tract and liver. Systemic bioavailability of orally administered therapeutic agents is primarily thought to be related to intestinal drug absorption and subsequent first phase metabolism in the liver. However, the human small intestine is increasingly considered an important part of the first pass extraction.

Therapeutic agents designed to have systemic activity must be absorbed from the site of administration to be effective. Furthermore, in order to allow passage through biological membranes, the therapeutic agent must be in solution. Since most therapeutic agents are administered in solid dosage forms, disintegration of the formulation must precede dissolution of the therapeutic agent in the surrounding medium. The rate of disintegration is affected by the nature of the formulation and physiological factors such as the rate of gastric emptying, transit time and gastrointestinal fluid pH. Once in solution, the therapeutic agent is susceptible to chemical and enzymatic degradation. Bioavailability may be further reduced by efflux mechanisms or first pass metabolism in the intestinal epithelium and/or liver.

Surprisingly, it has been demonstrated that oral absorption and systemic bioavailability of therapeutic agents can be achieved by formulating the therapeutic agent in lipid nanocarriers and then encapsulating in an enteric coating. It was further demonstrated that the oral absorption and bioavailability of insulin was improved, comparable to that of systemic insulin. Thus, the insulin dosage forms of the present disclosure are useful for treating or preventing diabetes. The methods of the present disclosure include orally administering such dosage forms to a subject in need of treatment or prevention of diabetes.

Diabetes (DM) is a chronic metabolic disease, currently estimated to affect 4.51 million people, and this figure is expected to increase significantly in the coming years. Diabetes is characterized by persistent hyperglycemia, which over time can lead to diabetic complications and ultimately death. Blood glucose levels on an empty stomach (BGL) above 7.0mmol/L and postprandial blood glucose levels above 11.1mmol/L indicate diabetes.

Insulin therapy is critical in the treatment of diabetes, where patients with diabetes require multiple injections of insulin per day. However, this mode of administration has a number of drawbacks, resulting in poor patient compliance.

In some examples, the dosage forms of the present disclosure may be used for oral administration of insulin or derivatives thereof to treat or prevent diabetes.

Specific embodiments and applications of the present disclosure will now be discussed in detail with reference to the accompanying examples. The discussion is in no way intended to limit the scope of the present disclosure.

The dosage forms described are also found to be useful in the treatment of diabetes, conditions mediated by bacterial infection, conditions mediated by human growth hormone, or conditions mediated by coagulation, and wherein the therapeutic agent is present in a therapeutically or prophylactically effective amount.

The dosage form is useful in a method of treating or preventing diabetes, a condition mediated by bacterial infection, a condition mediated by human growth hormone, or a condition mediated by blood coagulation, the method comprising administering the dosage form to a subject in need thereof and the therapeutic agent is present in a therapeutically or prophylactically effective amount.

A further use of the dose is for the treatment or prevention of diabetes, a condition mediated by bacterial infection, a condition mediated by human growth hormone, or a condition mediated by coagulation, and the therapeutic agent is present in a therapeutically or prophylactically effective amount.

Examples of conditions mediated by bacterial infections include gram positive pathogens, infections caused by methicillin-resistant staphylococcus aureus, infections caused by multidrug-resistant staphylococcus aureus, dermatitis, endocarditis, primary sclerosing cholangitis, endophthalmitis, gram negative pathogens, urinary tract infections, meningitis, intraperitoneal infections, pneumonia, sepsis or anthrax.

Examples of conditions mediated by human growth hormone include growth hormone deficiency, such as childhood, adult growth hormone deficiency, aids wasting, renal failure, turner syndrome, achondroplasia, prader-Willi syndrome (Prader-Willi syndrome), malgrowth or idiopathic short stature in children of less than gestational age.

Examples of conditions mediated by coagulation include the treatment or prevention of bleeding in patients with genetic factor X deficiency.

Examples

Example 1 materials and methods

Glyceryl monooleate (oleoyl-rac-glycerol) (> 99%) was purchased from Sigma Aldrich. Green Fluorescent Protein (GFP) (> 99%) and Red Fluorescent Protein (RFP) (> 99%) were derived from Biovision. Oral gavage and capsule No. 9 used in animal experiments were from Torpac, eudragit L100 from Evonik. Accu Chek BG monitor and test paper were purchased from Priceline pharmacy. ActRapid and Levemir (Novo Nordisk) are purchased from Chemist Warehouse.

Preparation of cubic lipid nano carrier

To prepare a GFP-encapsulated cubic-phase lipid nanocarrier, a known amount of glycerol monooleate (typically 50 mg) was added to a 100 μl syringe. GFP solution (4 mg/ml) in PBS was added to another 100. Mu.L syringe at a ratio of 60:40w/w lipid to protein solution. The contents of both syringes are mixed using a special syringe mixing attachment.

To prepare cubic-phase lipid nanocarriers comprising Actrapid insulin, a known amount of glycerol monooleate (typically 50 mg) was added to a 100 μl syringe. A PBS solution of Actrapid insulin (100 IU/ml) was added to another 100. Mu.L syringe at a ratio of 60:40w/v lipid to protein solution. The contents of both syringes are mixed using a special syringe mixing attachment.

To prepare cubic-phase lipid nanocarriers comprising Levemir insulin, a known amount of glycerol monooleate (typically 50 mg) was added to a 100 μl syringe. A PBS solution of Actrapid insulin (100 IU/ml) was added to another 100. Mu.L syringe at a ratio of 60:40w/w lipid to protein solution. The contents of both syringes are mixed using a special syringe mixing attachment.

The special mixer consisted of two 100. Mu.L (Hamilton Company, cat# 7656-01) or two 250. Mu.L (Hamilton Company, cat# 7657-01) airtight syringes and one syringe coupler made of two Removable Needle (RN) nuts (Hamilton, cat# 30902) and two gauge 22 removable needles (Hamilton, cat# 7770-02) (Cheng et al, 1998). Alternatively, a slightly different syringe connector may be purchased from Emerald Biosystems (cat#EB-LCP-SUNION) or Molecular Dimensions (cat#MD 6-17). The lipid mixer can rapidly and efficiently mix small volumes of lipid and aqueous solution (total volume 10-100. Mu.L with 100. Mu.L syringe and 25-250. Mu.L with 250. Mu.L syringe).

Preparation of enteric coated capsules

The diluted mixture was made using 342.9g acetone, 514.2g isopropyl alcohol and 42.9g Milli Q water and poured into a 3L beaker. 62.5g Eudragit L100 was mixed into the diluent suspension using a high torque mixer and mixed slowly for 60 minutes. 6.25g of triethyl citrate was added and stirred for an additional hour, after which the enteric coating mixture was prepared by passing through a 0.5mm sieve. The capsules were coated in the mixture using a dip pan. For GFP assay, each side of the capsule was briefly immersed twice and dried (thickness t=2). For the first phase of the insulin test, each side of the capsule was briefly immersed three times and dried (t=3). For the second phase of the insulin test, each side of the capsule was briefly immersed once and dried (t=1).

These capsules were then filled with 25 μl of GFP-loaded LCP as described above. The final amount of GFP in each capsule was 50. Mu.g. To deliver insulin, the capsule is filled with 25 μl of insulin-loaded LCP as described above. The final amount of insulin in each capsule was 1IU.

First stage test. Each capsule was immersed 3 times-coating thickness = 3 layers Eudragit L100 enteric coating.

And (5) testing in a second stage. Each capsule was immersed 1 time-coating thickness = 1 layer Eudragit L100 enteric coating.

Preparation of cubic phase insulin and glyceryl monooleate enteric coated preparation

The diluted mixture was made using 342.9g acetone, 514.2g isopropyl alcohol and 42.9g Milli Q water and poured into a 3L beaker. 62.5g Eudragit L100 was mixed into the diluent suspension using a high torque mixer and mixed slowly for 60 minutes. 6.25g of triethyl citrate was added and stirred for an additional hour, then passed through a 0.5mm sieve to make an enteric coating mixture. Gelatin capsules are coated in the enteric mixture. Half of each capsule was immersed in the enteric mixture using forceps and then air dried on tissue for 15-20 minutes. The latter half of the capsule was then immersed and air-dried on the tissue for 15-20 minutes. The film coating thickness is increased by dipping once, twice or three times and air-drying in between.

To prepare the lipid cubic phase of dispersed actipid insulin, a known volume of molten glycerol monooleate (50 μl) was added to a 100 μl syringe. PBS solution of Actrapid insulin (40. Mu.L, 100 IU/mL) was added to another 100. Mu.L syringe. The contents of both syringes are mixed using a special syringe mixing attachment. The resulting mixture was visually observed for turbidity due to the presence of excess water. To ensure that the cubic phase was formulated just under excess water, a small amount of lipid was then added and mixed using a syringe mixer until the formulated cubic phase was visually observed to be clear without any sign of turbidity.

The dispersed insulin lipid cubic phase produced in step 2 was directly injected from a 100 μl syringe into the capsule produced in step 1. For this purpose, the syringe mixing attachment is replaced with a high gauge needle.

Example 2: animal test 1 (GFP)

GFP/RFP Pre-test

Two Sprague Dawley rats were used in this section of the experiment. Each rat was first administered isoflurane until coma. Saphenous vein and first blood collection (100 μl) were then prepared. Then 500. Mu.L of 100. Mu.g/mL GFP was injected subcutaneously into rat 1, while 500. Mu.L of 100. Mu.g/mL RFP was injected subcutaneously into rat 2. These animals were then placed in two boxes, respectively. Blood collection (100 μl) was repeated every half hour for 120 minutes, then every hour until the 6 hour test ended. At the end of the test, blood was separated using a centrifuge at 1200rpm for 2 minutes. Thereafter, the plasma supernatant was removed and frozen at-40℃for the next day for fluorescence analysis on a CLARIOstar microplate reader (BMG Labtech).

GFP (subcutaneous injection)

Three Sprague Dawley rats were used in this section of the experiment. Each rat was first administered isoflurane until coma. Saphenous vein and first blood collection (100 μl) were then prepared. The rats were then subcutaneously injected with 500. Mu.L of 100. Mu.g/ml GFP and then placed in three cassettes, respectively. Blood collection (100 μl) was repeated every half hour for 120 minutes, then every hour until the 6 hour test ended. At the end of the test, blood was separated using a centrifuge at 1200rpm for 2 minutes. Thereafter, the plasma supernatant was removed and frozen at-40℃for the next day for fluorescence analysis on a CLARIOstar microplate reader (BMG Labtech).

GFP (oral capsule)

Four Sprague Dawley rats were used in this section of the experiment. Each rat was first administered isoflurane until coma. Saphenous vein and first blood collection (100 μl) were then prepared. Enteric capsules (t=2) containing GFP-loaded cubic phase (25 μl cubic phase containing 50 μg GFP) were then introduced into the rat stomach by oral gavage. The animals were then placed in four boxes, respectively. Blood collection (100 μl) was repeated every hour for 120 minutes, then every 30 minutes until 180 minutes, then every 60 minutes until the 6 hour test ended. At the end of the test, blood was separated using a centrifuge at 1200rpm for 2 minutes. Thereafter, the plasma supernatant was removed and frozen at-40 ℃ for the next day analysis.

Fluorescence analysis

Plasma samples were subjected to fluorescence analysis on a CLARIOstar microplate reader (BMG Labtech). 2 μl of plasma at each time point was thawed for 5 min and contained in LVis nanoplates and run in fluorescence mode at standard GFP excitation and emission wavelengths (488/510 nm). All measurements were performed at 25 ℃.

Example 2: animal test 2 (insulin)

Induction of diabetes by STZ injection

14 Sprague Dawley rats were used in this experiment. One week before each phase began, all rats were given isoflurane until coma. Each rat tail was then given an intravenous injection of 60mg/kg Streptozotocin (STZ) in sodium citrate buffer. All rats were then placed in pairs and monitored for 3 blood glucose per day over a period of 48 hours. The induction of diabetes can be confirmed in the morning with a blood glucose level >14 mmol/L.

At this point, 15IU of fast acting (actipid) insulin was administered by SC injection as needed until the start of the trial. Rats were then placed in pairs in two cassettes. Blood was collected 5, 360 and 1440 minutes after injection and BG levels were tested at each time point. This data was used to confirm that each rat had diabetes before the start of the test and to determine the average rise in blood glucose in the rats without any treatment.

Delivery of fast acting insulin (Actrapid) by subcutaneous injection (stages 1 and 2)

Four Sprague Dawley rats were used in stage 1 and four were used in stage 2. Isoflurane was administered first each until coma. Saphenous vein was then prepared and tested for baseline basal blood glucose. The rats were then subcutaneously injected with 1IU of fast acting insulin (Actrapid) and immediately thereafter placed on a heated pad until they regain consciousness, and then placed in pairs in cassettes. Blood (10 μl) was collected 15, 30, 60, 120 and 360 minutes after injection. BG testing was performed immediately at each time point using an Accu Chek BG monitor and test paper.

Delivery of slow acting insulin (Levemir) by subcutaneous injection (stages 1 and 2)

Four Sprague Dawley rats were used at stage 1 and four were used at stage 2. Isoflurane was administered first each until coma. Saphenous vein was then prepared and tested for baseline basal blood glucose. Rats were then subcutaneously injected with 1IU of slow acting insulin (Levemir) and immediately thereafter placed on a heated pad until they regain consciousness and then placed in pairs in cassettes. Blood (10 μl) was collected 30, 60, 120, 360 and 465 minutes after injection. BG testing was performed immediately at each time point using an Accu Chek BG monitor and test paper. One rat did not develop diabetes and one rat had kidney problems. In each case, AWO (animal welfare officer) was consulted and two rats were removed from the trial.

Delivery of fast acting insulin (Actrapid) by oral capsule (stage 1)

Four Sprague Dawley rats were used in this section of the experiment. Isoflurane was administered each time until coma. Saphenous vein and first blood sampling (10 μl) and BG testing were then prepared. Enteric capsules (t=3) containing a fast acting insulin-loaded cubic phase (25 μl of cubic phase containing 1IU of insulin) were then introduced into the rat stomach by oral gavage. They are then immediately placed on the heating pads until they regain consciousness and then placed in pairs in the box. Blood (10 μl) was collected at 75, 120, 180, 240, 300, and 360 minutes and BG test was performed at each time point using an acu Chek BG monitor and test paper.

Delivery of slow acting insulin (Levemir) by oral capsule (stage 1)

Two Sprague Dawley rats were used in this section of the experiment. Isoflurane was administered each time until coma. Saphenous vein and first blood sampling (10 μl) and BG testing were then prepared. Enteric capsules (t=3) containing a cubic phase loaded with slow acting insulin (25 μl cubic phase containing 1IU of insulin) were then introduced into the rat stomach by oral gavage. They are then immediately placed on the heating pads until they regain consciousness and then placed in pairs in the box. Blood (10 μl) was collected at 105, 135, 165, 210, 345 and 460 minutes and BG tested at each time point using an acu Chek BG monitor and test paper.

Delivery of fast acting insulin (Actrapid) by oral capsule (phase 2)

Four Sprague Dawley rats were used in this section of the experiment. Isoflurane was administered each time until coma. Saphenous vein and first blood sampling (10 μl) and BG testing were then prepared. Enteric capsules (t=1) containing a fast acting insulin-loaded cubic phase (25 μl of cubic phase containing 1IU of insulin) were then introduced into the rat stomach by oral gavage. They are then immediately placed on the heating pad until they regain consciousness and then placed in pairs in the box. Blood (10 μl) was collected at 45, 75, 120, 160, 220, 260, 300 and 360 minutes and BG tests were performed at each time point using an Accu Chek BG monitor and test paper.

Delivery of slow acting insulin (Levemir) by oral capsule (stage 2)

Two Sprague Dawley rats were used in this section of the experiment. Isoflurane was administered each time until coma. Saphenous vein and first blood sampling (10 μl) and blood glucose testing were then prepared. Enteric capsules (t=1) containing a cubic phase loaded with slow acting insulin (25 μl cubic phase containing 1IU insulin) were then introduced into the rat stomach by oral gavage. They are then immediately placed on the heating pads until they regain consciousness and then placed in pairs in the box. Blood (10 μl) was collected at 30, 60, 80, 125, 180, 280, 340, 400 and 460 minutes and BG tests were performed at each time point using the Accu Chek BG monitor and test paper.

Results

Oral delivery of green fluorescent protein as model drug

The proposed enteric capsule/cubic phase formulation for oral delivery of proteins was initially tested in preliminary animal experiments using fluorescent proteins. Measuring the level of fluorescence in plasma allows for easy measurement of drug delivery to the blood stream. Both Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP) were identified as possible model drugs. To determine which is more appropriate, background plasma fluorescence in Sprague Dawley rats was measured for GFP and RFP excitation and emission wavelengths, respectively. The mean plasma fluorescence of four rats was determined as: RFP excitation and emission wavelength (570/620 nm) was 31106RFU, GFP excitation and emission wavelength (488/509 nm) was 12123RFU. GFP was used as a model protein drug for initial animal experiments because of the much higher background fluorescence of RFP.

Three Sprague Dawley rats were administered 500. Mu.L 100. Mu.g/ml GFP by subcutaneous injection. Initially 100 μl blood was collected after 50-60 minutes, then every 30 minutes during 120 minutes, then every 60 minutes until a six hour time point. Due to the logical limitations of simultaneous injection testing of all rats, it is not possible to have an initial time point earlier than 50-60 minutes. Plasma was isolated from the collected blood samples, frozen, and fluorescence was measured over 24 hours. In fig. 3A, plasma fluorescence after SC injection is plotted as a function of time. At a first time point of about 50-60 minutes, an immediate increase in blood fluorescence can be seen; this condition decays over time within six hours of the test. The differences between rats are relatively low, possibly due to blood chemistry differences between individual rats, for example due to stress.

50 μg GFP was then delivered to four rats, which were encapsulated in MO lipid cubic phase (25 μl of 60wt% lipid; 40% w/w water containing 2mg/mL GFP) and contained in enteric vesicles. Due to the size of the enteric capsule relative to rats, the enteric capsule is delivered by oral gavage under anesthesia. The water content of the lipid cubic phase used (40 wt% water) was just under an excess of water. A slightly reduced hydration level was chosen because excessive water would lead to premature degradation of the enteric capsule. Blood was collected again periodically over 6 hours and fluorescence was measured, fig. 3B. Similar to that observed after SC injection, plasma fluorescence initially increases over time and then decays. Peak fluorescence measurements after oral delivery occur between 120 minutes and 180 minutes relative to peak delay observed after SC injection, as expected by the oral route of administration. Figure 3C provides the mean blood fluorescence after SC injection administration and capsule administration; error was calculated using standard deviations of all rats in each experimental group. Although the peak fluorescence measurement (50,000RFU) was on average slightly lower than that observed for SC injection (95,000), it was still consistent with efficient delivery of active protein by the oral route of administration. As expected, the subsequent blood fluorescence decay rate was found to be similar to that after SC injection (fig. 3C). The fluorescence difference between individual rats was observed, but not higher than that observed after SC injection.

A combined graph showing plasma fluorescence data for all rats after administration of GFP by SC injection or oral delivery in an enteric capsule loaded is provided in fig. 3D. The total area under the curve was calculated as an indication of the active GFP plasma level for each sample and is listed in table 3.

For each oral delivery sample, expressed as a percentage of average subcutaneous delivery, is shown in table 3. Efficacy of oral administration ranged from 41.7% to 86.5% relative to SC injection, with an average efficacy of 62.1% for oral delivery relative to delivery by SC injection.

Table 3. Total area under GFP fluorescence curves for all samples shown in fig. 3D (both subcutaneous and oral delivery). The average bioavailability (%) for oral delivery was calculated based on the average GFP fluorescence delivered by subcutaneous injection (set to 100%).

Oral delivery of Actrapid insulin

Based on the good bioavailability of GFP following oral administration, the second animal trial used fast acting (Actrapid) insulin as a commercially relevant protein drug. Quick acting (Actrapid) insulin 1IU was delivered to Sprague Dawley rats with diabetes mellitus by SC injection or oral (capsule) administration. Insulin delivery success can be directly determined by measuring the decrease in BG levels.

Diabetes in rats was initially induced by injection of STZ in 60mg/kg sodium citrate buffer, then observed for more than 48 hours and the first thing in the morning was to test blood glucose. The blood glucose level of each rat was generally in the range of 2mmol/L-8mmol/L prior to the induction of diabetes. Once blood glucose was found to exceed 14mmol/L, the rats were considered to have diabetes. During the pre-test period, 3 times daily blood glucose was measured and insulin was administered as needed depending on the blood glucose status of each rat. Figure 4 provides typical blood glucose readings during this period for four randomly selected rats. All rats and data were approved by animal welfare officials to confirm that diabetes had been successfully induced and all rats were insulin responsive.

During the pre-test, the diabetic rats had a highest blood glucose level in the range of 18-30mmol/L (this is the first thing in the morning due to their night-going nature). After administration of therapeutic doses of insulin (15 IU) by SC injection, blood glucose levels drop sharply within 5 minutes. The BG level remains below 14mmol/L for about 450 minutes. After about 450 minutes, BG levels began to rise in a reasonably linear fashion, indicating that most of the administered insulin had been cleared from the system. In four different rats and two different days, an increase in BG was observed with good reproducibility. The measured BG increase (0.0167 mmol/L/min) was used to predict BG increase in rats without any treatment (as shown by the dashed lines in fig. 5-9).

To test the efficacy of insulin delivery by SC injection, 20 μl of actipid insulin (50 IU/mL) was first administered to four diabetic rats. After a two-day recovery period, the same four rats were used to administer actipid insulin contained within an enteric coated capsule. 25. Mu.L of a lipid cubic consisting of 100IU/mL of 60% wt MO, 40% wt Actrapid insulin was used. A slightly reduced hydration level is chosen because excessive water can lead to premature degradation of the enteric capsule. Insulin is encapsulated therein at 1IU (equivalent to 0.0384 mg). Insulin can be encapsulated in the lipid cubic phase at this concentration without affecting its underlying cubic phase structure. Because of its small size and hydrophilicity, insulin should be contained within the aqueous channel region of the lipid cubic phase.

Delivery of fast acting (Actrapid) insulin to diabetic rats (stage 1)

Comparative experiments with rat 1 using perforated capsule shells

Fig. 5, rat 1 was orally administered with a perforated enteric coated capsule shell. The results of this rat are consistent with the requirements for successful delivery of the combination of enteric coated capsule shells and nanocarriers comprising lipid cubic phases, since it is clear that the cubic phases alone do not work after capsule perforation. Rat 1 had a less pronounced response to oral administration of insulin and blood glucose levels were continuously elevated.

Experiments with the use of rats 2-4 fully encapsulated

Fig. 6 plots BG levels for each of three rats after administration of actipid insulin by SC injection (1 IU) or lipid cubic phase contained in an enteric capsule (1 IU). As described in the section entitled "example 1-materials and methods", for this test, each capsule was coated three times in an enteric coating mixture. The predicted BG increase (based on the data in fig. 4) without any treatment is also plotted as a dashed line.

The initial BG level of rats 2-4 varied between 18mmol/L and 22mmol/L, consistent with diabetic rats. Immediately after administration of 1IU of actipid insulin by SC injection, a drop in BG levels was observed in all rats. BG levels in all four rats continued to drop for approximately 110 minutes and then rise again. The total drop in BG varied from 4.2mmol/L to 5.3mmol/L, with final BG levels in the range of 8.9mmol/L-17.2mmol/L, indicating successful administration of therapeutic levels of insulin. The rate of increase in BG after 360 minutes was similar to the rate predicted without insulin, indicating that all actipid insulin administered had been cleared from the animal. The observed BG reduction was consistent with other such animal trials.

For rats 2-4, BG levels initially increased continuously over a period of about 100 to 110 minutes following oral (capsule) administration of Actrapid insulin. The rate of increase is substantially the same as predicted without any treatment and is consistent with the longer period of insulin reaching the blood after oral administration. After this period, BG levels then begin to drop in a similar manner as observed for SC injections over a period of about 70-180 minutes. The overall decrease in BG was 3.6mmol/L in rat 2, 3.1mmol/L in rat 3 and 3.4mmol/L in rat 4, slightly less than the decrease observed with SC injection. After this point in time, the blood glucose level begins to increase, again at a similar rate as predicted.

Delivery of fast acting (Actrapid) insulin to diabetic rats (phase 2)

FIG. 7 coating process of enteric capsules was modified as described in the methods section for the second stage of F/A insulin use (rats 5-8). Specifically, the capsule is not immersed three times in the enteric coating, but only once. We expect this will result in a thinner coating, which may lead to premature decomposition of the released insulin. Furthermore, the elimination of two infusion steps may result in a more even coating between different capsules. However, the thickness and uniformity of the coating were not measured explicitly.

Blood glucose data after SC injection of 1IU of insulin (fig. 7) was very similar to that observed in the first phase. The initial blood glucose level is in the range of 22mmol/L-33 mmol/L. Immediately after administration of actipid insulin by SC injection, BG levels were observed to drop in all rats. BG levels in all four rats continued to drop for about 120 minutes and then rise again. The total drop in BG varies from 4.1mmol/L to 5.2mmol/L, with the lowest BG level being in the range 17mmol/L-26 mmol/L. The rate of BG increase after 120 minutes was similar to that predicted without insulin, indicating that all actipid insulin administered had been cleared from the animal. The observed blood glucose reduction was consistent with other such animal tests.

For rats 5-8, BG levels initially increased continuously over a period of about 60 minutes following oral (capsule) administration of Actrapid insulin. Notably, this is a shorter lead time (lead time) than the first stage (100-110 minutes), consistent with a thinner enteric coating (should break down earlier). After this period of time, BG levels began to decline in a similar manner as observed for SC injection and first phase oral administration. The total drop in BG was 6.2mmol/L in rat 5, 8.7mmol/L in rat 6, 11.4mmol/L in rat 7 and 4.6mmol/L in rat 8. This decrease was greater than that observed in the first stage, indicating that a thinner enteric coating resulted in improved absorption. After this point in time, the blood glucose level starts to increase, again at a similar rate as predicted. Thus, all future experiments were performed using capsules with such thinner enteric coatings (fig. 7).

Oral delivery of slow acting (Levemir) insulin

The ability of the lipid cubic phase to deliver slow acting insulin Levemir was also investigated. 20 μL of 50IU/ml Levemir insulin was first administered to four rats by SC injection. After a two-day recovery period, the same four rats were then used to administer Levemir insulin encapsulated in a cubic phase contained within an enteric capsule. The lipid cubic phase consisted of 60% wt MO, 40% wt Levemir insulin at a concentration of 100 IU/ml. In general, each rat was orally administered the same dose of 1IU of insulin. Figure 8 plots BG levels after administration of Levemir insulin by SC injection or by inclusion in the lipid cubic phase within an enteric capsule. The predicted subsequent BG increase without treatment is plotted in dashed lines in each single graph in fig. 8. Note that the results of both stages 1 and 2 are provided in fig. 8. For rats 9 and 10, the results were from stage 1, in which the capsules were immersed 3 times in the enteric coating. For rats 11-14, the results were from stage 2, where the capsules were immersed only once in the enteric coating.

The initial blood glucose level is in the range of 19mmol/L to 25 mmol/L. Immediately after administration of 1IU of Levemir insulin by SC injection, a drop in BG levels was observed in all rats. BG levels in all six rats decreased continuously for about 75 minutes and then remained stable for 480 minutes. The total drop in BG varies from 1mmol/L to 2mmol/L, with the lowest BG level being in the range of 18mmol/L to 25 mmol/L.

For rats 9 and 10, BG levels initially increased continuously over a period of about 105 minutes after oral administration of Levemir insulin (t=3) and then began to decrease. For rats 11-14, (t=1) BG levels increased only over a period of 30 minutes, then BG decreased was observed. The rate of increase during this initial period is substantially the same as the rate of increase predicted without any treatment. Likewise, the difference in lead time of insulin before release from the capsule and the observed BG decrease were consistent with the level of enteric coating. The shorter lead time compared to the three layers seen in the first stage of the test is associated with capsules having only one enteric coating.

After the BG initially increased, a decrease in level was then observed. We note that the data after SC-injected fast-acting or slow-acting insulin or capsule administration of fast-acting insulin is much noisier than BG data. Nonetheless, a significant drop in BG levels was observed for all rats, and BG levels were still significantly below predicted values over the 480 minute study period. The total decrease in BG was: 1.4mmol/L for rat 1, 2.1mmol/L for rat 2, 6.1mmol/L for rat 3, 2.8mmol/L for rat 4, 8.2mmol/L for rat 5 and 4.3mmol/L for rat 6. For rats 11, 13 and 14 with thinner enteric coatings, the decrease in BG was actually greater than that observed after SC injection at longer time points (> 30-100 minutes).

We note that the duration of action of slow acting insulin is up to 14 hours. However, limitations in animal testing (due to animal ethics-limitations on how long an animal can be kept under test) mean that BG cannot be analyzed for these longer periods of time. It is therefore not possible to compare the two methods of administration during this period of time.

Fig. 9 plots the average blood glucose levels for the following cases: a) fast acting insulin-rats 2-4, B) slow acting insulin-rats 9 and 10, C) fast acting insulin-rats 5-8 and D) slow acting insulin-rats 11-14. All data were normalized and the initial BG reading was set to zero. For the quick results, rat 1 was removed from the average as the capsule was perforated during induction. The area between each plot and the predicted increase in blood glucose was calculated and is provided in table 3. This was used to determine the total decrease in blood glucose for SC injection and oral delivery (as a measure of efficacy) compared to the standard increase in blood glucose in diabetic rats.

Table 4 the total BG reduction was calculated from the area between the plot of the rapid-acting insulin SC injected and encapsulated rapid-acting insulin and the predicted average rise in blood glucose (dashed line). The total BG reduction was calculated from the area between the plot of slow insulin SC injected and encapsulated slow insulin and the predicted average rise in blood glucose (dashed line).

In summary, the combination of enteric coated formulations using cubic lipid nanocarriers achieved effective oral delivery of model therapeutic agents. Experiments with GFP initially showed that the enteric capsule/lipid cube combination allowed for successful oral delivery of the model therapeutic, with efficacy of about 62% when oral delivery was compared to SC injection of GFP. The test also allows the coating level of the enteric coated capsules to be tested, testing the slightly thicker enteric coating obtained by impregnating the capsules twice. However, delivery efficacy can only be measured indirectly using fluorescence.

Subsequent animal studies using insulin demonstrated that cubic phase enteric coated capsules/lipid nanocarriers as a combination successfully delivered insulin and its derivatives. Both the fast-acting and slow-acting forms of insulin have proven successful delivery. Delivery was tested using two different levels of coating-the thicker coating was obtained by dipping the capsule three times, while the thinner coating was obtained by dipping the capsule once. For thicker coatings, the efficacy of oral delivery of fast acting insulin was 56% relative to SC injection, while slow acting insulin was 52%. The thinner the coating, the shorter the time for the drug to reach the blood, the higher the efficacy, 93% for fast acting insulin and 158% for slow acting insulin. It is not possible to test slow acting insulin over the whole period of 16 hours and therefore the efficacy value may be slightly higher than that achieved over the whole period.

Example 3 preparation of dosage forms of small molecule and protein therapeutics using cubic phase lipids

A material.

Glycerol Monooleate (MO) (97%, sigma), eudragit L100 (Evonik), capsule S9 (Torpac), meropenem (Sigma), vancomycin (Sigma), human coagulation factor 10 (analytical image), human growth hormone (MyBioSource). Male Sprague Dawley rats were from ARC animal facility (Australian Peltier). HGH, HCFOX, vancomycin and meropenem Elisa kits were all derived from BMA.

Capsule preparation 1- (Eudragit L100)

To prepare the lipid cubic phase encapsulated with therapeutic agent, a known amount of glycerol monooleate (typically 50 mg) is added to a 100 μl mL syringe. PBS solution of protein/hormone (pH adjusted to 4 using HCL) was used at 38:62w/w drug solution: the ratio of lipids was added to another 100 μl mL syringe. The contents of both syringes are mixed using a special syringe mixing attachment.

The diluted mixture was made using acetone (268 mL), isopropanol (400 mL) and Milli Q-water (43 mL) and poured into a 3L beaker. Eudragit L100 (62.5 g) was mixed into the diluent using a high torque mixer and mixed slowly for 60 minutes. Triethyl citrate (6.25 g) was added and stirred for an additional hour and then passed through a 0.5mm sieve to produce an enteric coating mixture. The capsules were coated once in the mixture using a dip pan. After the enteric coating has dried, each capsule portion is placed in a capsule holder and the enteric mixture is transferred into and out of the respective capsule portion using a pipette. As described above, the capsules were filled with 25 μl of drug-loaded LCP, resulting in a final amount of 1U per capsule. The mass change of the capsule geometry was solved by radius = ((surface area/(2 pi r)) -length)/2, and the thickness of the inner and outer coating was calculated to be about 160 μm.

Capsule preparation 2- (Eudragit L100-55)

To prepare the lipid cubic phase encapsulated with therapeutic agent, a known amount of glycerol monooleate (typically 50 mg) is added to a 100 μl mL syringe. PBS solution of therapeutic agent (pH adjusted to 5 using HCL) was used at 38:62w/w drug solution: the ratio of lipids was added to another 100 μl mL syringe. The contents of both syringes are mixed using a special syringe mixing attachment.

The diluted mixture was made using acetone (268 mL), isopropanol (400 mL) and Milli Q-water (43 mL) and poured into a 3L beaker. Eudragit L100-55 (62.5 g) was mixed into the diluent using a high torque mixer and mixed slowly for 60 minutes. Triethyl citrate (6.25 g) was added and stirred for an additional hour and then passed through a 0.5mm sieve to produce an enteric coating mixture. The capsules were coated once in the mixture using a dip pan. After the enteric coating has dried, each capsule portion is placed in a capsule holder and the enteric mixture is transferred into and out of the respective capsule portion with a pipette. As described above, the capsules were filled with 25 μl of drug-loaded LCP. The mass change of the capsule geometry was solved by radius = ((surface area/(2 pi r)) -length)/2, and the coating thickness of the inner and outer portions was calculated to be about 160 μm.

Delivery of protein hormone therapeutics

Human coagulation factor X delivery by subcutaneous injection

Four male Sprague Dawley rats were used as controls. Each rat was anesthetized with isoflurane and then saphenous vein was prepared. Rats were injected with 0.5mg/kg of human coagulation factor X by SC injection, immediately after which they were placed on a heating pad until they restored consciousness, and then they were placed in pairs in cassettes. Blood collection (150 μl) was performed via saphenous vein at 0, 30, 70, 120, 180, 240 and 360 minutes post injection. The blood concentration of proteins in blood was measured using a human coagulation factor X-related ELISA kit.

Delivery of human coagulation factor X by oral capsule

Eight male Sprague Dawley rats were used. Each rat was anesthetized with isoflurane, then saphenous vein was prepared and a first blood sampling (150 μl) was performed. An enteric capsule having a polymeric coating of about 160 μm thickness containing 0.5mg/kg of human coagulation factor X encapsulated in a MO lipid cubic phase was then introduced into the rat stomach by oral gavage. They are then immediately placed on the heating mat until they regain consciousness and then are placed in pairs. Blood collection (150 μl) was performed via saphenous vein at 0, 35, 50, 120, 180, 240 and 360 minutes post injection

As shown in fig. 11, the drug concentration was either not increased or only slightly increased in all four rats during the first 35 minutes of the test. The drug concentration then rose sharply at 50 minutes to a maximum in the range of 3.7-5.2 ng/mL. Then reduced to 0ng/ml over the next 4 hours and reached baseline drug concentration at the 300 minute cutoff. The maximum drug plasma concentration achieved was 47% after SC injection. The overall bioavailability was 88.52% after SC injection.

Delivery of human growth hormone by subcutaneous injection

Four male Sprague Dawley rats were used as controls. Each rat was anesthetized with isoflurane and then saphenous vein was prepared. Rats were injected with 1mg/kg of human growth hormone by SC injection, immediately after which they were placed on a heated pad until they restored consciousness, and then placed in pairs in cassettes. Blood collection (150 μl) was performed via saphenous vein at 0, 30, 70, 120, 180, 240 and 360 minutes post injection. The blood concentration of proteins in blood was measured using a human growth hormone-related ELISA kit.

Delivery of human growth hormone by oral capsule

Eight male Sprague Dawley rats were used. Each rat was anesthetized with isoflurane, then saphenous vein was prepared and a first blood sampling (150 μl) was performed. The enteric capsule, having a polymeric coating of about 160 μm thickness, containing 1mg/kg of human growth hormone encapsulated in the MO lipid cube phase, was then introduced into the rat stomach by oral gavage. They were then immediately placed on the heating pads until they regained consciousness and then placed in pairs. Blood collection (150 μl) was performed via saphenous vein at 0, 35, 50, 120, 180, 240 and 360 minutes post injection

As shown in fig. 10, the drug concentration was either not increased or only slightly increased in all four rats during the first 35 minutes of the test. The drug concentration then rises sharply at 50 minutes to a maximum in the range of 19-24 ng/ml. The drug concentration was then reduced to 0ng/ml over 3 hours and reached baseline drug concentration at 240 minutes. Fig. 10 (C) plots the area under the drug response curve (AUC) for the data provided in (a). (D) The area under the drug response curve (AUC) of the data provided in (B) is plotted. It represents the total delivered protein/hormone and is a component of determining overall biodistribution as a time factor. After SC injection, the total amount of protein delivered increased over time, reaching a plateau at about 180 minutes. After administration by oral capsule, a short lag phase of 30 minutes was observed before the total protein delivered began to increase and reached a steady level around 240 minutes. The differences in results between the different rats after oral delivery are also smaller. The maximum drug plasma concentration achieved was 81% after SC injection. The overall bioavailability was 87.2% after SC injection.

Delivery of small molecule antibiotic therapeutic agents

Subcutaneous injection delivery of meropenem

Four male Sprague Dawley rats were used as controls. Each rat was anesthetized with isoflurane and then saphenous vein was prepared. Rats were injected with 1mg/kg of meropenem by SC injection, immediately after which they were placed on a heated pad until they restored consciousness, and then placed in pairs in cassettes. Blood collection (150 μl) was performed via saphenous vein at 0, 30, 70, 120, 180, 240 and 360 minutes post injection. The blood concentration of proteins in blood was detected using a related ELISA kit for meropenem.

Delivery of meropenem by oral capsule

Eight male Sprague Dawley rats were used. Each rat was anesthetized with isoflurane, then saphenous vein was prepared and first blood collection (150 μl) was performed. The enteric capsule, having a polymer coating about 160 μm thick, containing 1mg/kg meropenem encapsulated in the MO lipid cube phase, was then introduced into the rat stomach by oral gavage. They are then immediately placed on the heating mat until they regain consciousness and then are placed in pairs. Blood collection (150 μl) was performed via saphenous vein at 0, 35, 50, 120, 180, 240 and 360 minutes post injection.

As shown in fig. 13, the drug concentration was either not increased or only slightly increased in all four rats during the first 35 minutes of the test. The drug concentration then rises sharply over 50 minutes to a maximum value in the range of 19-23 ng/ml. It then fell to 0ng/ml within 2 hours and reached the baseline drug concentration for 180 minutes. The maximum drug plasma concentration achieved was 55% after SC injection. The overall bioavailability was 58% after SC injection.

Delivery of vancomycin by subcutaneous injection

Four male Sprague Dawley rats were used as controls. Each rat was anesthetized with isoflurane and then saphenous vein was prepared. Rats were given 15mg/kg vancomycin by SC injection, immediately after which they were placed on a heated pad until they restored consciousness, and then placed in pairs in cassettes. Blood collection (150 μl) was performed via saphenous vein at 0, 30, 70, 120, 180, 240 and 360 minutes post injection. The blood concentration of proteins in blood was tested using the relevant ELISA kit for each protein.

Delivery of vancomycin by oral capsule

Eight male Sprague Dawley rats were used. Each rat was anesthetized with isoflurane, then saphenous vein was prepared and first blood collection (150 μl) was performed. An enteric capsule having a polymeric coating about 160 μm thick containing 15mg/kg vancomycin encapsulated in a MO lipid cube was then introduced into the rat stomach by oral gavage. They are then immediately placed on the heating mat until they regain consciousness and then are placed in pairs. Blood collection (150 μl) was performed via saphenous vein at 0, 35, 50, 120, 180, 240 and 360 minutes post injection.

As shown in fig. 12, the drug concentration was either not increased or only slightly increased in all four rats during the first 35 minutes of the test. The drug concentration then rises sharply over 50 minutes to a maximum value in the range 790-960 ng/ml. The concentration was then reduced to 0ng/ml over 2 hours and the baseline drug concentration was reached over 180 minutes. The maximum drug plasma concentration achieved was 57% after SC injection. The overall bioavailability was 73.93% after SC injection.

Example 4 structural analysis of therapeutic Agents with mesophase forms of lipids

Small angle X-ray scattering (SAXS)

Synchrotron SAXS experiments were performed on australian synchrotron to determine the structure of the lipid mesophase used in each capsule. Excess cubic phase from animal experiments was brought onto the SAXS/WAXS beam line and the data shown in FIGS. 14-16. HGH and HCFOX have the concentration of 1mg/mL and vancomycin has the concentration of 15mg/mL.

FIG. 14 is a 1DSAXS of human growth hormone (1 mg/ml) encapsulated in MO at a water phase content of 38%. The Bragg peaks of v 2, v 3, v 4, v 6, v 8, v 9, v 10, v 11 of the QIID cubic phase (crystal space group Pn3 m) are indicated by broken lines. Calculated lattice parameters of QIID phase are

FIG. 15 is a 1D SAXS of human coagulation factor X (1 mg/ml) encapsulated in MO at a water phase content of 38%. The Bragg peaks of v 2, v 3, v 4, v 6, v 8, v 9, v 10, v 11 of the QIID cubic phase (crystal space group Pn3 m) are indicated by broken lines. The lattice parameter of the QIID phase was calculated to be 103.0A.

FIG. 16 is a 1DSAXS of vancomycin (15 mg/ml) encapsulated in MO at a water phase content of 38%. Two coexisting QIIG phases (crystal space group Ia3 d) -were observed with the Bragg peaks at the ratios of ∈6, ∈8, ∈14 at (A) And (B)/(B)>Is indicated in (a).

Drawing/data/experimental convention

All data were analyzed on a Clariostar microplate reader to determine standard curve concentrations of the relevant drug.

The concentrations provided in FIGS. 10-13 are measured concentrations of drug in plasma (measured using a specific Elisa kit) in ng/ml. In each case, the initial drug concentration measured at time 0 has been set to zero and the other data is scaled relative thereto.

EXAMPLE 5 study of insulin and lipid nanocarriers in cubic phase form

Glycerol monooleate (oleoyl-rac-glycerol) (MO) (> 99% purity), phytantriol (PT) (> 99% purity), pluronic F127 (> 99%), human recombinant insulin (> 99%), octadecylrhodamine B chloride (> 99%), chymotrypsin (> 99%), fluorescein Isothiocyanate (FITC) -labeled insulin (> 99%) and Phosphate Buffered Saline (PBS) (> 99%) were purchased from Sigma Aldrich (australia).

Preparation of cubic phase

An aqueous solution of insulin (various concentrations between 0.5mg/ml and 50 mg/ml) in Phosphate Buffered Saline (PBS) was mixed with molten phytantriol or molten glycerol monooleate to form a bulk cubic phase (bulk cubic phase) with insulin. This was achieved by filling a 0.5mL syringe with insulin solution (MO 50wt%, PT sample 30 wt%) and a 0.5mL syringe with molten lipid (MO 50wt%, PT sample 70 wt%) and mixing through the syringe connector as described. The resulting mixture was visually observed for turbidity due to the presence of excess water, which was consistent with the known excess water point for both lipids (MO 48%, PT 28%). To ensure that the two cubic phases were formulated just under excess water, a small amount of lipid was then added and mixed using a syringe mixer until the formulated cubic phases were visually observed to be clear without any signs of turbidity. The water content of the final cubic phase was about 48% (MO) and 28% (PT) according to the known MO and PT phase diagrams.

Small angle X-ray scattering (SAXS)

A 200nL aliquot of this cubic phase was dispensed onto the surface of a Corning 96-well plate using a molquito LCP machine equipped with a humidity chamber (TTPLabtech, UK). The plate was sealed with a 200lm plastic seal (molecular size). SAXS data was obtained on the SAXS/WAXS beam line of the australian synchrotron, using a 96-well plate setup of our laboratory custom design. The wavelength of the X-ray beam is(10.000.+ -. 0.002 keV), size 250. Mu.m.times.120. Mu.m, flux 5X 10.) ¹² Photon s ^-1 . The sample detector used was a Pictatus 1-M900 mm from the sample. The temperature was maintained at 25 ℃ using a Peltier-control system. Automated data acquisition of a single plate takes approximately 10 minutes. The axess package developed by london empire institute of technology was used for SAXS data analysis. Integrating the 2D diffraction images to generate a 1D map of intensity versus scattering vector q; calibration was performed using silver behenate, d= 58.38A as a standard. For the QIID phase (crystal space group Pn3 m) employed by MO and PT at all insulin concentrations, lattice parameters were calculated based on known peak positions using p (110), (111), (200), and (211) Bragg peaks (ratios v 2, v3, v4, v6). The lattice parameter is determined from the peak position q using the following formula:

The error was calculated from SAXS measurements of three (3) different samples.

Circular Dichroism (CD)

The SR-CD measurement is performed using the CD beam line of synchrotron radiation source ASTRID2 of ISA at the center of the storage ring facility of university of ohus (denmark) in the 170-350nm region. A Suprasil quartz cell (Hellma GmbH & co., germany) with a nominal optical path length of 0.1mm (actual calibrated optical path length = 102.3 and 97.7 mm) requires a sample volume of about 30 μl for measurement at 37 ℃ in a temperature-regulated sample rack. For enzymatic degradation experiments run in the far UV region, 20. Mu.L of 0.02mg/mL chymotrypsin solution was injected into the sample holder to enclose 10. Mu.L of the host cubic phase encapsulated with 0.2mg/mL insulin. For enzymatic degradation experiments performed in the near UV region, 20. Mu.L of 0.05mg/mL chymotrypsin solution was injected into the sample holder to enclose 10. Mu.L of the subject cubic phase encapsulated with 0.5mg/mL insulin. Baseline measurements (cubic phase without insulin) were obtained in 1nm wavelength increments, an average time of 2.15 seconds and 6 scans. The sample measurements for each sample were obtained in 1nm wavelength increments, 1 second average time and 1 scan. The data was slightly smoothed using a 5-point moving average in Matlab. The online service dichoroweb was used to perform secondary structural content analysis on CD spectra using the control routine and SP175 reference set.

Fluorescence Recovery After Photobleaching (FRAP)

FRAP was performed on a Nikon confocal microscope. To measure the pure cubic phases of MO and PT, octadecylrhodamine B chloride (100 mg/mL) was combined with MO and PT as lipids: the aqueous phases (MO 50:50 and PT 70:30) were mixed in the proportions stated. The areas of the cubic phase were selectively fluorescence bleached using bleaching and background areas of interest (ROIs) and the change in fluorescence recovery curve over time was recorded. Octadecylrhodamine B chloride is used with excitation/emission wavelengths of 553/627nm. To measure insulin in the cubic phase, either MO or PT cubic phases were loaded with FITC-labeled insulin (5 mg/mL). The areas of the cubic phase were selectively fluorescence bleached using bleaching and background areas of interest (ROIs) and the change in fluorescence recovery curve over time was recorded. For FITC-labeled insulin, excitation/emission wavelengths of 490/525nm, respectively, were used. The open source Matlab code snap_analysis_2p5 is used to determine the diffusion coefficient μ from FRAP data.

Release from the principal cubic phase

An aqueous solution of insulin in PBS (5 mg/mL) was mixed with molten phytantriol or molten glycerol monooleate to form a main cubic phase (100 mg) with encapsulated insulin as described in the formulation section of the cubic phase. The mixture was then poured into a 1.5mL Eppendorf tube and centrifuged at 1200RPM for 1 minute to obtain a flat surface. The height of the surface is measured to determine the diameter for subsequent calculation of the surface area. 150 μl of water was added to the top of the cubic phase and immediately removed at time t=0. 150 μl of supernatant was removed every 30 minutes over 12 hours for analysis and replaced with another 150ml of water. The final reading was taken at 22 hours. The concentration of insulin in the supernatant at each time point was calculated using absorbance at 280nm (a 280 analysis) on Nanodrop 2000 (Thermo Fisher).

Structural study of insulin lipids in lipid nanocarriers in cubic phase form

Synchrotron SAXS is used to characterize the structure of the lipid cubic phase formed by MO and PT after encapsulation of insulin in a range of concentrations up to 10 mg/mL. The phases employed and the associated lattice parameters at 25 ℃ are shown in figure 18. Samples were run in triplicate. Figure 19 provides a representative diffraction pattern of MO and PT cubic phases encapsulating insulin. MO forms a main cubic phase of Q _II D phase, lattice parameter ofConsistent with the literature. At any of the study concentrations (up to 10mg/mL insulin), the addition of insulin did not significantly affect the underlying nanostructure. As the insulin concentration increases, the lattice parameter remains reasonably constant (fig. 18), while the Bragg peaks remain well defined and do not broaden with the variation of the insulin concentration used (fig. 19). PT also employs Q in excess water _IID The phase has smaller lattice parameter of +.>Insulin in PT nanocarriers causes an increase in lattice parameter in the form of cubic phase, which is proportional to insulin concentration. When the concentration reached 10mg/mL, the lattice parameter increased to +.>The increase is 14%. The error line was observed to increase with increasing insulin concentration, which was for protein (opposite phase) Destructive effects) are common. For PT, at higher protein concentration [ ]>7 mg/mL) also observed a second Q _IID Cubic phase. Based on the average intensities of v 2 and v 3. Second Q _IID The cubic phase is the minor phase. Second Q _IID The lattice parameter of the cubic phase is slightly higher than that of the original phase, which may represent an uneven distribution of proteins.

The increased destructive effect of insulin on PT cubic nanostructures may reflect an increased geometric mismatch between insulin and the aqueous channel in which it resides. In aqueous solution, insulin is known to exist predominantly in dimeric form, measured as a hydrodynamic diameterTherefore, the dimer is considered to be easily suited for MO cubic phase +.>Is consistent with the original cubic lattice parameter that the system retains at all insulin concentrations studied. In contrast, insulin dimer is smaller in the PT cube +.>Adaptation within the channel will result in considerable strain in the cubic lattice, which can be relieved by the expansion of the system and the observed increase in lattice parameter with increasing protein concentration.

Diffusion of insulin in lipid nanocarriers in cubic phase form

The diffusion coefficient of proteins within a lipid matrix depends on the size of the water channel and the presence of "bottlenecks" in the cubic phase, which are known to be directly related to the observed drug release rate. Thus, the diffusion coefficient of FITC-labeled insulin, whether in solution or encapsulated in the lipid cubic phase, was measured using FRAP, table 5.

Table 5 also measures the diffusion coefficient of octadecylrhodamine B chloride in MO and PT cubic phases using FRAP.

Table 5 lists the diffusion coefficients of octadecylrhodamine B chloride in each cubic phase, which are closely related to the corresponding lipid self-diffusion coefficients. In this regard, we noted that octadecylrhodamine B chloride was measured in MO (0.97 x10 ^-6 cm ² s ^-1 ) And PT (0.59 x 10) ^-6 cm ² s ^-1 ) The diffusion coefficient of (a) was highly similar to the lipid self-diffusion coefficients of MO and PT measured in previous studies using NMR (1.2X10, respectively ^-6 cm ² s ^-1 And 0.3X10 ^-6 cm ² s ^-1 ). The diffusion coefficients of lipids and insulin within the bulk lipid cubic phase and at the water interface are determined by FRAP. Diffusion coefficient of free insulin measured using FRAP (6.6x10 ^-6 cm ² s ^-1 ) Is equivalent to the diffusion coefficient in water (1.5x10 ^-6 cm ² s ^-1 ) And a diffusion coefficient (1.4x10 ^-6 cm ² s ^-1 ) And (5) reasonable and consistent.

The diffusion coefficient measured within the cubic matrix is only slightly lower than that measured in water. This may reflect the fact that the continuous network of aqueous channels in the cubic phase allows easy diffusion of insulin in three dimensions, especially considering that insulin dimers are not significantly larger than the aqueous channel radius. We note that the bulk PT cubic phase (4.17 x10 ^-6 cm ² s ^-1 ) The diffusion coefficient of endoinsulin is higher than that of MO (2.29X10 ^-6 cm ² s ^-1 ). This is a somewhat counterintuitive result, as smaller PT aqueous channel sizes are believed to hinder diffusion. Previous studies of glucose diffusion at 37℃have shown that the diffusion coefficient of glucose in the cubic phase formed by MO (1.9X10 ^-6 cm ² s ^-1 ) Higher than in PT (7.3X10) ^-7 cm ² s ^-1 ). However, we noted reasonable variation of the previously published values (diffusion of glucose in MO was also reported with a diffusion coefficient of 3.6x10 ^-7 cm ² s ^-1 ) Although the sample was formulated in 30% w/w aqueous phase, well below the point of MO water excess (48%). The partial location of insulin in such "nano pockets" within the PT matrix may result in a slight increase in the diffusion coefficient of insulin in PT (and an increase in the release rate described in the next section). The diffusion coefficient was measured at the center of the main cubic phase and at the water interface. For both lipids, the diffusion coefficient at the interface is about 15% higher than the diffusion coefficient inside the body. This may be due to the large concentration gradient of insulin between the bulk phase and the surrounding water.

Insulin release from lipid nanocarriers in the host cubic phase

The cubic insulin encapsulation efficiencies of MO and PT were measured to be 92.6% and 83.9%, respectively. The lipid nanocarrier mesophase containing insulin was then exposed to a pool of water and the concentration of released insulin was measured over a 24 hour period. Fig. 20A provides the percentage of insulin released from MO over time and fig. 20C provides the percentage of insulin released from PT over time. For MO and PT cubic phases, insulin release was initially fast and the rate gradually decreased over time as shown in fig. 20A and 20C, respectively. Insulin release from PT is generally faster, especially in the early stages, with 20% release from PT achieved within-2 hours, and-4 hours for MO. This is consistent with the faster diffusion coefficient of insulin in PT measured. On a longer time scale, about 50% was released from MO and 60% was released from PT within 24 hours. To study the release mechanism of the mesophase, the release profile was fitted against the Ritger-Peppas model:

In the first 7 hours (17 time points). M is M _t 、M ₀ K and n are the release amount at time t, the initial amount of encapsulation, the fitting coefficient and the fitting index, respectively. This allows for multiple release mechanisms to couple (e.g., diffusion control, zero order), where the diffusion index n represents the release mechanism and interface geometry, and the fitting coefficient k reflectsCharacteristics of the matrix and solute. The determined fitting parameters are shown in table 6. The calculated indices of MO and PT were 0.45 and 0.41, respectively, consistent with diffusion control or Fickian release (diffusion index less than 0.5).

Table 6 fitting parameters determined from insulin release studies. n is the fitting index and k is the fitting coefficient used in the Ritger-Peppas model. D is the diffusion coefficient determined by the Higuchi model.

The diffusion-controlled release of a thin film or slab can be similarly described by the Higuchi equation, which is defined as:

therein Q, C ₀ And D is the release concentration per unit area, the initial concentration per unit volume, and the diffusion coefficient, respectively. The diffusion control specifies that the release profile for v t should be linear. As shown in fig. 20B and D, the release showed a reasonable correlation with the linear fit, further indicating that the release was mainly diffusion controlled. The diffusion coefficient of insulin in MO and PT was determined using Higuchi equation (Drelease) and is shown in table 6. The determined parameters are compared to those found during FRAP analysis (DFRAP). DFRAP and Drelease values of insulin released from MO are 2.89x10 ^-6 cm ² S and 4.61x10 ^-6 cm ² And/s. For PT, DFRAP and Drelease values are 4.36x10, respectively ^-6 cm ² S and 6.85x10 ^-6 cm ² /s。

The lipid nanocarriers in cubic phase form can protect the encapsulated insulin from enzymatic degradation.

The ability of cubic matrices to protect encapsulated insulin from enzymatic degradation was investigated. Chymotrypsin (typically present in the small intestine) is used as model enzyme. Synchrotron CD was used to determine the evolution of insulin secondary structure in solution and after exposure to chymotrypsin in MO or PT based cubic phases.

For insulin in solution, fig. 21 (left), the initial spectrum consisted of a larger maximum at 200nm and a dual minimum at 208 and 222nm, consistent with a predominantly alpha-helical structure. The dichoroweb analysis (table 6) gave a 89% alpha helical structure. Upon addition of chymotrypsin, an immediate change in secondary structure is observed. It was observed that the maximum around 200nm was reduced stepwise over a 30 minute time scale, while the dual minima were gradually replaced by a single minimum at a lower wavelength, consistent with destruction of the alpha helix. After 12 minutes of enzyme addition, the percentage of alpha-helices decreased to 41% and the disordered structure increased to 41%, tables 7 and 8.

Tables 7 and 8 below are the dichoroweb analyses of synchrotron CD spectra of insulin in water (0.2 mg/mL) at various time points after chymotrypsin addition (0.02 mg/mL).

/>

The dichoroweb analysis cannot be performed on a longer time scale due to the extensive degradation of the protein structure. 30 minutes after chymotrypsin addition, the CD spectrum consisted of a single minimum at about 208nm, consistent with a predominantly disordered structure, fig. 21. The change in secondary structure of insulin encapsulated in phytantriol over a 132 minute time scale after chymotrypsin addition is shown. Figure 22 provides selected spectra of insulin in PT at 2, 30 and 130min to help visualize the differences between the two samples. Although a decrease in intensity of the maxima and the double minima around 200nm was observed, we noted that the change in insulin secondary structure was significantly reduced when encapsulated in phytantriol. Although the dichoroweb analysis of these spectra was not reliable due to the presence of phytantriol, the spectrum of insulin in PT remained largely a-helix after 30 minutes of chymotrypsin addition, while the insulin in water was largely disordered, fig. 21 and 22. Even 2 hours after chymotrypsin addition, the spectrum of insulin encapsulated in PT still shows a maximum near 200nm and a double minimum, consistent with retention of the α -helical structure (fig. 21 and 22), indicating the ability of the lipid cubic phase to protect insulin from enzymatic attack on a physiologically relevant time scale. Similar analysis is not possible for glycerol monooleate because the MO forms cubes that are more opaque than PT cubes, resulting in significant noise in the lower wavelength data. However, a near-ultraviolet spectrum associated with protein tertiary structure was obtained for insulin in solution and MO (fig. 23). Figure 24 provides selected spectra of insulin in solution and MO at different time points to help visualize the differences between the two samples. In solution, the tertiary structure of insulin changes immediately after chymotrypsin addition, with a minimum decrease at 270nm, again consistent with disruption of the alpha helix secondary structure. 9 minutes after the enzyme addition, this minimum value had substantially disappeared. Although a similar decrease in the minimum intensity of insulin was also observed in glycerol monooleate, the observed changes were more gradual and the time span was longer, again indicating the protective properties of the lipid matrix. Even 132 minutes after chymotrypsin addition, a minimum at 270nm was observed, FIG. 24.

Degradation of insulin by chymotrypsin can occur through two different mechanisms. Insulin can diffuse out of the bulk cubic phase and interact with chymotrypsin in the surrounding fluid. Alternatively, chymotrypsin may diffuse into the host cubic phase and pass through the aqueous channel network. We note that this protection occurs in the first two hours. As shown by the black lines in fig. 20A and C, only about 15% and about 25% of insulin was released from MO and PT, respectively, during this period. Thus, the rate of diffusion of the enzyme through the main cubic phase (depending on the size, charge and hydrophobicity of the enzyme) is the dominant factor limiting enzyme degradation.

Cubic mesophase nanocarriers provide significant protection for the supported proteins for up to 2 hours. We note that during this time some insulin may be released from the cubic phase, possibly resulting in rapid solution-based enzymatic degradation of the released protein. However, the release data obtained in this study suggests that only about 20% of the insulin should be released over this 2 hour period, while the majority of the loaded insulin remains within the protective lipid nanocarriers. This illustrates the potential of the lipid cubic phase to protect water-soluble proteins, small molecules, and peptide-based drugs with a range of molecular weights from the enzymatic damage environment of the human gastrointestinal tract.

Claims

1. A dosage form for oral delivery of a therapeutic agent to a subject, the dosage form comprising:

(i) A lipid nanocarrier formulation comprising a therapeutic agent and a lipid in the form of an mesophase; and

(ii) Enteric coating encapsulating the lipid nanocarrier formulation.

2. The dosage form of claim 1, wherein the mesophase may comprise an inverse bicontinuous cubic phase, an original cubic phase, a double rhombohedral cubic phase, a spiral icosahedron cubic phase, a hexagonal phase, an inverse hexagonal phase, a cubic structure, or a hexagonal structure.

3. The dosage form of claim 1 or 2, wherein the lipid nanocarrier comprises a lipid selected from the group consisting of: mono-, di-or tri-substituted glycerols, charged lipids, long chain lipids, branched lipids and glycolipids.

4. The dosage form of claim 3, wherein the charged lipid is dioleoyl-3-trimethylammoniopropane (DOTAP) which is present in an amount up to and including 10% of the lipid nanocarrier formulation.

5. The dosage form of any one of claims 1 to 4, wherein the lipid nanocarrier comprises a lipid of formula I:

6. The dosage form of any one of claims 1 to 4, wherein the lipid nanocarrier comprises a lipid of formula III:

7. The dosage form of any one of claims 1 to 6, wherein the lipid nanocarrier comprises a lipid selected from the group consisting of: glycerol monooleate, phytantriol and glycerol monopalmitoleate.

8. The dosage form of claim 7, wherein the glycerol monooleate is present at about 40% to 80% by weight of the formulation.

9. The dosage form of claim 7, wherein the phytantriol is present at about 60% to 75% by weight of the formulation.

10. The dosage form of any one of claims 1 to 9, wherein the therapeutic agent is selected from insulin or a derivative thereof, a steroid hormone, an antimicrobial agent such as an antibiotic, a protein such as a hormone, and a peptide such as a neuropeptide.

11. The dosage form of claim 10, wherein the insulin or derivative thereof is selected from the group consisting of insulin glargine (Lantus, basaglar, toujeo), insulin detention (Levemir), insulin deglutide (Tresiba), NPH (eurine N, norand reion insulin N), fast acting insulin, and short acting insulin.

12. The dosage form of claim 10 or 11, wherein the insulin or derivative thereof is present at 0.01% to 1% by weight of the lipid nanocarrier formulation.

13. The dosage form of any one of claims 1 to 12, wherein the nanocarrier comprises an aqueous channel having a size of 1nm to 17 nm.

14. The dosage form of any one of claims 1 to 13, wherein the enteric coating is soluble in the range of about pH 4.5 to pH 7.2.

15. The dosage form of claim 14, wherein the enteric coating is soluble in the range of about pH 5.0 to pH 6.0.

16. The dosage form of any one of claims 1 to 15, wherein the lipid nanocarrier formulation is encapsulated in an enteric coating having a thickness in the range of 0.07mm-0.4 mm.

17. The dosage form of any one of claims 1 to 16, wherein the lipid nanocarrier formulation is aqueous.

18. The dosage form of claim 17, wherein the water content of the lipid nanocarrier formulation is from 1% to 70% by weight of the lipid nanocarrier formulation.

19. The dosage form of claim 17, wherein the lipid nanocarrier formulation has a water content up to and including a range of 48% by weight of the lipid nanocarrier formulation, and wherein the lipid is glycerol monooleate, or has a water content up to and including a range of 48% by weight of the lipid nanocarrier formulation, and wherein the lipid is phytantriol.

20. The dosage form of any one of claims 1 to 19, wherein the dosage form is a capsule comprising a fill and a shell encapsulating the fill, the fill comprising the lipid nanocarrier formulation, the shell comprising an enteric coating.

21. The dosage form of claim 20, wherein the shell is coated with an enteric coating on at least one of the shell surface facing the filler and the outer shell surface.

22. The dosage form of claim 21, wherein the enteric coating on the filler-facing shell surface and on the outer shell surface each independently has a thickness in the range of 30 μιη to 380 μιη.

23. The dosage form of any one of claims 1 to 22, wherein the lipid nanocarrier formulation further comprises a swelling agent.

24. The dosage form of claim 23, wherein the water content of the lipid nanocarrier formulation ranges up to and including 70% by weight of the lipid nanocarrier formulation.

25. A method of preparing the dosage form of any one of claims 1 to 24, comprising the steps of:

a) Providing a lipid nanocarrier formulation; and

b) The lipid nanocarrier formulation is encapsulated in an enteric coating.

26. The method of claim 25, wherein the lipid nanocarrier formulation is prepared by contacting the lipid, the therapeutic agent, and an aqueous solvent under conditions sufficient to promote formation of a lipid mesophase.

27. The method of claim 26, wherein the lipid to aqueous solvent ratio is about 60:40w/w.

28. The method of any one of claims 25 to 27, wherein high pressure homogenization is used to promote self-assembly of lipids into an mesophase.

29. The method of any one of claims 25 to 28, wherein an enteric coating is applied to at least one of the shell surface and the outer shell surface facing the filling.

30. The dosage form of any one of claims 1 to 24 for use in the treatment or prevention of diabetes, a condition mediated by a bacterial infection, a condition mediated by human growth hormone, or a condition mediated by blood coagulation, and wherein the therapeutic agent is present in a therapeutically or prophylactically effective amount.

31. A method for treating or preventing diabetes, a condition mediated by bacterial infection, a condition mediated by human growth hormone, or a condition mediated by coagulation comprising administering to a subject in need thereof the dosage form of any one of claims 1 to 24 and wherein the therapeutic agent is present in a therapeutically or prophylactically effective amount.

32. Use of a dosage form according to any one of claims 1 to 24 in the manufacture of a medicament for the treatment or prophylaxis of diabetes, a condition mediated by a bacterial infection, a condition mediated by human growth hormone, or a condition mediated by coagulation, and wherein the therapeutic agent is present in a therapeutically or prophylactically effective amount.