Nanoparticle dispersions comprising therapeutic agents

Abstract

Disclosed herein are dense nanolipid fluid (DNLF) dispersions including desirable properties for incorporation of a bioactive agent, such as a peptide, into the lipid phase of the dispersion to biologically deliver the agent for typical purposes thereof. Also disclosed herein is a continuous process for preparing the DNLF dispersion comprising forming a coarse ground base stock and passing the base stock through a twin screw extruder. The dispersions disclosed herein can exhibit a particle size of less than 150 nm under stable storage conditions while forming a layered structure upon exposure to heat and / or evaporation of an aqueous component of the dispersion.

Description

[0001] Related applications

[0002] This application is filed as a PCT international patent application on November 21, 2023, and claims the benefit of and priority to: U.S. Provisional Application Serial No. 63 / 384,585, filed on November 21, 2022; U.S. Patent Application 63 / 384,584, filed on November 21, 2022; U.S. Provisional Application Serial No. 63 / 486,132, filed on February 21, 2023; and U.S. Provisional Application Serial No. 63 / 511,992, filed on July 5, 2023, each of which is incorporated herein by reference in its entirety. Background Art

[0003] Peptide compounds, water-soluble drugs, and hydrophilic bioactive compounds are difficult to administer effectively to living organisms due to poor bioavailability. Oral administration can result in poor bioavailability due to peptide degradation in the gastrointestinal tract or interference with various factors, such as congenital defects in the digestive or absorptive systems, intestinal disease / resection, drug interactions, or chronic alcohol consumption. Peptides and hydrophilic compounds are also generally difficult to absorb through the stratum corneum of the skin. Poor transdermal bioavailability may be due to an inability to penetrate the stratum corneum of the skin.

[0004] Peptides are an important class of drugs that act as agonists and antagonists of cellular functions, hormones, neurotransmitters, growth factors, antimicrobial agents, etc., activating and regulating biological functions, including signal transduction, namely the transcription and translation of cellular DNA.

[0005] Important biologically active peptides include oligopeptides comprising a single amino acid residue, oligopeptides comprising from 2 to about 25 amino acid residues linked in a polypeptide chain, and proteins comprising greater than about 25 amino acid residues. Peptides may comprise only amino acid residues, or they may be chemically modified, for example by lipidation, including acylation of peptides and proteins with long chain saturated lipids.

[0006] Peptides also undergo degradation under conditions commonly used for the synthesis of lipid nanoparticles. -1At relatively low shear rates, insulin shows unfolding and aggregation of globular structures under Couette flow [Bekard IB, Dunstan DE. Shear-induced deformation of bovine insulin in Couette flow. J Phys Chem B. 2009 Jun 25; 113(25):8453-7. doi:10.1021 / jp903522e. PMID:19534559], which is about three orders of magnitude lower than the shear stress encountered in ultrahigh pressure homogenization. The shear sensitivity of polypeptides limits the use of typical nanoemulsion and solid lipid nanoparticle particle pulverization processes, restricting lipid nanoparticle synthesis to methods such as the so-called double emulsion technique [Sarmento B, Martins S, Ferreira D, Souto EB. Oral insulin delivery by means of solid lipid nanoparticles. Int J Nanomedicine. 2007; 2(4): 743-9. PMID: 18203440; PMCID: PMC2676823], which exhibit relatively low shear processes. Consequently, lipid nanoparticle dispersions containing proteins such as insulin typically do not have a particle size less than 100 nanometers.

[0007] The present disclosure relates in part to the preparation of nanoparticle dispersions comprising hydrophilic and amphiphilic bioactive agents for oral administration.It is within this context that the present invention is made.

[0008] U.S. Patent Application No. 16 / 748,399, "METHOD OF PREPARING NANOPARTICLES BY HOT-MELT EXTRUSION," which is incorporated herein by reference in its entirety, describes a method for preparing concentrated lipid nanoparticle dispersions containing a lipid content of 25% to 60%, wherein "lipid content" means the sum of the concentrations of surfactant, water-immiscible oil, hydrophobic drug, and hydrophobic bioactive agent, wherein a "hydrophobic bioactive ingredient" is defined as a chemical compound or mixture of compounds that has an effect on living organisms, tissues, or cells and has a logP value (i.e., the logarithm of the octanol / water partition coefficient) greater than 1, and wherein "lipid nanoparticles" means particles having a diameter of less than 150 nm that comprise a surfactant, a water-immiscible oil, and one or more hydrophobic bioactive ingredients. Such highly concentrated dispersions of lipid nanoparticles comprising a hydrophobic bioactive agent in water, which contain a lipid content between 25% and 60%, are known as dense nanolipid fluid (DNLF) dispersions.

[0009] Nanolipid dispersions comprising hydrophobic bioactive ingredients, i.e., dispersions of lipid particles having a diameter less than 150 nm in water, can be used to increase the bioavailability of hydrophobic bioactive ingredients. Nanolipid dispersions comprising hydrophobic bioactive ingredients can be difficult to prepare, especially those having a lipid content greater than 25%, i.e., DNLF dispersions. DNLF dispersions provide advantages over nanolipid dispersions having a lipid concentration less than 25%, including the ability to administer a useful amount of hydrophobic bioactive agents in low volumes (such as oral and sublingual administration). DNLF dispersions can also be used for dilution to administer when the therapeutically effective amount of the hydrophobic bioactive agent is low or the volume of administration is high.

[0010] The ability of DNLF dispersions to adopt a lamellar structure is an important property related to bioavailability. The ability of DNLF dispersions to adopt a lamellar structure upon heating, evaporation, or contact with other materials can facilitate absorption into other lamellar materials, including skin and cell membranes. Potential lamellar structure, i.e., the tendency to form a lamellar structure upon physical change, can be identified by heating a DNLF dispersion and observing evidence of lamellar structure. Such evidence includes a decrease in conductivity, which appears as a negative peak in a conductivity versus temperature graph, or the appearance of optical birefringence.

[0011] In certain aspects, the present disclosure relates to formulations of stable nanoparticle dispersions comprising bioactive compounds that typically have poor oral bioavailability. The present disclosure also relates to treatment methods comprising oral administration of nanoparticles comprising drugs that are typically susceptible to degradation in the gastrointestinal tract, such as peptides. In certain aspects, DNLF dispersions with improved properties while incorporating a peptide compound of interest are contemplated herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 is a graph of conductivity versus temperature for a mill-base composition comprising caffeine compared to a standard (Example 10).

[0013] Figure 2 is a graph of dasatinib plasma concentration versus time after administration of Examples 16 to 19.

[0014] Figure 3 is a graph comparing the relationship between the plasma concentration of dasatinib and time after administration of Examples 17 and 20.

[0015] Figure 4 is a graph comparing the relationship between the plasma concentration of dasatinib and time after administration of Examples 17 and 21.

[0016] Figure 5 is a graph of the particle size distribution of a DNLF dispersion before and after lipolysis (Example 22).

[0017] Figure 6 is a graph of the particle size of DNLF of Example 22 before and after lipolysis, expressed as the volume percentage of particles having a smaller diameter than a certain diameter.

[0018] Figure 7 is a graph showing the pH static titration results of Example 23.

[0019] Figure 8 is a graph of the particle size of DNLF before and after lipolysis (Example 23).

[0020] Figure 9 is a graph of the particle size of DNLF of Example 23 before and after lipolysis, expressed as the volume percentage of particles having a smaller diameter than a certain diameter.

[0021] Figure 10 is a graph comparing the pH steady-state titration of DNLF without orlistat and DNLF with orlistat (Example 28).

[0022] Figure 11 The results of the pH static titration of Example 30 are shown. Summary of the Invention

[0023] The present invention generally provides nanolipid dispersions comprising peptides and hydrophilic bioactive compounds, which can be used for administration to mammals by oral, sublingual, buccal, dermal, subcutaneous and intravascular routes of administration. In some aspects, the nanolipid dispersions can comprise water, water-immiscible oils, surfactants and a mixture of a bioactive agent or a bioactive agent selected from peptides (e.g., amino acids, oligopeptides, polypeptides, proteins), hydrophilic bioactive agents, hydrophobic bioactive agents, small molecules.

[0024] It is contemplated herein that the nanoparticle dispersion for peptide bioactivator is delivered to mammals. In some aspects, nanoparticle dispersion can include one or more bioactivators (for example, hydrophilic small molecules, hydrophilic or amphipathic peptides) of 0.01 % by weight to 8.5 % by weight, one or more high hydrophilic-lipophile balance (HLB) surfactants of 1.6 % by weight to 11.9 % by weight, one or more low hydrophilic-lipophile balance (HLB) surfactants of 2.6 % by weight to 12.0 % by weight, one or more water-immiscible oils of 13.9 % by weight to 41.8 % by weight, and the water of 39.1 % by weight to 58.1 % by weight. In other respects, high hydrophilic-lipophile balance (HLB) surfactant can include ester type polyethoxylated high hydrophilic-lipophile balance (HLB) surfactant, and low hydrophilic-lipophile balance (HLB) surfactant includes phospholipid low hydrophilic-lipophile balance (HLB) surfactant. In other aspects, the high hydrophilic-lipophilic balance (HLB) surfactant can include an ether-type polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactant.

[0025] In a further aspect, the dispersion may comprise an ether polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactant and an ester polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactant. In such aspects, the weight ratio of the ether polyethoxylated high hydrophilic-lipophilic balance HLB surfactant to the ester polyethoxylated high hydrophilic-lipophilic balance HLB surfactant is greater than 1:1. Alternatively, the dispersion comprising the ether polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactant may further comprise a non-polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactant (e.g., sodium lauryl sulfate).

[0026] In certain aspects, the bioactive agent can be hydrophilic or amphiphilic (e.g., the bioactive agent is not hydrophobic, and / or the bioactive agent has a logP of less than 1). The bioactive agent can be a peptide (e.g., a peptide having a molar mass in the range of 1 kDa to 1,000 kDa), or alternatively, a small molecule. In certain aspects, the bioactive agent can be insulin.

[0027] In other aspects, the lipophilic phase of the nanoparticle dispersion (e.g., surfactant, water-immiscible oil, hydrophobic therapeutic agent, etc.) is resistant to lipolysis (e.g., less than 25% of the ester bonds are hydrolyzed in one hour in the presence of 500 units / mL of porcine pancreatic lipase; alternatively, it is non-digestible). In certain aspects, the nanoparticle dispersion may include a lipase inhibitor (e.g., orlistat). In other aspects, the high HLB surfactant is inert to lipolysis (e.g., non-digestible). In further aspects, the nanoparticles in the nanoparticle dispersion may have a net positive charge.

[0028] Encapsulated nanoparticle dispersions are also contemplated herein. In some aspects, the capsule comprises an encapsulating polymer surrounding the nanoparticle dispersion. In other aspects, the encapsulating polymer comprises a carboxylic acid, and the pH of the nanoparticle dispersion is less than the pKa (e.g., at least one pH unit) of the encapsulating polymer carboxylic acid group.

[0029] Also contemplated are methods for treating a disorder comprising orally administering any of the nanoparticle dispersions or capsules disclosed herein to a patient in need thereof.

[0030] definition

[0031] As used herein, the term "lipid" refers to fats and fat-derived materials that are insoluble in water except as micellar solutions or dispersions, but soluble in organic solvents, including surfactants, water-immiscible oils, and hydrophobic bioactive ingredients.

[0032] As used herein, the term "dense nanolipid fluid (DNLF) dispersion" refers to a dispersion of lipid nanoparticles in water having a lipid content between 25% and 60%, wherein the lipophilic content refers to the sum of the concentrations of surfactant, water-immiscible oil, hydrophobic drug, and hydrophobic therapeutic agent.

[0033] As used herein, the term "nanolipid dispersion" refers to a dispersion of lipid particles in water having a volume average particle size of less than 150 nm.

[0034] As used herein, the term "enteral administration" refers to the administration of food or drugs via the gastrointestinal tract of a human.

[0035] As used herein, the term "therapeutic agent" refers to a chemical compound, complex, or composition that exhibits a desired effect in a biological context (ie, when administered to a subject).

[0036] As used herein, the term "drug" refers to a chemical compound regulated as a drug by the U.S. Food and Drug Administration.

[0037] As used herein, the term "oral administration" refers to the process of delivering a drug through the mouth to the digestive tract.

[0038] As used herein, the term "orally administered" refers to a process in which a therapeutic agent is held or applied to the oral area and diffuses directly into the bloodstream through the oral mucosa.

[0039] As used herein, the term "sublingual administration" refers to a process in which a therapeutic agent is held or applied to the sublingual area and diffuses directly into the bloodstream through the oral mucosa.

[0040] As used herein, the term "dermal administration" refers to the process of applying a therapeutic agent to the skin.

[0041] As used herein, the term "HLB" refers to the hydrophile-lipophile balance, which is an empirical expression of the relationship between the hydrophilic ("water-loving") and hydrophobic ("water-hating") groups of a surfactant.

[0042] As used herein, the phrase "low hydrophilic-lipophilic balance (HLB) surfactant" refers to a surfactant having a hydrophilic-lipophilic balance (HLB) value of less than about 10.

[0043] As used herein, the phrase "high hydrophilic-lipophilic balance (HLB) surfactant" refers to a surfactant having a hydrophilic-lipophilic balance (HLB) value of about 14 or greater.

[0044] As used herein, the phrase "anionic high hydrophilic-lipophilic balance (HLB) surfactant" refers to a surfactant having a hydrophilic-lipophilic balance (HLB) value equal to or greater than about 14, which dissociates in water to produce an anion comprising a hydrophobic group covalently bonded to an anionic group (such as a sulfate or carboxylate group) plus a cation selected from the group consisting of a hydrogen ion and an alkali metal ion.

[0045] As used herein, "cryoprotectant" refers to a compound used to delay ice formation when cooling a composition containing water.

[0046] As used herein, "preservative" refers to a substance or chemical added to products such as foods, beverages, and pharmaceuticals to prevent decomposition caused by microbial growth or by undesirable chemical changes.

[0047] As used herein, the term "essential oil" refers to a volatile oil derived from the leaves, stems, flowers, or twigs of a plant or a synthetically prepared compound having the same chemical properties. Essential oils typically have the smell or flavor of the plant.

[0048] As used herein, the term "immiscible" refers to liquids that will not mix or remain mixed with each other, although under certain conditions, such as elevated temperatures, they may mix, but any such mixture will generally be thermodynamically unstable and will generally separate into distinct phases at lower temperatures.

[0049] As used herein, the term "water-immiscible oil" refers to a compound or mixture of compounds that is immiscible or insoluble in water and does not disperse in water in the absence of a surfactant, the compound comprising hydrophobic groups, such as water-insoluble hydrocarbon chains, and does not comprise hydrophilic groups, such as ionizable groups, hydroxyls, amines, carboxylic acids, poly(ethylene oxide).

[0050] As used herein, "latent lamellar structure" refers to a lamellar structure that is not observable in a dispersion comprising surfactant, oil, and water but becomes observable when the dispersion is subjected to heating or evaporation.

[0051] As used herein, the phrase "bioactive ingredient" or "bioactive agent" refers to a chemical compound or mixture of compounds that has an effect on living organisms, tissues, or cells (eg, skin care agents such as hyaluronic acid and its derivatives).

[0052] As used herein, the phrase "lipid nanoparticle" refers to particles less than 150 nm in diameter that comprise a surfactant, a water-immiscible oil, and one or more hydrophobic bioactive components.

[0053] As used herein, the phrase "vesicular nanoparticle" refers to a nanoparticle having a vesicular structure, ie, comprising a lipid bilayer and encapsulating an aqueous core.

[0054] As used herein, when referring to nanoparticle formulations, the phrase "edible" describes a formulation that is suitable for and designed for oral administration. Thus, in certain aspects, an edible nanoparticle dispersion may comprise or consist of ingredients approved by the FDA for oral consumption. However, it should be understood that the edible formulations referred to herein may also comprise additional ingredients suitable for oral ingestion that have not been approved by the FDA for oral consumption.

[0014] The edible nanoparticle dispersion has a vesicular structure, i.e., comprises a lipid bilayer and encapsulates an aqueous core.

[0055] As used herein, when referring to nanoparticle dispersions, the phrases "digestible" and "non-digestible" describe dispersions that retain their essential structural characteristics under lipolysis conditions. A digestible dispersion refers to a nanoparticle dispersion comprising a lipid phase having an ester-based surfactant. A non-digestible dispersion refers to a nanoparticle dispersion comprising a lipid phase lacking a surfactant that undergoes lipolysis (e.g., an ether-based surfactant).

[0056] As used herein, an agent can be characterized as hydrophilic or hydrophobic based on an agent having a logP less than 1 or greater than or equal to 1, respectively. Individually, an agent can be characterized as amphipathic, i.e., comprising both a hydrophilic portion and a hydrophobic portion, regardless of their overall logP value. For example, a peptide can generally be considered to be amphipathic, wherein both hydrophilic and hydrophobic amino acids are included in the amino acid sequence of the peptide. When conjugated to a hydrophobic group by a covalent bond, the peptide can also be considered to be amphipathic. For example, palmitoyl tripeptide 1 (PubChem CID 156595485) is a condensation product of the amine group of glycine of a glycine-histidine-lysine tripeptide with hydrophobic palmitic acid, and semaglutide (PubChem CID 56843331) is a condensation product of a peptide of 31 amino acid residues with hydrophobic stearic acid. Due to the presence of a hydrophobic portion and a hydrophilic portion in the compound, each can be characterized as amphipathic. Even individual amino acids containing hydrophobic side chains (e.g., phenylalanine, leucine, isoleucine, tyrosine, tryptophan, valine, methionine, proline) can be considered amphipathic because a hydrophilic carboxyl / amino moiety is also attached to the alpha carbon.

[0057] The logP value of insulin (PubChem compound identifier (CID) number 118984375) is reported as -13.1, indicating that insulin is highly hydrophilic. Therefore, insulin is soluble in water at low pH (e.g., 0.1N HCl). However, peptides are also amphoteric and can therefore act as either an acid or a base depending on conditions such as pH. For example, the isoelectric point of insulin is pH 5.4. When the pH of dissolved insulin approaches the isoelectric point (starting from about pH 4.3), insulin becomes insoluble and therefore exhibits hydrophobicity. When the pH increases to physiological pH 7.4, insulin becomes water-soluble again and exhibits hydrophilicity.

[0058] Despite this behavior, it will be understood in the context of the present disclosure that insulin is characterized as hydrophilic based on its logP being less than 1, and as hydrophobic or amphipathic based on the properties of its chemical structure. It is noteworthy that the hydrophilicity (logP) of a peptide can depend on the density of ionizable groups, peptide chain length, and peptide chain conformation. Peptides can also form hydrophobic ion pairs (HIPs). For example, if anionic sodium lauryl sulfate is added to a uniform, clear solution of a cationic, hydrophilic insulin in 0.1N HCl, a hydrophobic precipitate is formed.

[0059] Unless otherwise stated, all amounts based on percentages are based on the weight of the relevant component relative to the total weight of the relevant composition. DETAILED DESCRIPTION

[0060] Dense nanolipid dispersions containing various bioactive compounds are contemplated and described herein, including hydrophilic small molecules and amphiphilic compounds (e.g., peptides, modified amino acids, palmitoyl tripeptide-1, etc.), and combinations thereof. Also disclosed herein are methods for preparing the dispersions and methods for administering the nanoparticle dispersions to treat diseases. In certain aspects, the nanolipid dispersions can be lipolytically resistant, for example, by the addition of lipolysis inhibitors, zwitterionic agents, or sustained particle size.

[0061] In certain aspects, it is noted that the nanolipid dispersion itself or any of its components (e.g., a high hydrophilic-lipophilic balance (HLB) surfactant, a low hydrophilic-lipophilic balance (HLB) surfactant, a water-immiscible oil, or a combination thereof present in an amount greater than 0.1%) may be listed as a food additive permitted under Part 172 of Title 21 of the U.S. Food and Drug Administration Code of Federal Regulations for direct addition to food for human consumption.

[0062] Also disclosed herein are encapsulated nanolipid dispersions.Methods of treating diseases using the nanolipid dispersions disclosed herein are also contemplated.

[0063] Nanolipid dispersions

[0064] Disclosed herein are nanolipid dispersions (e.g., dense nanolipid fluid (DNLF) dispersions) that can be used to incorporate any number of bioactive and non-bioactive substances of interest without destroying the properties of the dispersion. In some aspects, the dispersions disclosed herein can incorporate oil, water, and surfactants in various amounts as described herein. In some aspects, the dispersions disclosed herein can allow for the incorporation of larger amounts of any bioactive and non-bioactive substances or combinations thereof relative to, for example, an otherwise identical dispersion in which the lipid nanoparticles present in the dispersion comprise vesicular lipid nanoparticles (or an otherwise identical nanoparticle dispersion in which the lipid nanoparticle content is greater than 10%, greater than 25%, greater than 50%, or greater than 80% vesicular nanoparticles).

[0065] In certain embodiments, DNLF dispersions can improve the stability and biodelivery of bioactive agents for conventional, typical, and recognized clinical purposes, and have improved efficacy relative to administration forms that suffer from relatively reduced bioavailability. For example, the DNLF dispersions disclosed herein can provide advantages over nanolipid dispersions having a lipid concentration of less than 25%, including the ability to administer useful amounts of bioactive agents at low administration volumes (e.g., buccal and sublingual administration). DNLF dispersions can also be used as diluted compositions for administration where the therapeutically effective amount of the bioactive agent is low or the administration volume is high.

[0066] In some aspects, the nano-lipid dispersions disclosed herein (including DNLF dispersions) can include water, water-immiscible oils, surfactants, and optional bioactivators, such as peptides, biomolecules, or small molecules. In some aspects, the nanoparticle dispersions can further include a structure-promoting additive that can impart necessary stability during the formation of the mesophase between the crude mixture of components and the formation of a stable nanoparticle dispersion, such as during the mechanical shearing and kneading performed in a twin-screw extruder. In some aspects, the dispersions envisioned herein can further include additives for the preservation and stability of the dispersions and other common uses.

[0067] Surprisingly, non-hydrophobic (i.e., hydrophilic or amphiphilic) bioactive agents can be incorporated into the lipid phase of nanoparticle dispersions. For example, a peptide can be hydrophilic and / or have a net positive charge at physiological pH, which can be paired with anionic hydrophobic substances (e.g., lauryl sulfate) so that the peptide ion pairs become hydrophobic during preparation and associate in the lipid phase of the dispersion. In some aspects, the incorporated peptides are recovered from the dispersion primarily in their native state, indicating that their incorporation into the dispersion does not cause damage to the secondary, tertiary, or quaternary structure of the peptide due to unfolding. This is particularly surprising in the case of peptides that contain an external hydrophilic group for solvation in an aqueous medium and transfer the hydrophobic group to the internal portion of the peptide when solvated. Therefore, unexpectedly, even hydrophilic and amphiphilic peptides can be alternately incorporated and dissociated in the lipid phase without affecting their overall structure. Incorporating the peptide into the lipid phase of the dispersion provides a protective effect on the peptide because it becomes difficult for the peptide to interact with hydrophilic enzymes that can cleave the peptide bond. The incorporation of hydrophilic and amphiphilic peptides as disclosed herein may also result in the unexpected benefit of helping such compounds cross biological membranes as part of a lipid phase dispersed structure, which may allow administration of much larger peptide compounds through the skin and other membranes beyond previously accepted limitations.

[0068] The molecular weight of the bioactive agent suitable for nanoparticle dispersions disclosed herein is generally unrestricted. For example, peptides suitable for incorporation into nanoparticle dispersions described herein can generally be of any size, for example, ranging from single peptides (amino acids), oligopeptides (generally ranging from 2 to 25 amino acids), including dipeptides, tripeptides, tetrapeptides, etc., to polypeptides (generally comprising more than about 50 amino acids), to small proteins comprising approximately 50 to 500 amino acids, and large proteins comprising more than 500 amino acids. The peptide sequences suitable for incorporation into dispersions disclosed herein can also be characterized by their molecular weight, and in some aspects can be in the range of 1 kDa to 2,000 kDa, 3 kDa to 1,000 kDa, 5 kDa to 800 kDa, 10 kDa to 500 kDa, 25 kDa to 100 kDa. Similarly, the size of the small molecules suitable for dispersions is only subject to practical limitations and the definition of the compound class. Thus, the nanoparticle dispersions herein may comprise a bioactive agent that is a small molecule having a molecular weight of less than about 300 g / mol, less than about 500 g / mol, less than about 1000 g / mol, or less than about 1,500 g / mol; alternatively, in the range of about 100 g / mol to about 1,000 g / mol or about 250 g / mol to about 750 g / mol.

[0069] The bioactive agent suitable for incorporation into the nanoparticle dispersions described herein can also comprise small molecules. In some aspects, the bioactive agent can be selected from spermidine, caffeine, acetaminophen, nicotinamide, or a combination thereof. This paper also contemplates small molecules for treatment that can be found in relevant FDA approvals.

[0070] The peptides and other bioactive agents suitable for incorporation into the dispersions disclosed herein can have any number of physiological purposes or classifications. In some aspects, the bioactive agent can be a peptide selected from a signaling peptide, an antibody, an AA peptide, a hormone, or a combination or derivative thereof. In some aspects, the peptides suitable for use in the dispersions disclosed herein can include lipid derivatives, such as lipidated peptides. For example, in some aspects, the dispersion can include insulin as a bioactive agent in an amount that is any amount or range of amounts contemplated for the bioactive agent herein. Insulin is a human hormone composed of 51 amino acids and is approximately 5.8 kDa. In other aspects, the dispersion can include a tyrosine kinase inhibitor (e.g., nilotinib, dasatinib) or a skin care agent (e.g., hyaluronic acid).

[0071] Contemplated peptides suitable for incorporation into dispersions as described herein may include abaloparatide, abarelix, aclerastide, afamelanotide, albiglutide, albusomatropin, anamorelin, angiopeptin, angiotensin, avexitide, aviptadil, bacitracin, bleomycin, bovine serum albumin, bleomycin, Bremelanotide, Carbetocin, Carfilzomib, Colistin, corticorelin, Cyclosporine A, Dalbavancin, Degarelix, Dulaglutide, Enfuvirtide, Etelcalcetide, Exenatide, Goserelin, Icatibant, Lanreotide, Gastrosporine Ghrelin, leptin, linaclotide, liraglutide, lixisenatide, lucinactant, macimorelin, mifamurtide, nesiritide, octreotide, oritavancin, pasireotide, peginesatide, plecanatide, polymyxin B , Pramlintide, Sandostatin, selepressin, Semaglutide, Serum albumin, Setmelanotide, Somapacitan, Somatostatin, Lanreotide, Taltirelin, Taspoglutide, Teduglutide, Teriparatide, Tesamorelin, Thymalfasin,Thymosin beta-4, tirzepatide, ularitide, vapreotide, ziconotide, and combinations thereof.

[0072] Peptide-based cosmeceuticals may also be incorporated into the dispersions disclosed herein and, in certain aspects, may include acetyl dipeptide-3 aminohexanoate, acetyl dipeptide-1 cetyl ester, acetyl hexapeptide-1, acetyl hexapeptide-3, acetyl hexapeptide-30, acetyl hexapeptide-37, acetyl hexapeptide-38, acetyl hexapeptide-39, acetyl hexapeptide-49, acetyl hexapeptide-51 amide, acetyl hexapeptide-8, acetyl octapeptide-3, acetyl tetrapeptide-11, acetyl tetrapeptide-15, acetyl tetrapeptide-2, acetyl tetrapeptide-22, acetyl tetrapeptide-3, acetyl tetrap ...3, acetyl tetrapeptide-3, acetyl tetrapeptide-3, acetyl tetrapeptide-3, acetyl tetrapeptide-3, acetyl tetrapeptide-3, acetyl tetrapeptide-3, acetyl tetrapeptide-3, acetyl tetrapeptide-3, acetyl tetrapeptide-3, acetyl tetrapeptide-3, acetyl tetrapeptide-3, acetyl tetrapeptide-3, acetyl tetrapeptide-3, Acyl Tetrapeptide-40, Acetyl Tetrapeptide-5, Acetyl Tetrapeptide-9, Acetyl Hexapeptide-3, β-Glucan, Biotinyl Tripeptide-1, Hexanoyl Tetrapeptide-3, Collagen, Copper Peptide (GHK-Cu), Copper Tripeptide-34, Decapeptide-10, Decapeptide-12, Decapeptide-18, Decapeptide-4, Diaminopropionyl Tripeptide-33, Dipeptide-2, Elastin, Glutathione, Heptapeptide-6, Heptapeptide-7, Hexapeptide-10, Hexapeptide-11, Hexapeptide-12, Hexapeptide-2, Hexapeptide-3, Hexapeptide-42, Hexapeptide-9, Keratin, Manganese Tripeptide- 1. Myristoyl Hexapeptide-16, Myristoyl Hexapeptide-23, Myristoyl Hexapeptide-4, Myristoyl Pentapeptide-17, Myristoyl Pentapeptide-17, Myristoyl Pentapeptide-8, Myristoyl Pentapeptide-8, Myristoyl Tetrapeptide-12, Myristoyl Octapeptide-2, Nonapeptide-1, Octapeptide-2, Oligopeptide-10, Oligopeptide-20, Oligopeptide-24, Oligopeptide-34, Oligopeptide-51, Oligopeptide-54, Oligopeptide-68, Palmitoyl Dipeptide-5, Palmitoyl Dipeptide-6, Palmitoyl Glutathione, Palmitoyl Hexapeptide, Palmitoyl palmitoyl hexapeptide-12, palmitoyl pentapeptide-3, palmitoyl pentapeptide-4, palmitoyl tetrapeptide-7, palmitoyl tripeptide, palmitoyl tripeptide-1, palmitoyl tripeptide-3, palmitoyl tripeptide-36, palmitoyl tripeptide-38, palmitoyl tripeptide-5, palmitoyl tripeptide-8, pentapeptide-18, pentapeptide-18, pentapeptide-25, pentapeptide-3, tetrapeptide-21, tetrapeptide-26, tetrapeptide-30, trifluoroacetyl tripeptide-2, tripeptide-1, tripeptide-10 citrulline, tripeptide-29, tripeptide-32, tripeptide-9 citrulline, or a combination thereof.

[0073] Suitable bioactive agents also include compounds wherein the pH of the nanodispersion is less than the isoelectric point of the bioactive agent. As mentioned above, the isoelectric point of insulin is pH 5.4. In aqueous mixtures with a pH similar to the isoelectric point of insulin, starting from about pH 4.3, insulin becomes insoluble and therefore exhibits hydrophobicity. As the pH decreases, insulin again adopts hydrophilic behavior. Surprisingly, in aspects disclosed herein, it was found that even if the pH of the nanoparticle dispersion is lower than the isoelectric point of the bioactive agent, the bioactive agent can be incorporated into the lipid portion of the nanoparticle dispersion. In some aspects, the pH of the nanoparticle dispersion can be less than the isoelectric point of the bioactive agent (less than 0.1, less than 0.5, less than 1, less than 1.5, less than 2.5, less than 3; alternatively, in the range of less than about 0.5 to less than about 5, less than about 1 to less than about 4, less than about 2 to about 3).

[0074] In certain aspects, the DNLF dispersion comprises zwitterionic lipid nanoparticles having separated positively and negatively charged ions.

[0075] Having separated positively and negatively charged ions can, but does not necessarily, make lipid nanoparticles and macromolecules (such as peptides and proteins) hydrophilic. Because the association of positively and negatively charged ions can occur in a manner such that ion pairs are buried within hydrophobic groups, the presence of charged ions does not always result in particles or peptides acquiring hydrophilic, water-soluble, or water-dispersible characteristics. For example, in a hydrophobic ion pair formed by the association of positively charged ions in a peptide with negatively charged ions in an anionic surfactant, the charged ions are positioned so that the complex is hydrophobic. For peptides close to their isoelectric points, the charged ions can also be positioned so as not to impart hydrophilicity. Therefore, peptides close to their isoelectric points can be considered hydrophobic. In the case where the lipid nanoparticle surface is not hydrophilic enough for stable dispersibility due to the presence of charged ions, hydrophilicity and dispersion stability can be provided by the polyethoxylated chain hydrophilic groups. In the case where the hydrophilicity of the peptide is not enough to dissolve, they can be dispersed by encapsulation in lipid nanoparticles, whether as a hydrophobic ion pair or individually.

[0076] In some aspects, bioactive agents can be biomolecules composed of or comprising nucleotides, including but not limited to compounds comprising naturally occurring nucleotides (adenosine, guanine, tyrosine, cytosine) and their common synthetic derivatives. Bioactive agents disclosed herein can include nucleic acid sequences, such as DNA and RNA, both of which include naturally occurring, synthetic, and fragments thereof.

[0077] In some aspects, the nanoparticle dispersion can include any suitable amount of water that results in a stable nanoparticle dispersion having suitable properties (e.g., a particle size less than 150 nm, a non-vesicular and potential lamellar structure, sufficient incorporation of bioactive agents and additives, an acceptable ratio of water-immiscible oil to bioactive agents, etc.). Without being bound by theory, it is believed that in some aspects the amount of water suitable for the dispersion can be minimized to provide a dispersion with an excess or large amount of lipid phase to increase the capacity of the bioactive agents and additives within the dispersion. In some aspects, the nanoparticle dispersion can include water in an amount ranging from 10% to 90% by weight, 20% to 80% by weight, 25% to 75% by weight, 35% to 65% by weight, 40% to 60% by weight, 40% to 55% by weight, 45% to 60% by weight, or 50% to 60% by weight. In other aspects, the nanoparticle dispersion can include less than 75 wt%, less than 65 wt%, less than 55 wt%, less than 50 wt%, less than 45 wt%, less than 40 wt%, or less than 35 wt% water. Certain aspects can include water in an amount ranging from 39.1 wt% to 58.1 wt%.

[0078] Dispersion disclosed herein can further comprise water-immiscible oil.Not bound by theory, water-immiscible oil can be provided, for the purpose of carrying bioactive components in dispersion and across biofilm, which may be necessary to realize the effective incorporation of bioactivator in nanoparticle dispersion and the delivery of bioactivator to biological entity or in biological entity to carry out appropriate treatment respectively. Therefore, it may be advantageous to increase the relative amount of water-immiscible oil and surfactant in the dispersion, so that the capacity of bioactivator as described above is maximized. It may also be advantageous to maximize the amount of lipid phase in the dispersion to maximize the peptides degraded in aqueous environment, such as proteolytic enzymatic degradation of insulin in the gastrointestinal tract. In some aspects, dispersion can be included in the water-immiscible oil of the amount in the scope of 5 % by weight to 65 % by weight, 10 % by weight to 45 % by weight, 15 % by weight to 40 % by weight or 20 % by weight to 35 % by weight. In certain aspects, the amount of water-immiscible oil may be greater than 10 wt%, greater than 20 wt%, greater than 30 wt%, greater than 35 wt%, greater than 40 wt%, greater than 45 wt%, greater than 50 wt%, or greater than 55 wt%.

[0079] Generally, the water-immiscible oil is not limited to any particular chemical structure or combination and can be an oil or combination of oils that contains hydrophobic groups such as water-insoluble hydrocarbons. Typically, the water-immiscible oil does not contain hydrophilic groups such as ionizable groups, hydroxyls, amines, carboxylic acids, or poly(ethylene oxide), however, there may be exceptions (e.g., in the case of 2-butyl-1-octanol) based on the overall properties of the oil (e.g., the water-immiscible oil exhibits non-amphiphilic characteristics in testing).

[0080] In certain aspects, the water-immiscible oils may include cocoyl caprate / caprylate, alkyl alcohols such as Isofol 12 (2-butyl-1-octanol), benzyl alcohol, diisopropyl adipate, capric / caprylic triglyceride oil, isopropyl myristate, limonene, medium chain triglyceride oil, mineral oil, omega 3 fatty acids, oleyl alcohol, isohexadecane, isododecane, C 13 to C 15 Alkanes or combinations thereof. In other aspects, suitable water-immiscible oils useful in practicing the invention disclosed herein may include medium chain triglyceride oils, coconut oil, isopropyl palmitate, isopropyl myristate, methyl caprate, ethyl myristate, ethyl oleate, mineral oil, orange essential oil, cyclopentasiloxane, poly(dimethylsiloxane), hexadecane, propylene glycol dicaprylate, isododecane, isoeicosane, isohexadecane, soy biodiesel, jojoba oil, cocoyl caprylate, C 10 to C 13 Alkanes, squalane, sunflower oil, diacetylated monoglycerides, clove essential oil, and limonene.

[0081] Those skilled in the art will appreciate that water and water-immiscible oils as described above cannot be combined to form nanoparticle dispersions as disclosed herein alone and readily separate into different phases. Therefore, surfactants are needed to stabilize dispersions comprising water and water-immiscible oils as dense nanolipid fluid dispersions and to prevent phase separation of water and water-immiscible oils. Surfactants as disclosed herein can be characterized by their structure and their general lipophilicity.

[0082] In certain aspects, the dispersions disclosed herein can comprise a high hydrophilic-lipophilic balance (HLB) surfactant, a low hydrophilic-lipophilic balance (HLB) surfactant, or both. Typically, the high hydrophilic-lipophilic balance (HLB) surfactant can include a polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactant, an ether-type polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactant, an ester-type polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactant, or a non-polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactant.

[0083] Suitable polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactants may include, for example, one or more ether-type polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactants, each independently having a hydrophilic-lipophilic balance (HLB) value equal to or greater than about 14; one or more ester-type polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactants, each independently having a hydrophilic-lipophilic balance (HLB) value equal to or greater than about 14; one or more non-polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactants, each independently having a hydrophilic-lipophilic balance (HLB) value equal to or greater than about 14; one or more low hydrophilic-lipophilic balance (HLB) surfactants, each independently having a hydrophilic-lipophilic balance (HLB) value less than about 10; one or more phospholipid low hydrophilic-lipophilic balance (HLB) surfactants, each independently having a hydrophilic-lipophilic balance (HLB) value less than about 10; and combinations thereof.

[0084] Suitable one or more ether-type polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactants, each independently having a hydrophilic-lipophilic balance (HLB) value equal to or greater than about 14, may include, for example, surfactants derived from the addition of about 20 to about 100 moles of ethylene oxide to fatty alcohols such as lauryl alcohol, cetyl alcohol, oleyl alcohol, stearyl alcohol, and isotridecyl alcohol, which may be referred to as alcohol ethoxylates, polyoxyethylene alkyl ethers, and polyoxyethylated fatty alcohols, including laureth-23, ceteth-20, ceteareth-20, ceteareth-25, ceteareth-30, oleth-20, steareth-20, steareth-40, and steareth-100, which are available, for example, as commercial products, including L23, CS20, 020, S20 and S100. Particularly preferred ether-type polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactants include, for example: laureth-23, ceteareth-20, ceteareth-30 and steareth-40. Preferably, one or more ether-type polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactants are selected from the group consisting of laureth-23, laureth-30, steareth-100, steareth-20, steareth-40, ceteareth-20 and ceteareth-30.

[0085] Suitable one or more ester-type polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactants each independently having a hydrophilic-lipophilic balance (HLB) value equal to or greater than about 14 may include, for example, (1) ester-type polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactants derived from the addition of 20 to 100 moles of ethylene oxide to saturated or unsaturated fatty acids, which may be referred to as polyethylene glycol carboxylates, poly(ethylene oxide) carboxylates, and poly(ethylene oxide) carboxylates, including poly(ethylene oxide) laurate, poly(ethylene oxide) oleate, and poly(ethylene oxide) stearate, such as PEG-20 laurate, PEG-20 oleate; PEG-20 stearate, PEG-32 stearate, PEG-40 stearate, and PEG-100 stearate. Ester-type polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactants are available, for example, as commercial products including S40 and S100; (2) Ester-type polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactants derived from the addition of 20 to 100 moles of ethylene oxide to fatty acid sorbitan esters, which can be referred to as polyoxyethylene sorbitan carboxylates, such as polysorbate 20, polysorbate 40, polysorbate 60 and polysorbate 80, which are available, for example, as commercial products, including 20. 40. 60 and 80; (3) Ester-type polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactants derived from the addition of 20 to 100 moles of ethylene oxide to fatty acid glycerides, which can be referred to as polyethylene glycol glycerol carboxylates, such as PEG-30 cocoglyceryl, poly(oxyethylene)glyceryl monolaurate and poly(oxyethylene)glyceryl monostearate, which are available, for example, as commercial products including Jeechem GL-30 and Jeechem GC-30; and (4) ester-type polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactants derived from the addition of 20 to 100 moles of ethylene oxide to castor oil or hydrogenated castor oil, such as PEG-25 castor oil ethoxylate, PEG-40 castor oil ethoxylate, PEG-60 castor oil ethoxylate, hydrogenated PEG-25 castor oil ethoxylate, hydrogenated PEG-40 castor oil ethoxylate, and hydrogenated PEG-60 castor oil ethoxylate, which are available, for example, as commercial products, including RH40 and RH60. Preferably, the one or more ester-type polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactants are selected from the group consisting of: PEG 100 stearate; PEG 20 stearate; PEG 30 glyceryl cocoate; PEG 32 stearate; polysorbate 20 and polysorbate 80. Thus, in certain aspects, the ester-type polyethoxylated surfactant may comprise at least 40 ethoxylate groups per molecule.

[0086] Suitable one or more non-polyethoxylated high HLB surfactants, each independently having a HLB value of about 14 or greater, may include, for example, (1) fatty ether mono-, di- and oligosaccharides containing ether linkages between fatty alcohols and mono-, di- and oligosaccharides, alkyl glucosides and alkyl polysaccharides such as decyl glucoside, cocoyl glucoside, alkyl glucosides having (C8-C 14 ) poly (D-glucopyranose) ethers of linear primary alcohols and xylitol decanoate / octanoate, which are available, for example, as commercial products, include 2000UP and Giorbis GiO TM -103; (2) polyglycerol fatty acid monoesters, such as triglycerol monolaurate, tetraglycerol monolaurate, triglycerol monooleate, tetraglycerol monooleate, triglycerol monostearate, tetraglycerol monostearate; (3) monoesters and diesters of glycerol with linear or branched long-chain (greater than about 8 carbon atoms) fatty acids, which are further esterified with short-chain monocarboxylic acids, such as glycerol monostearate lactate; (4) saturated or unsaturated, linear or branched aliphatic C8 to C 22 Alkyl sulfonate and sulfate compounds, for example octanesulfonic acid, sulfate esters with lauryl alcohol and their salts such as sodium lauryl sulfate; (5) saturated or unsaturated, linear or branched aliphatic C8 to C 22 Ethoxylated alkyl sulfonates and sulfate compounds, for example, the product of sulfate esters with four moles of ethylene oxide added to lauryl alcohol, and salts thereof such as sodium lauryl ether sulfate; (6) with saturated or unsaturated, linear or branched aliphatic C8 to C 22Sulfosuccinates of alcohols, such as bis(2-ethylhexyl) sulfosuccinate and lauryl poly(ethylene oxide) sulfosuccinate, or mixtures of these surfactants; (7) esters of lactic acid or lactic acid oligomers with fatty acids and their salts, such as sodium stearoyl-2-lactylate; (8) sulfonates of benzene, cumene, toluene and alkyl-substituted aromatic compounds and their salts, such as dodecylbenzenesulfonic acid, or mixtures of these surfactants; (9) alcohol ethoxylates, alcohol propoxylates, alcohol ethoxylates (10) long chain (greater than about 8 carbon atoms) acylamino acids, such as acylglutamates, acylpeptides, acylsarcosinates, acyltaurates, their salts and mixtures of these surfactants; (11) saturated or unsaturated, linear or branched aliphatic C8 to C 22Alkylamidopropyl (dimethylammonium) acetate compounds, such as cocamidopropyl betaine, lauroamidopropyl betaine and stearamidopropyl betaine, and mixtures of these surfactants; (12) sophorolipids, which consist of a hydrophobic fatty acid tail of a hydroxylated 16 or 18 carbon atom fatty acid connected to a hydrophilic sophorose head via a 13-glycosidic bond, including free acid (open) and internally esterified (lactone) forms and acetylated forms (acetylated at the 6'- and / or 6" positions). Sophorolipids useful in the practice of the present invention include, for example, mixtures of products produced by yeasts, such as Candida bombicola, Candida apicoia, Starmerella bombicola and Candida asp. NRRL Y-2720 (identified by Price et al., Carbohydrate Research, 348 (2012) 33-41) and mixtures of chemically modified products; and (13) rhamnolipids, including monorhamnolipids composed of one or two 3-(hydroxyalkanoyloxy)alkanoic acid tails and a single rhamnose head, and dirhamnolipids composed of one or two 3-(hydroxyalkanoyloxy)alkanoic acid tails and two rhamnose heads, including mixtures of compounds produced by Pseudomonas and Burkholderia bacterial species, for example, Pseudomonas aeruginosa and Burkholderia plantarii. Preferably, the one or more non-polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactants can be selected from the group consisting of: coco-glucoside, decyl glucoside, xylitol caprylate / caprylate, and sodium laureth sulfate.

[0087] Typically, nanoparticle dispersions disclosed herein can include any amount of high hydrophilic-lipophilic balance (HLB) surfactants that are suitable for keeping dispersion structure characteristics. In some aspects, the amount of high HLB surfactant can be in the scope of about 0.1 % by weight to about 20 % by weight, about 1 % by weight to about 15 % by weight, about 1.5 % by weight to about 15 % by weight, or about 2 % by weight to about 10 % by weight. In some aspects, the amount of high HLB surfactant can be in the scope of 1.6 % by weight to 11.9 % by weight. Similarly, nanoparticle dispersions can be included in the low HLB surfactants of the amount in the scope of about 0.1 % by weight to about 20 % by weight, about 1 % by weight to about 15 % by weight, about 1.5 % by weight to about 15 % by weight, or about 2 % by weight to about 10 % by weight. In some aspects, the amount of high HLB surfactant can be in the scope of 2.6 % by weight to 12.0 % by weight.

[0088] Suitable one or more low hydrophilic-lipophilic balance (HLB) surfactants each independently having a hydrophilic-lipophilic balance (HLB) value of less than about 10 may include, for example, (1) fatty acid esters comprising sugar residues, including sorbitan monolaurate, sorbitan monopalmitate, sorbitan stearate, sorbitan oleate, sorbitan isostearate, sorbitan sesquioleate, sorbitan trioleate, and sorbitan tristearate, which are available, for example, as commercial products, including 120. 20. 60. 80. 83 and 85; (2) fatty acid glycerides, for example, glyceryl monooleate, glyceryl monostearate, glyceryl dioleate, glyceryl distearate, which are available, for example, as commercial products including Jeechem GMS-O and Jeechem GMIS; (3) fatty alcohol ethoxylates, fatty alcohol propoxylates, and fatty alcohol ethoxylate propoxylates, for example, oleth-2, ceteareth-2, and lauryl alcohol 3 mol ethoxylate / 6 mol propoxylate, which are available, for example, as commercial products including L4, O5, S2 and L 306; and (4) saturated or unsaturated, linear or branched aliphatic C8 to C 22 Carboxylic acid functional compounds, including fatty acids derived from the saponification of plant and animal fats and oils, such as caprylic acid, coconut fatty acid, oleic acid, ricinoleic acid, stearic acid and carboxylic acid terminated short chain (e.g., n=4) polymers of ricinoleic acid and mixtures of these surfactants. Preferably, the one or more low hydrophilic-lipophilic balance (HLB) surfactants are selected from the group consisting of stearic acid, caprylic acid, glyceryl monostearate, sorbitan oleate and sorbitan stearate.

[0089] Suitable one or more phospholipid low HLB surfactants, each independently having a HLB value of less than about 10, can include, for example, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol, as well as compositions comprising mixtures of these substances, such as lecithin. Phospholipid products are available, for example, as commercial products, including 90G, 90H, XTRA-A, PC 75 and 65. Preferably, the one or more phospholipid low hydrophilic-lipophilic balance (HLB) surfactants are selected from the group consisting of phosphatidylcholine and lecithin.

[0090] In certain aspects, it is noted that the nanolipid dispersion itself or any of its components (e.g., a high hydrophilic-lipophilic balance (HLB) surfactant, a low hydrophilic-lipophilic balance (HLB) surfactant, a water-immiscible oil, or a combination thereof present in an amount greater than 0.1%) may be listed as a food additive permitted under Part 172 of Title 21 of the U.S. Food and Drug Administration Code of Federal Regulations for direct addition to food for human consumption.

[0091] The use of relatively long-chain poly(ethylene oxide) surfactants in a phase inversion process caused by temperature changes is described in U.S. Patent No. 6,221,370. Typically, the phase inversion process requires a polyethoxylated surfactant, which has the property of decreasing hydrophilicity as the temperature increases. According to U.S. Patent No. 6,221,370. Examples F1 to F3, compositions comprising an ester-type polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactant (palmitic acid / stearic acid + 30 mol ethylene oxide) were processed using a phase inversion temperature (PIT) emulsification method to obtain a dispersion that was stable after storage at 40°C for 4 weeks. If an ether-type polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactant (cetostearyl alcohol + 30 mol ethylene oxide, Example F4) was used instead of the ester-type polyethoxylated surfactant, the processed dispersion was unstable.

[0092] It was previously discovered that ibuprofen-containing compositions comprising ether-type polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactants can yield concentrated nanoparticle dispersions that are stable at 40°C through a phase inversion process, but compositions containing ibuprofen and ester-type polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactants yield unstable non-nanoparticle dispersions of large particle size.

[0093] These results are in contrast to those disclosed in U.S. Pat. No. 6,221,370. The inclusion of ibuprofen (an organic compound having a carboxylic acid group) in a polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactant appears to eliminate the need for an ester group (a carboxylic acid residue). It was subsequently discovered that the requirement for an ether-type rather than an ester-type polyethoxylated surfactant to obtain a stable dispersion is not limited to compositions containing ibuprofen; it also applies to ibuprofen-free compositions containing other hydrophobic drugs having carboxylic acid groups and carboxylic acid residues, including diclofenac (carboxylic acid), aspirin (carboxylic acid and ester), and lidocaine (ester), as well as ibuprofen-free compositions containing, for example, drugs that do not have carboxylic acid residues (hydrocortisone).

[0094] As disclosed herein, nanoparticle dispersions with advantageous characteristics can also be prepared in the absence of a hydrophobic drug. In a preferred embodiment of the present invention, concentrated nanoparticles are prepared without ibuprofen and S-ibuprofen. Surprisingly, a modified twin-screw extruder can be used to prepare stable, highly concentrated nanoparticle dispersions through a reaction characterized by phase inversion, wherein the composition contains an ether-type polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactant but does not contain an ester-type polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactant, as opposed to the stable PIT emulsions prepared according to U.S. Patent No. 6,221,370. By using an ether-type polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactant and a modified twin-screw extruder, highly concentrated nanoparticle dispersions with a volume average particle size of less than 150 nm can be prepared, which can include peptide bioactive agents, such as those disclosed herein, as well as hydrophobic drugs, oil-soluble vitamins and provitamins, and plant extracts. In certain aspects, highly concentrated dispersions are possible regardless of whether the composition includes, for example, ibuprofen or other compounds having a carboxylic acid group residue.

[0095] Due to their commercial importance, the phase behavior of ether polyethoxylated surfactants has been studied in detail, and a key feature of these surfactants is their self-assembly and structuring behavior in aqueous compositions. The structuring behavior produces many different phases, depending on the concentration, temperature, and the presence or absence of water-immiscible oils. At higher concentrations, ether polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactants form phases including liquid crystals (LCs), hexagonal phases, and cubic phases that exhibit significant gel-like properties and very high viscosities (see, e.g., Kunieda et al., Highly Concentrated Cubic-Phase Emulsions: Basic Study on D-Phase Emulsification using Isotropic Gels. J Oleo Sci 50(8):633-639. January 2001). If carbonyl (C═O) groups are inserted into ether polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactants, the tendency towards highly structured phases and high viscosity can be significantly reduced (see, e.g., Spiering et al., Changes in Phase Behavior from the Substitution of Ethylene Oxide with Carbon Dioxide in the Head Group of Nonionic Surfactants. ChemSusChem. 2019 Nov 25. doi: 10.1002 / cssc.201902855.), however, the presence of carbonyl groups (from carboxylic acid residues) in ester polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactants is apparently the reason why compositions containing ester polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactants fail to produce nanoparticle dispersions when processed using a modified twin-screw extruder (compare the examples disclosed in U.S. Appl. No. 16 / 748,399, e.g., Examples 10 to 13 with Examples 5 to 9 and Example 18 with Example 17).

[0096] Although important for the development of nanostructures and nanoparticles, the phase-rich behavior of ether-type polyethoxylated surfactants can be problematic for processing due to the formation of a highly viscous phase. Since the surfactant-oil-water mixture passes through a complex, highly viscous mesophase, it is necessary to provide a constant positive mechanical flow during the process by kneading or masticating. For this reason, simply cooling the surfactant-oil-water mixture with conventional plate or concentric tube heat exchangers results in a coagulated, large particle size emulsion, rather than a nanoparticle dispersion.

[0097] In certain aspects, the total amount of surfactants (e.g., high HLB and low HLB surfactants) in the dispersions disclosed herein can be in the range of 2 wt % to 30 wt %, 5 wt % to 28 wt %, or 15 wt % to 25 wt %. In other aspects, the total amount of surfactants can be less than about 40 wt %, less than about 35 wt %, less than about 30 wt %, less than about 28 wt %, less than about 25 wt %, less than about 22 wt %, less than about 20 wt %, less than about 15 wt %, or less than about 10 wt %.

[0098] However, it has also been found that certain combinations of high and low HLB surfactants can produce stable nanolipid dispersions with special and advantageous properties as disclosed herein. In certain aspects, the dispersion can comprise an ester-type polyethoxylated high HLB surfactant (e.g., PEG100 stearate, PEG20 stearate, PEG30 cocoglyceryl, PEG32 stearate, polysorbate 20, polysorbate 80) and a phospholipid low HLB surfactant (e.g., phosphatidylcholine and lecithin). In other aspects, the dispersions contemplated herein can comprise an ether polyethoxylated high HLB surfactant (e.g., Laureth-23, Laureth-30, Steareth-100, Steareth-20, Steareth-40, Ceteareth-20, and Ceteareth-30) and an ester polyethoxylated high HLB surfactant (e.g., PEG 100 Stearate, PEG 20 Stearate, PEG 30 Cocoglyceryl, PEG 32 Stearate, Polysorbate 20, Polysorbate 80). In such aspects, the weight ratio of the ether polyethoxylated high HLB surfactant to the ester polyethoxylated high HLB surfactant can be greater than 1:1, greater than 2:1, or greater than 4:1, for example, in the range of 1:1 to 4:1 or 1:1 to 2:1. Alternatively, the dispersion may comprise an ether-type polyethoxylated high HLB surfactant, and may further comprise a non-polyethoxylated high HLB surfactant (eg, sodium lauryl sulfate).

[0099] The dispersions disclosed herein may also contain one or more water-soluble polymers or gums, including: (1) polysaccharides such as dextrins, gums including maltodextrins, cyclodextrins, hyaluronic acid, xanthan gum, guar gum, and water-dispersible or water-soluble starches; (2) water-soluble cellulose derivatives including cellulose ethers such as methylcellulose, carboxymethylcellulose, hydroxypropylmethylcellulose, and carboxyethylcellulose; (3) polyacrylic acid and its salts including carbomer 940 and sodium carbomer such as Neutragel DA (product of 3V Sigma USA, Georgetown SC); (4) acrylate / vinyl cross-linked polymers such as Rapidgel EZ1 (product of 3V Sigma USA); and (5) poly(vinyl pyrrolidone). Preferably, the one or more water-soluble polymers or gums are selected from the group consisting of methylcellulose, sodium carbomer, and acrylate / vinyl cross-linked polymers.

[0100] Nanoparticle dispersion can also comprise for example one or more cryoprotectants, and it prevents the freezing of nanoparticle dispersion, or prevents nanoparticle dispersion from decomposing during freezing.Preferred cryoprotectants are water miscible or water-soluble compounds, comprise glycerol, propylene glycol, ethylene glycol, diethylene glycol ethyl ether, sucrose, sorbitol, trehalose and dimethyl sulfoxide (DMSO).Preferably, one or more cryoprotectants are selected from the group consisting of the following: diethylene glycol, dimethyl sulfoxide, ethylene glycol, glycerol, propylene glycol, sorbitan and trehalose.

[0101] Nanoparticle dispersions can also include, for example, one or more additives, such as antioxidants, chelating agents, acidulants, and biocides. Useful antioxidants include, for example, butylated hydroxytoluene (BHT) and mixed tocopherols. Useful chelating agents include, for example, phosphates, ethylenediaminetetraacetic acid (EDTA) and its salt, sodium phytate, and nitrilotriacetic acid. Useful biocides include, for example, phenoxyethanol and caprylyl glycol, such as Optiphen (product of Ashland Chemicals) and JeechemCAP-4 (product of Jeen). Preferably, one or more additives are selected from the group consisting of: tetrasodium EDTA, citric acid, butylated hydroxytoluene (BHT), and sodium chloride.

[0102] The observation that the addition of a structure-promoting additive allows the use of ester-type polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactants in the practice of the present invention provides support for the conclusion that structure formation during phase transition is responsible for the fine particle size in extruded dispersions. It has been found that the insufficient structure-forming properties of ester-type polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactants can be compensated for by adding a low-structure-forming HLB surfactant as an additive. Particularly useful low-structure-forming HLB surfactants are phospholipids. Examples 19 to 23 of U.S. Patent No. 11,504,327 show that by adding phospholipids to compositions having ester-type polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactants, these surfactants alone would not produce nanoparticle dispersions. It is possible to obtain useful and stable nanoparticle dispersions. Useful phospholipids include, for example, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, and lecithin.

[0103] In the context of the present disclosure, compounds such as ibuprofen can also be considered as structure-promoting additives, and can be exemplified by ibuprofen. Without being bound by theory, checking the structure of ibuprofen shows that, along with the phase of mixture being converted into the nano-lipid dispersion with particle size less than 150nm from crude mixture, ibuprofen may interact with the surfactant in the mixture. Specifically, ibuprofen includes a hydrophobic alkyl group relative to a carboxylic acid group in a phenyl ring, and the carboxylic acid group is moderately hydrophilic when pH 5 or lower, and more hydrophilic when higher pH, wherein the carboxylic acid group is neutralized at physiological pH. These opposite groups can allow ibuprofen to regulate and promote the interaction between surfactant, water and the water-immiscible oil in the mixture. Therefore, in some aspects, the dispersion envisioned herein can be included in the ibuprofen (or acetaminophen, or other structure-promoting additives) of the amount in the range of approximately 0.01 % by weight to 10 % by weight, 0.05 % by weight to 5 % by weight or 0.1 % by weight to 3 % by weight. Of course, since ibuprofen (and other structure-promoting additives) may also be biologically active and may be considered separate biologically active agents as defined herein, for purposes of this disclosure, it will be understood that ibuprofen and / or acetaminophen will be considered structure-promoting additives in a dispersion in which the other biologically active agents are also present. Alternatively, in certain aspects, the dispersion may be substantially free of ibuprofen (or free of lidocaine, or free of acetaminophen), wherein the combination of surfactants remains stable upon formulation and maintains the desired properties as described below.

[0104] Dispersion disclosed herein can advantageously maintain its stability and other desired characteristics, while adapting to the load of the increase of bioactive compounds disclosed herein, particularly relative to the dispersion comprising vesicle nanoparticles. Without being bound by theory, it is believed that by identifying a stable dispersion with an increased amount of hydrophobic lipid component, dispersion is allowed to incorporate the bioactive substance of increased amount in the lipid phase of dispersion. In addition, the lipid phase dispersion allows dispersion to incorporate bioactive agent into the entire volume of the particle lipid phase as the property of non-vesicle lipid particles, rather than being confined to the lipid layer present in the entire vesicle and other lipid membrane structures (e.g., lamellar etc.). Equally, without being bound by theory, it is expected that nano lipid dispersion disclosed herein can be formed completely or mostly by the lipid particles dispersed in the aqueous medium comprising hydrophilic components.

[0105] In view of the above discussion, certain aspects disclosed herein may include an amount of bioactive agent that may significantly exceed an otherwise identical nanoparticle composition comprising otherwise identical components but in another nanoparticle form. In certain aspects, it is contemplated that the dispersion may include up to 2 wt%, 5 wt%, 10 wt%, 20 wt%, 30 wt%, or 40 wt% of the bioactive agent relative to the total weight of the dispersion. Alternatively, the dispersion may include an amount of bioactive agent in the range of about 0.01 wt% to about 40 wt%, about 0.01 wt% to about 20 wt%, or about 0.1 wt% to about 10 wt%, about 1 wt% to about 10 wt%, or about 3 wt% to about 8 wt%.

[0106] In some embodiments, the amount of bioactivator can be expressed relative to the amount of water-immiscible oil in the dispersion. Especially, the relatively high weight ratio of water-immiscible oil and bioactivator can have the bioactive compound encapsulated in the protective nanoparticle and away from the proven effect of the lipophilic surface adjacent to the hydrophilic component, wherein interaction may occur between the hydrophilic component of the bioactivator and the environment. Therefore, the relatively high dispersion of the weight ratio of water-immiscible oil and bioactive component can show the protective effect to bioactive substance, prevents bioactive substance from degrading due to environmental factors and the enzyme (such as may occur when taking in or being applied to skin) that may be present in the hydrophilic part. Also expect that the significantly excessive water-immiscible oil relative to bioactive component can improve the biological delivery of bioactive substance, for example, by increasing the skin penetration of nano lipid particles. As mentioned above, the non-vesicle, the non-lamellar structure of nano lipid dispersion also may contribute to protective effect. In certain aspects, the weight ratio of the water-immiscible oil to the bioactive agent can range from 10,000:1 to 1:1, 1,000:1 to 10:1, 800:1 to 25:1, 500:1 to 50:1, 500:1 to 10:1, 200:1 to 10:1, or 100:1 to 10:1.

[0107] It is known that the ability of nanoparticles to penetrate the skin is related to the particle size, with smaller particles generally having better permeability. When it comes to skin penetration, the appropriate amount of particle size in the particle distribution is the weight average particle size, because it defines the average value based on the mass fraction of nanoparticles with a specific diameter, and it is important to have the mass fraction of nanoparticles with a diameter small enough to effectively penetrate the skin. In contrast, the unspecified majority of particles measured by mass fraction can have a diameter much larger than the number average particle size, making the number average particle size a poor measure of permeability. In certain aspects, the nanoparticle dispersion disclosed herein may have a volume average particle size of less than 150 nm, less than 100 nm, less than 60 nm, or less than 50 nm; alternatively, in the range of about 10 to about 150 nm, about 20 nm to about 120 nm, about 30 nm to about 100 nm, or about 30 nm to about 80 nm.

[0108] Dispersion disclosed herein can also be characterized by their lipid content. Without being bound by theory, it is believed that the lipid content increased can provide extra ability to carry and protect bioactivators as disclosed herein. In some aspects, dispersion disclosed herein can comprise a lipid content greater than 25 % by weight, greater than 30 % by weight, greater than 35 % by weight, greater than 40 % by weight, greater than 45 % by weight, greater than 50 % by weight, greater than 55 % by weight or greater than about 60 % by weight. In other aspects, dispersion can comprise a lipid content in the scope of 25 % by weight to 55 % by weight or 35 % by weight to 50 % by weight. In some aspects, dispersion can comprise a lipid content greater than 25 % by weight and a particle size less than 100 nm. In alternatives, dispersion can comprise a lipid content in the scope of 35 % by weight to 50 % by weight and a particle size less than 60 nm.

[0109] Another important property of highly permeable nanoparticles is the polydispersity of the hydrophilic-lipophilic balance (HLB) surfactant value. The HLB polydispersity promotes the formation of a lamellar structure of the nanoparticles, otherwise the nanoparticles do not have the prepared lamellar structure when they are applied to the skin. The lamellar structure has been shown to be important for promoting the skin penetration of nanoparticles, for example in liposomes and niosomes. The HLB polydispersity can be calculated as the HLB weight mean square deviation, WMSD HLB , which is the sum of the products of the weight fraction of the ith surfactant species multiplied by the square of the deviation of the hydrophilic-lipophilic balance (HLB) of the ith surfactant species from the weight average hydrophilic-lipophilic balance (HLB) divided by the weight average hydrophilic-lipophilic balance (HLB), for dispersions of nanoparticles without a hierarchical structure as prepared, it is desirable to have a WMSD greater than about 1.5 HLBThe hydrophilic-lipophilic balance (HLB) polydispersity supports the formation of lamellar structures after skin application. HLB At WMSD values, nanoparticles with aqueous cores may be formed, including undesirable multilayer structures. In some nanoparticle dispersions, nanoparticles may have oily cores, and WMSD HLB Between about 1.5 and about 4.5, between about 1.75 and about 3.5, and between about 2 and about 3.

[0110] In certain aspects, the DNLF dispersions disclosed herein may have a latent lamellar structure, i.e., a tendency of nanoparticle dispersions that do not have a lamellar structure to form a lamellar structure. In certain aspects, the dispersions disclosed herein may adopt a lamellar structure by evaporation or heating. The phase transition from a non-lamellar dispersion to a lamellar dispersion characterized by latent lamellar characteristics can be observed by the development of optical birefringence or an increase in electrical impedance in the sample under appropriate conditions (e.g., evaporation, heating, or both). Optical birefringence can be observed by observing cross-polarized films. For heated and stirred nanoparticle dispersions, when heated at a rate between 1°C and 4°C per minute and the conductivity is measured using an open-pore geometry electrode (such as the 013005MD 4-cell conductivity electrode available from ThermoScientific), the electrical impedance can be observed as a negative peak in a graph of conductivity versus temperature and a corresponding positive peak in a graph of the first derivative of conductivity versus temperature.

[0111] In some aspects, the transition between the non-lamellar structure and the lamellar structure can be characterized by the temperature at which the phase transition begins (i.e., the phase transition onset temperature) and the temperature at which the phase transition is completed (i.e., the phase transition end temperature). The phase transition onset temperature is the temperature at which the conductivity drops to less than 80% of the maximum conductivity when heated. The phase transition end temperature is determined to be the highest temperature at which the conductivity drops to less than 10% of the maximum conductivity. In some aspects, the potential lamellar structure can be characterized in that the nanoparticle dispersion adopts a lamellar structure when heated to a temperature in the range of 40°C to 95°C, 45°C to 90°C, 50°C to 85°C, 60°C to 80°C, or 65°C to 75°C (e.g., the phase transition onset temperature). In some aspects, evaporation can also be combined with gentle heating or induced without heating at all.

[0112] Alternatively, the disclosed nanoparticle dispersion comprising a latent lamellar structure is characterized in that the dispersion exhibits a positive peak in a plot of the first derivative of normalized conductivity versus temperature, wherein the positive peak has a relative humidity of greater than about 0.05° C. within a temperature range of about 45° C. to 80° C. (or any of the phase inversion onset temperatures described above). -1 , greater than about 0.1℃ -1 or greater than about 0.3°C -1 The peak amplitude of .

[0113] Furthermore, the nanoparticle dispersions disclosed herein and comprising a latent lamellar structure can be characterized in that they exhibit optical birefringence when the dispersion is viewed through a crossed polarizer film when heated to a temperature in the range of 60°C to 95°C (or any of the phase inversion onset temperatures described above).

[0114] In some aspects, the dispersion may become relatively unstable after phase inversion, and phase separation can be observed within the scale of many hours to several days. However, this does not constitute a problem for the use prepared as a ready-to-use therapeutic agent. Non-lamellar dispersions disclosed herein (e.g., nanolipid dispersions with potential lamellar features before phase inversion) show excellent stability under storage conditions. For example, in some aspects, the peptide compound DNLF dispersions maintain high kinetic stability so that they do not phase separate within 28 days when stored at a temperature between 18 ° C and 23 ° C, and do not phase separate within 7 days when stored at 40 ° C. On the other hand, nanolipid dispersions can be stable for 5 days at 40 ° C with respect to phase separation.

[0115] Separately, the nanoparticle dispersions disclosed herein can exhibit stability with respect to retaining the bioactive agent in the nanoparticle dispersion. In certain aspects, the bioactive agent does not form crystals within the nanoparticle dispersion for more than about 1 month, more than about 2 months, more than about 3 months, 8 months, more than about 12 months, more than about 18 months, or more than about 24 months at a temperature of about 18°C ​​to about 22°C; alternatively, no crystals are formed within the range of 1 month to 24 months, or within the range of 3 months to 12 months. The nanoparticle dispersions disclosed herein can remain free of crystals, even at a concentration greater than the solubility of the bioactive agent in the aqueous phase of the dispersion, even outside the environment of the nanoparticles. Surprisingly, the characteristics of supersolubility still apply even if the bioactive agent has a logP of less than 1, and thus demonstrates significant solubility in aqueous solutions.

[0116] Method for preparing nanoparticle dispersion

[0117] DNLF dispersions will not form spontaneously within 2 weeks after low shear mixing of the oil phase with the water phase. Low shear mixing includes mixing with a magnetic stirrer and a magnetic stirrer, as well as stirring with a stirring element such as a blade, paddle, or spiral paint mixer, such as a Red 5 Gallon Drywall Mud / Paint Mixer Model 4041 or equivalent. Surprisingly, it was found that a modified twin-screw extruder in which the majority of the screw elements are conveying elements with a small portion of mixing or kneading screw elements can provide sufficient mechanical kneading and mastication during the phase change to provide concentrated nanoparticles in the extrudate.

[0118] We have further discovered that highly concentrated dispersions having up to 60 wt% dispersed nanoparticles and a volume average particle size as low as about 25 nm can be prepared by a single-step process using a twin-screw extruder modified to provide a temperature gradient of greater than about 35° C. for the surfactant-oil-water composition conveyed therethrough. The processing time for converting the coarse non-nanoparticle dispersion to the final concentrated nanoparticle dispersion can be less than 2 minutes, which is remarkable considering that time and temperature cycling have been shown to be important for preparing nanoparticle dispersions by phase inversion methods (Heurtault et al., Pharm. Res. 19, 875, 2002 and Anton et al., Int J Pharm. 2007 Nov 1;344(1-2):44-52).

[0119] Some aspects may include forming a coarse ground base to be processed in an extruder by preparing an aqueous mixture and a lipid mixture, the aqueous mixture and the lipid mixture each comprising a hydrophilic and hydrophobic component as described above, respectively. The coarse ground base may be formed by adding the aqueous mixture and the lipid mixture to produce a single mixture, which may then be processed in an extruder.

[0120] In certain aspects, the coarse ground base can be processed using a pilot-scale twin-screw extruder with a screw diameter of 24 mm or 27 mm at a rate of up to about 380 grams per minute. Preferably, the coarse ground base can be processed in the extruder using a temperature gradient, which includes, for example, the temperature of the composition when it is a microemulsion. The temperature gradient during processing can be provided by preheating the composition before introduction into the extruder or by heating in one or more first temperature-controlled zones of the extruder, or both, plus cooling the composition in one or more subsequent temperature zones of the extruder. In preferred embodiments, the process ΔT, defined as the difference between the maximum temperature of the composition upon introduction into the extruder and the extruder zone temperature minus the minimum temperature of the cooled extruder zone, is greater than about 35°C, greater than about 50°C, and greater than about 60°C.

[0121] Surprisingly, a modified twin-screw extruder can be used to prepare stable, highly concentrated nanoparticle dispersions by a reaction characterized by phase inversion, wherein the composition contains an ether-type polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactant but does not contain an ester-type polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactant, as opposed to the stable PIT emulsions prepared according to U.S. Patent No. 6,221,370. By using an ether-type polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactant and a modified twin-screw extruder, highly concentrated nanoparticle dispersions having a volume average particle size of less than 150 nm can be prepared, including a variety of active ingredients such as hydrophobic drugs, oil-soluble vitamins and provitamins, and plant extracts, regardless of whether the composition contains structure-promoting additives such as ibuprofen or other compounds having carboxylic acid residues.

[0122] Nanoparticle dispersions containing bioactive agents as oral formulations with improved bioavailability

[0123] Where feasible, drugs and therapeutic agents are often administered in oral dosage forms because such dosage forms are easier for patients to administer themselves (e.g., compared to injectable drugs). However, orally administered drugs encounter several challenges before reaching the desired location in the body. Such challenges may include extreme pH environments, poor absorption, and large amounts of the drug being processed as waste by the body. Therefore, some drugs have poor bioavailability when administered in oral dosage forms.

[0124] U.S. Patent No. 11,504,327, entitled "Method of Preparing Nanoparticles by Hot-Melt Extrusion," which is incorporated herein by reference in its entirety, describes a method for preparing a concentrated lipid nanoparticle dispersion containing between 25% and 60% lipid content. It is disclosed and demonstrated herein that such highly concentrated lipid nanoparticle dispersions may also contain hydrophilic drugs and bioactive agents as described in U.S. patent application 63 / 384,585, filed on November 21, 2022, or peptides as described in U.S. patent application 63 / 384,584, filed on November 21, 2022, each of which is also incorporated herein by reference in its entirety.

[0125] Surprisingly, oral administration of the lipid nanoparticle dispersions disclosed herein can produce excellent Tmax and Cmax characteristics, which are more typical of injection. In certain aspects, peak plasma concentrations occur less than one hour after oral gavage to Jackson Labs C57BL / 6J mice. In one embodiment, the lipid nanoparticle dispersion provides a plot of plasma concentration versus time, wherein d(plasma concentration) / dt is negative at every time greater than one hour after oral gavage to Jackson Labs C57BL / 6J mice.

[0126] In some aspects, oral administration of the nanoparticle dispersion produces peak plasma concentration in the patient less than one hour (or 10 minutes to 1 hour, or less than 30 minutes, or less than 15 minutes, or 5 to 30 minutes) after administering nanoparticles or capsules to the patient. In other aspects, a graph of plasma concentration versus time, wherein at each time point after administering nanoparticles or capsules for more than one hour (e.g., 1 to 24 hours), d (plasma concentration) / dt is a negative value.

[0127] Those skilled in the art will appreciate that the nanoparticle dispersions disclosed herein are applicable to numerous well-known methods of treating and using known bioactive agents. For example, the use of insulin in treating type 1 and type 2 diabetes is well known, despite the fact that no previously known effective oral formulations exist. Based on the ability to achieve suitable Cmax and Tmax values ​​after oral administration, it is expected that the nanoparticle dispersions disclosed herein comprising insulin as a bioactive agent will be applicable to methods of treating type 1 and type 2 diabetes. Similarly, based on the ability to achieve Cmax and Tmax values ​​comparable to existing treatment methods, the treatment methods of other bioactive compounds contemplated herein rely on methods recognized in the art that can be improved by increasing oral bioavailability.

[0128] It has also been observed that nanoparticle dispersions can be absorbed intact from the GI tract, and thus can provide a protective effect on the bioactive agent by oral administration of a dispersion containing the bioactive agent. In this way, it is expected that the dispersions disclosed herein can exhibit a rate of drug absorption that is greater than (e.g., more than 1.5 times greater, more than 2 times greater; alternatively, in the range of 1 to 5 times greater) the rate at which the drug is released from the lipid phase of the nanoparticle dispersion in the GI tract. Ultimately, the amount of drug released from the nanoparticle dispersion by digestion is less than the amount of drug absorbed from the GI tract. Therefore, most of the bioactive agent can be absorbed into the body under milder physiological conditions.

[0129] The formulation comprising the nanoparticle dispersion disclosed above may further comprise an inactive ingredient that has been used in a drug product approved by the U.S. Food and Drug Administration (FDA) for a specific route of administration. When an inactive ingredient has been approved for a route of administration, the inactive ingredient is not considered new and may require less extensive review when seeking approval for a DNLF dispersed drug product.

[0130] In one aspect, all inert ingredients in the topical DNLF dispersion drug product appear in the FDA Inert Ingredient Guide (IIG) for the drug or drugs approved for topical administration.

[0131] In one aspect, all inert ingredients in the topical DNLF dispersion drug product appear in the FDA Inert Ingredient Guide (IIG) for one or more drugs approved for oral administration.

[0132] In certain aspects, DNLF dispersions for oral administration include an ether polyethoxylated high HLB surfactant. Ether polyethoxylated high HLB surfactants lack ester bonds and are therefore less susceptible to hydrolytic cleavage of the bond between the hydrophobic and hydrophilic surfactant groups, which can lead to loss of surfactant performance, increased particle size, and phase separation. Several ether polyethoxylated high HLB surfactants have been approved for use in orally administered pharmaceuticals, including ceteareth-20 (CAS No. 68439-49-6), steareth-40 (CAS No. 9005-00-9), poloxamer 124, poloxamer 188, poloxamer 331, and poloxamer 407. Ether polyethoxylated high HLB surfactants with polyethoxylated chain lengths greater than about 20 ethoxylate units and molecular weights less than 2500 daltons, such as ceteareth-20 and steareth-40, are particularly suitable for extrusion of DNLF dispersions.

[0133] In one aspect, the DNLF dispersion comprises an ether polyethoxylated high HLB surfactant that appears in the FDA Inert Ingredient Guide (IIG) for one or more drugs approved for oral administration.

[0134] In one aspect, the DNLF dispersion comprises an ether polyethoxylated high HLB surfactant that appears in the FDA Inert Ingredient Guide (IIG) for use with one or more drugs approved for oral administration, wherein the ether polyethoxylated high HLB surfactant has a polyethoxylated chain length of greater than about 20 ethoxylate units and a molecular weight of less than 2500 Daltons.

[0135] In one aspect, the DNLF dispersion for oral administration comprises an ether-type polyethoxylated high HLB surfactant selected from ceteareth-20 and steareth-40.

[0136] Anti-lipolytic nanolipid dispersion

[0137] In addition to those discussed above, further improvements in the bioavailability of bioactive agents are contemplated herein.

[0138] Biopharmaceutical Class II and Class IV drugs have poor solubility in the aqueous environment of the GI tract and are mostly eliminated by excretion rather than absorption. A common strategy to improve the absorption and bioavailability of Class II and Class IV drugs is to increase effective solubility by creating supersaturated solutions. For example, the solubility of a Class II or Class IV drug can be increased by providing the compound in very fine particle form or in the form of an amorphous solid dispersion (ASD), in which the drug is molecularly dissolved in a water-soluble polymer.

[0139] Another method of producing drug supersaturated solution in GI tract is to digest lipid in drug delivery (LBDD) system based on lipid.Medicine is formulated as preparation based on lipid, such as oil solution or suspension, emulsion, self-emulsifying drug delivery system (SEDDS), self-microemulsifying drug delivery system (SMEDDS) or self-nanoemulsifying drug delivery system (SNEDDS), solubility and the dissolution rate of lipophilic drugs can be increased, and the formation of dissolved substances is promoted, thereby absorption occurs.Almost no evidence shows that the construct (for example, micelle or emulsion droplet) of dissolving has the potentiality of direct absorption, and general viewpoint is, moves to the free drug as intermicellar solution from the lipid reservoir of dissolving by medicine, absorbs free drug subsequently from intermicellar phase, thereby promotes drug absorption.The digestion of lipid forces medicine to leave oil phase and enter in intermicellar solution, and is considered to be key for absorbing medicine from lipid formulations.

[0140] The lack of evidence for direct absorption of dispersed lipid particles and nanoparticles may be due to the inability to prepare particles small enough to be readily absorbed directly while also being sufficiently resistant to digestion to persist in the intestinal lumen long enough to be absorbed. Typically, particles small enough to undergo rapid absorption (e.g., by caveolin-mediated endocytosis) are digested so rapidly that the absorption results are essentially the same as other methods of producing supersaturated aqueous drug solutions.

[0141] For drugs administered in an LBDD system, the time required to reach maximum plasma drug concentration (Cmax), Tmax, is typically one hour or longer. It's reasonable to speculate that digestion of the LBDD lipids and subsequent release of the drug from the micelles by bile salt solubilization requires an hour or more. Plasma concentration profiles of drugs administered orally as LBDD formulations typically show increasing drug concentrations during the first 1 to 10 hours, followed by a decrease in concentration due to drug metabolism and elimination. In contrast, intravenous bolus administration of a drug produces a curve of continuously decreasing plasma concentrations.

[0142] The present invention provides drugs encapsulated in lipid nanoparticles that are directly absorbed from the intestinal lumen and exhibit a drug plasma Tmax value of 30 minutes or less when the lipid nanoemulsion is orally administered. The present invention provides lipid nanoemulsions that exhibit monotonically decreasing drug plasma concentrations after oral administration, which begin to monotonically decrease 30 minutes or less after oral administration.

[0143] The lipid nanoemulsions of the present invention may comprise lipids that lack ester bonds and are therefore not subject to enzymatic decomposition by lipases, or they may comprise compounds that are lipase inhibitors, or they may comprise surfactants that prevent lipases from crossing the oil-water interface. The lipid nanoemulsions of the present invention have a sufficiently small volume average particle size to allow direct absorption by endocytosis, particularly caveolin- and clathrin-mediated endocytosis. In caveolin- and clathrin-mediated endocytosis, pits or invaginations of approximately 60 nm to 100 nm in diameter are formed in the cell wall, which produce endocytic vesicles that become increasingly efficient vehicles for particle absorption as the diameter of the absorbed particles decreases to below approximately 80 nm.

[0144] The present invention provides a lipid nanoparticle dispersion comprising a hydrophobic therapeutic agent encapsulated in nanoparticles composed of a lipophilic material, wherein the nanoparticles have a volume average particle size of less than 100 nm, wherein the lipophilic material is resistant to lipolysis, meaning that less than 25% of the ester bonds in the lipophilic material are hydrolyzed within one hour in the presence of porcine pancreatic lipase at a concentration of 50 units / ml, wherein "lipophilic material" includes all surfactants, water-immiscible oils, hydrophobic drugs and hydrophobic therapeutic agents.

[0145] In one embodiment, the lipid nanoparticle dispersion comprises a high HLB polyethoxylated surfactant that is inert to lipolysis. In one embodiment, the lipid nanoparticle dispersion comprises a high HLB polyethoxylated surfactant that lacks an ester group. In one embodiment, the lipid nanoparticle dispersion comprises an ether-type high HLB polyethoxylated surfactant.

[0146] In one embodiment, the lipid nanoparticle dispersion comprises a lipase inhibitor. In one embodiment, the lipase inhibitor can be selected from the group consisting of anandamide, diacylglycerol lipase, orlistat, 2-arachidonoylglycerol, cannabinoids, endocannabinoids, fatty acid amidase, acylglycerol lipase, and combinations thereof. In some aspects, the lipase inhibitor is orlistat. In one embodiment, the lipid nanoparticle dispersion comprises a lipophilic material content greater than 25% by weight.

[0147] The nanoparticle dispersions disclosed herein may also comprise positively charged lipid nanoparticles. In one embodiment, the lipid nanoparticle dispersion comprises lipid nanoparticles having a zeta potential greater than 1.0 millivolts; alternatively, within the range of 0.5 to 5 mV or 1 mV to 3 mV. Thus, in certain aspects, the nanoparticle dispersions disclosed herein may comprise zwitterionic surfactants. Zwitterionic surfactants comprise separated positively and negatively charged ions that are conformationally disallowed from associating in a manner that becomes hydrophobic. The presence of charged ions on the surface of lipid nanoparticles can effectively reduce bending forces in cell membranes and allow for easier wrinkling to promote endocytosis; alternatively, zwitterionic groups on the surface of lipid nanoparticles can mimic the improved attachment, cell membrane penetration, and absorption observed for capsid viruses.

[0148] Thus, in certain aspects, the nanoparticle dispersion comprises an amphiphilic compound having a betaine group. In one aspect, the nanoparticle dispersion comprises cocamidopropyl betaine. In one aspect, the nanoparticle dispersion comprises an amphiphilic compound having a phosphorylcholine group. In one aspect, the DNLF dispersion comprises a phosphatidylcholine compound.

[0149] Lipolysis resistance can be characterized by the change of particle size because ester bond is hydrolyzed in dispersion.In some aspects, nanoparticle dispersion is in the lipolysis solution containing calcium, one or more bile salts and 0.4% lipase containing 2.6% DNLF dispersion, pH is about 6.8 after 60 minutes, and the particle of more than 80% (or more than 60% or more than 70% or more than 90%) by volume in dispersion has the diameter less than 100nm.In further aspect, less than 10% (for example, less than 5%, less than 2%; Alternatively, in the range of 1 to 10%) lipid ester bond is in the lipolysis solution containing calcium, one or more bile salts and 0.4% lipase at pH is about 6.8 after 60 minutes by lipolysis.

[0150] However, even in the case of lipolysis, it is observed that the nanoparticle dispersions disclosed herein can maintain a reduced particle size. For example, it can be seen that after 30% or more of the lipid nanoparticle ester bonds are hydrolyzed by lipase, more than 80% of the particles by volume can have a diameter of less than 100 nm.

[0151] Encapsulated nanolipid dispersions

[0152] The high water activity of nanolipid dispersions can cause degradation of gelatin capsules. Overnight at temperatures between 4°C and 50°C, gelatin capsules filled with DNLF become too soft and difficult to swallow. One solution is to fill the gelatin capsules with DNLF shortly before swallowing, but this approach is inconvenient and carries the risk of incorrectly measuring the required dose.

[0153] Provides a solution to systemic malabsorption by delivering drugs and therapeutics in dense nanolipid fluid (DNLF) dispersions, which are highly concentrated lipid nanoemulsions in a continuous aqueous phase.

[0154] It has been found that DNLF dispersions can be encapsulated in so-called enteric polymers with carboxylic acid functionality, even if such polymers can plasticize by absorbing some of the water in the DNLF dispersion, if the pH of the DNLF dispersion is below the pKa value of the polymer's carboxylic acid groups. Furthermore, DNLF dispersions are stable to encapsulation in carboxylic acid-functional enteric polymers, even if some of the water in the DNLF dispersion is lost due to absorption in the capsule wall.

[0155] The present invention generally provides capsules comprising a DNLF dispersion. In certain aspects, the DNLF dispersion can comprise a hydrophobic active compound, a hydrophilic active compound, and / or a peptide encapsulated in a capsule comprising a carboxylic acid-functional polymer. In certain aspects, the capsule can comprise a carboxylic acid-functional polymer and an encapsulated DNLF dispersion.

[0156] In another aspect, the DNLF dispersion can be encapsulated in a carboxylic acid-functional polymer. In one embodiment, the pH of the DNLF dispersion is less than the pKa value of the carboxylic acid groups of the encapsulating polymer. In one embodiment, the pH of the DNLF dispersion is more than one pH unit lower than the pKa value of the carboxylic acid groups of the encapsulating polymer. In one embodiment, the encapsulating polymer is a product of vinyl polymerization.

[0157] In one embodiment, the encapsulating polymer comprises acrylate monomer units. In one embodiment, the encapsulating polymer comprises methacrylate monomer units. In one embodiment, the encapsulating polymer comprises vinyl acetate monomer units. In one embodiment, the encapsulating polymer comprises acrylic acid monomer units. In one embodiment, the encapsulating polymer comprises methacrylic acid monomer units. In one embodiment, the encapsulating polymer comprises 4-hydroxyvinyl phthalate monomer units. In one embodiment, the encapsulating polymer comprises modified cellulose. In one embodiment, the encapsulating polymer comprises cellulose esterified with acetic acid and phthalate. In one embodiment, the encapsulating polymer comprises cellulose esterified with acetic acid and succinate. In one embodiment, the encapsulating polymer is a methyl acrylate-methacrylic acid copolymer. In one embodiment, the encapsulating polymer is a methyl methacrylate-methacrylic acid copolymer. In one embodiment, the encapsulating polymer is an ethyl acrylate-methacrylic acid copolymer. In one embodiment, the encapsulating polymer is a vinyl acetate-4-hydroxyvinyl phthalate copolymer.

[0158] In one embodiment, the encapsulating polymer is hydroxypropyl methylcellulose acetate succinate. In one embodiment, the encapsulating polymer is cellulose acetate phthalate. In one embodiment, the encapsulating polymer is Eudragit L30D-55.

[0159] In one embodiment, the capsule comprises a surface layer comprising carnauba wax, dimethicone, and an organomodified silicone. In one embodiment, the capsule comprises a surface layer comprising carnauba wax.

[0160] In one embodiment, the capsule comprises an innermost layer composed of a carboxylic acid functional polymer, a middle layer of gelatin, and a surface layer comprising carnauba wax.

[0161] Examples

[0162] Unless otherwise indicated, all numbers used in the specification and claims expressing the amounts of ingredients, properties (such as molecular weight, reaction conditions), etc. should be understood as being modified in all cases by the term "about". Therefore, unless otherwise indicated, the numerical parameters listed in the following specification and the appended claims are approximate values ​​that may vary depending on the desired properties sought to be obtained by the present invention. At the very least, and not attempting to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be interpreted in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0163] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0164] Table 1. Abbreviations, descriptions, and sources of materials

[0165]

[0166]

[0167] The particle size distribution of the extruded DNLF dispersions prepared in the following examples was measured within two days of preparation using a NanoFlex dynamic light scattering (DLS) instrument (Microtrac Instruments, York PA) on samples diluted approximately 50 times with deionized water. Particle polydispersity was estimated as the volume average particle size (D v ) and the number average particle size (D n ) ratio.

[0168] General Procedures

[0169] The following examples were performed under the following general procedures, with modifications as described throughout the examples.

[0170] Preparation of coarse emulsion millbase.

[0171] The lipid components comprising surfactant and water-immiscible oil are combined and heated until the solid components melt or dissolve to obtain a liquid having a clarity range of clarity to a medium turbidity. By dissolving the peptide in 0.1N HCl, then adding an aqueous solution of sodium lauryl sulfate, a suspension of hydrophobic precipitate is obtained, which is then mixed with the mill base of the remaining components previously formed to convert the water-soluble peptide into lipophilic, water-insoluble hydrophobic ion pairs. The water-soluble component is dissolved in the water in a separate container. Aqueous solution is added to the warm mixture of the lipid components and stirred to obtain the mill base in the form of an opaque emulsion.

[0172] Preparation of dense nanolipid fluid (DNLF) dispersions

[0173] The DNLF dispersions were prepared in a 27 mm twin-screw extruder or an 11 mm twin-screw extruder.

[0174] In an example using a 27 mm twin-screw extruder, a 50 g sample of the crude emulsion base was heated in a 2 quart pyrex jar using a microwave to between 90° C. and 98° C. The heated emulsion base was then poured by hand into the inlet of a Leistritz ZSE 27 / GL-36D extruder modified as described in Example 57 of U.S. Patent Application Serial No. 16 / 748,399, which is incorporated herein by reference in its entirety. The temperature of the extruded DNLF dispersion was maintained below 10° C. throughout the process.

[0175] In the example using an 11 mm twin-screw extruder, a 50 g sample of the crude emulsion mill base was first preheated in bulk to approximately 60° C. in a 250 mL pyrex beaker using a microwave oven and then preheated to between 80° C. and 90° C. using a handheld torch. The heated sample was then introduced into the inlet of a modified Thermo Fisher Process 11 extruder. Typically, the extruder was loaded with zone temperatures of Zone 2 = approximately 50° C., Zone 3 = 35° C., Zone 4 = 9° C., Zone 5 = 9° C., Zone 6 = 7° C., Zone 7 = 9° C., Zone 8 = 8° C., and screw speed = 200 rpm.

[0176] Alternatively, the crude emulsion mill base was heated using a heat exchanger consisting of a 50.0 cm long, 3 mm ID x 4 mm OD copper tube coiled to an inner diameter of 13 mm. The tube was located in a 25 mm radius x 75 mm long cylindrical cavity approximately 8 mm from the end of a 225 mm long, 38 mm square aluminum beam. The aluminum beam was heated by placing the end opposite the cylindrical cavity on a hot plate stirrer. The extruder was modified to provide barrel cooling by removing the port plug from the top of the barrel and adding a copper heat exchanger consisting of a 2 inch wide x 0.75 inch thick x 12 inch long copper rod bolted to the top (exit) of the last 12 inches of the barrel. The copper block was cooled by circulating water / propylene glycol transversely through nine horizontal channels in a three-channel region. The cold coolant passed through the first three channels, returned through the next three channels, and again through the side opposite the side it first entered through the last three channels before returning to the cooler. The coolant temperature was maintained at 5°C.

[0177] The underlying lamellar character was determined by observing birefringence.

[0178] The potential lamellar structure of the DNLF dispersion sample was tested by heating while measuring conductivity and observing birefringence. Approximately 10 g of sample DNLF dispersion was added to a 30 mL beaker with a magnetic stirrer and heated at a rate of 2°C to 3°C / minute while stirring with a hot plate stirrer. The sample was observed through two orthogonally oriented polarizing films. When observed through the polarizing film, the potential lamellar structure was obvious when birefringence (i.e., a pattern of alternating darker and lighter areas or colored areas in the stirred sample) was observed in the stirred sample, while when observed without the polarizing film, the sample was transparent, isotropic, and featureless at the same time.

[0179] The potential lamellar structure of lipid nanoparticle dispersions was examined by heating on a hot plate / magnetic stirrer at a rate of approximately 10°C / minute while simultaneously recording temperature and conductivity. In instances where samples were too viscous to stir, they were carefully heated using a microwave oven in defrost mode until the viscosity was low enough to allow stirring. Samples that formed transparent microemulsions upon heating were examined for birefringence.

[0180] For heated and stirred nanoparticle dispersions, changes in electrical impedance can also be observed as negative peaks in a graph of conductivity versus temperature and corresponding positive peaks in a graph of the first derivative of conductivity versus temperature when heated at a rate between 1°C and 4°C per minute and the conductivity is measured with an open geometry electrode (such as the 013005MD 4-cell conductivity electrode available from ThermoScientific).

[0181] Oral bioavailability testing.

[0182] In male Jackson Labs C57BL 6J mice, 10 mg / kg dasatinib was administered by oral gavage, and high performance liquid chromatography-mass spectrometry (HPLC-MS) was used to measure the change in plasma concentration over time after administration to test the oral bioavailability of lipid nanoparticle dispersions. Before administration, the dasatinib concentration was brought to 1.25 mg / L by dilution with deionized water. For 20 g mice, the administration volume was 160 μL. Plasma samples were collected from three mice and combined for HPLC-MS analysis at each time point.

[0183] Examples 1 to 12. Preparation of extruded DNLF dispersions containing hydrophilic and / or amphiphilic bioactive agents for topical administration.

[0184] The disclosure of previous embodiment has shown that hydrophobic compound is mixed in the lipid phase of nano lipid dispersion with the form of stable and applicable topical application.Surprisingly, at least as shown in following example 1 to 12, stable DNLF dispersion-s can also form with the hydrophilicity of significant concentration or amphipathic reagent.Even more surprisingly, the heating and extrusion process that is used to prepare nano lipid dispersion is enough gentle, to comprise peptide and other small molecules that are sensitive to harsh conditions (comprising high shear, temperature and pH).As shown in example 4, the dispersion-s that comprises insulin by twin screw extruder processing, to produce the initial insulin concentration loss is only 30% DNLF dispersion-s.As shown in the figure, DNLF dispersion-s can be applicable to the preparation that comprises the hydrophilicity and / or amphipathic bioactivator that are suitable for topical application.

[0185] Example 1. Insulin DNLF for topical administration.

[0186] A coarse emulsion mill base was prepared by heating a mixture of the oil phase components consisting of 7.2% laureth-23, 5.5% PEG32 stearate, 3.6% sorbitan oleate, 1.5% dioleoylphosphatidylcholine (Phospholipon 90G, a product of Lipoid USA), 2.0% lauric acid, 25.0% isopropyl myristate, 6.0% limonene, 3.0% medium chain triglyceride oil, and 6.5% ibuprofen, and adding 0.2% sodium citrate dihydrate and 39.6% deionized water. In a separate beaker, 106.0 mg of recombinant human insulin (available from Sigma Aldrich, catalog number 91077C) was dissolved in 20.2 g of 0.1 N HCl. To the resulting clear insulin solution, 0.69 g of 10% sodium lauryl sulfate was added with stirring to give a white suspension. To this white suspension was added 182.0 g of the crude emulsion to give a crude extrudable emulsion premix. The crude extrudable emulsion premix contained 0.0475% insulin, 5.8% laureth-23, 4.5% PEG 32 stearate, 2.9% sorbitan oleate, 1.2% dioleoylphosphatidylcholine, 1.7% lauric acid, 20.4% isopropyl myristate, 4.9% limonene, 2.4% medium chain triglyceride oil, 5.3% ibuprofen, 0.033% HCl, 0.2% sodium citrate dihydrate, and 50.7% deionized water. The ratio of water-immiscible oil to insulin was 583 to 1.

[0187] Prior to extrusion, the crude emulsion (150 g) was placed in a 250 mL pyrex beaker and heated in a microwave oven for 60 seconds to a temperature of approximately 65° C. It was then heated with a handheld torch for approximately 2 minutes while stirring with a stick thermometer until it reached a temperature of 85° C. The hot crude emulsion was poured into the inlet of a modified Process 11 extruder with zone temperatures of approximately 50° C. for zone 2, 35° C. for zone 4, 9° C. for zone 5, 9° C. for zone 6, 7° C. for zone 7, 9° C. for zone 8, and 8° C. at a screw speed of 200 rpm. The processing rate was approximately 40 g / min, and the entire batch was extruded in less than 4 minutes, yielding an insulin DNLF dispersion as a translucent beige liquid with a viscosity of approximately 10,000 cSt (determined relative to the consistency of honey).

[0188] The volume average particle size, number average particle size and polydispersity index were 53.1 nm, 42.4 nm and 0.1151, respectively, and the lipid concentration was 49.2%. The pH was 4.6.

[0189] The sample (23.4 g) was placed in a 30 mL beaker and heated on a hot plate while monitoring the conductivity and temperature. When heated to 71°C, the sample became transparent and birefringent, indicating that the DNLF dispersion had a latent lamellar structure. The d (normalized conductivity) / dT plot showed a 0.05°C -1 The phase inversion start and end temperatures were 56°C and 69°C, respectively.

[0190] DNLF dispersion sample (0.41g) is diluted with 3.63g 4% bovine serum albumin PBS buffer, and the sample of dilution is diluted twice with 1 to 100 using 4% bovine serum albumin PBS buffer, and total dilution is 1 to 100,000.Insulin concentration based on formulation data is 5.3 parts per billion by weight.Use the human insulin ELISA kit from Crystal Chem (catalog number (Cat. No.) 90095), use the standard curve measurement insulin concentration prepared by 1.0, 2.0, 3.0, 4.0 and 5.0ppb standard substances, and this standard substance is prepared by identical Sigma Aldrich (catalog number (Cat. No.) 91077C) human recombinant insulin.According to this measurement, the concentration of insulin is 1.2ppb, is equivalent to 23% insulin recovery.

[0191] In a separate measurement, a sample of DNLF dispersion (1.00 g) was diluted with 10.0 g of 10% Triton X-100 (4-tert-octylphenol ether with 9 moles of poly(ethylene glycol), a product of Union Carbide) to obtain a clear solution. The clarity of this solution, compared to the turbid appearance of DNLF dispersions diluted with deionized water or phosphate-buffered saline, indicated that the Triton X-100 was disrupting the lipid nanoparticles and promoting the release of insulin. The DNLF plus 10% Triton X-100 solution was diluted twice in a 1 to 100 dilution using 4% bovine serum albumin PBS buffer, for a total dilution of 1 to 110,000. The insulin concentration based on the formulation data was 4.7 ppb. When measured using the human insulin ELISA assay described above, the insulin concentration was 1.1 ppb, equivalent to an insulin recovery of 23%. This experiment showed that the amount of insulin in the DNLF dispersion that survived the extrusion process was 23% and that insulin was efficiently released from the DNLF lipid nanoparticles simply by dilution with bovine serum albumin phosphate-buffered saline at physiological pH 7.4.

[0192] Example 2. Bovine serum albumin DNLF for topical administration.

[0193] A crude emulsion was prepared by heating a mixture of the oil phase components consisting of 7.2% laureth-23, 5.5% PEG 32 stearate, 3.6% sorbitan oleate, 1.5% dioleoylphosphatidylcholine (Phospholipon 90G, a product of Lipoid USA), 2.0% lauric acid, 25.0% isopropyl myristate, 6.0% limonene, 3.0% medium chain triglyceride oil, and 6.5% ibuprofen, and adding 0.2% sodium citrate dihydrate and 39.6% deionized water. In a separate beaker, 0.50 g of bovine serum albumin (available from Sigma Aldrich, catalog number A9647) was dissolved in 20.0 g of 0.1 N HCl. To the resulting clear bovine serum albumin solution, 0.68 g of 10% sodium lauryl sulfate was added with stirring to give a white suspension. To this white suspension was added 183.4 g of the crude emulsion to give a crude extrudable emulsion premix. The crude extrudable emulsion premix contained 0.0223% bovine serum albumin, 5.8% laureth-23, 4.5% PEG 32 stearate, 2.9% sorbitan oleate, 1.2% dioleoylphosphatidylcholine, 1.7% lauric acid, 20.4% isopropyl myristate, 4.9% limonene, 2.4% medium chain triglyceride oil, 5.3% ibuprofen, 0.033% HCl, 0.2% sodium citrate dihydrate, and 50.4% deionized water. The ratio of water-immiscible oil to bovine serum albumin was 124 to 1. The crude emulsion (150 g) was placed in a 250 mL pyrex beaker and heated in a microwave oven for 60 seconds to a temperature of approximately 65°C. The mixture was then heated with a handheld torch for approximately 2 minutes while stirring with a stick thermometer until it reached a temperature of 85°C. The hot coarse emulsion was poured into the inlet of a modified Process 11 extruder with zone temperatures of approximately 50°C for Zone 2, 35°C for Zone 4, 9°C for Zone 5, 9°C for Zone 6, 7°C for Zone 7, and 8°C for Zone 8, and a screw speed of 200 rpm. The processing rate was approximately 40 g / min, and the entire batch was processed in less than 4 minutes, yielding an insulin DNLF dispersion as a translucent, beige liquid with a viscosity similar to honey.

[0194] The volume average particle size, number average particle size and polydispersity index were 58.7 nm, 44.3 nm and 0.0623, respectively, and the lipid concentration was 49.2%. The pH was 4.2.

[0195] The sample (22.6 g) was placed in a 30 mL beaker and heated on a hot plate while measuring conductivity and temperature. When heated to 72°C, the sample was transparent and birefringent, and a plot of d (normalized conductivity) / dT showed a 0.17°C drop at 70.5°C. -1The positive peak of magnitude 1.5 indicates that the DNLF dispersion has a potential lamellar structure. The phase inversion start and end temperatures are 64°C and 73°C, respectively.

[0196] A sample of DNLF dispersion (1.00 g) was diluted with 10.0 g of 10% Triton X-100 (4-tert-octylphenol ether with 9 moles of poly(ethylene glycol), a product of Union Carbide) to obtain a translucent dispersion. The diluted dispersion was diluted twice with 4% bovine serum albumin in PBS buffer at a ratio of 1 to 100, for a total dilution of 1 to 110,000. Insulin concentration was measured using a human insulin ELISA kit from CrystalChem (Cat. No. 90095) using a standard curve prepared from 1.0, 2.0, 3.0, 4.0, and 5.0 ppb standards, which were prepared from the same Sigma Aldrich (Cat. No. 91077C) human recombinant insulin. Based on this measurement, the concentration of insulin in the DNLF dispersion was 0.27 ppb (baseline value - empty lipid nanoparticles encapsulating bovine serum albumin).

[0197] Example 3. Cyclosporin A DNLF for topical administration.

[0198] A crude emulsion was prepared by heating a mixture of oil phase components consisting of 0.8% cyclosporin A, 6.0% laureth-23, 4.7% PEG 40 stearate, 3.1% sorbitan oleate, 1.5% soy lecithin, 11.5% isopropyl myristate, 8.7% acetates of mono- and diglycerides, 5.2% limonene, and 5.0% ibuprofen, and adding 0.2% sodium chloride and 53.4% ​​deionized water. The ratio of water-immiscible oil to cyclosporin A was 33 to 1. The millbase was pumped at a rate of 12.5 mL / min through a heat exchanger maintained at a temperature between 86°C and 91°C into the inlet of a modified Thermo Fisher Process 11 extruder with a screw rotating at 300 rpm, resulting in a translucent, milky white liquid with a viscosity similar to that of vegetable oil. The volume average particle size, number average particle size and polydispersity index were 94.4 nm, 43.1 nm and 0.396, respectively.

[0199] The sample (23.2 g) was placed in a 30 mL beaker and heated on a hot plate while measuring conductivity and temperature. A plot of d(normalized conductivity) / dT showed a 0.12°C drop at 84.8°C. -1 The positive peak of magnitude 1.5 indicates that the DNLF dispersion has a potential lamellar structure. The phase inversion start and end temperatures are 73℃ and 78℃, respectively.

[0200] Example 4. Nonapeptide-1DNLF dispersion for topical application, comprising nonapeptide-1, laureth-23, PEG40 stearate, sodium lauryl sulfate, sorbitan oleate, soy lecithin, lauric acid, isopropyl myristate, cold-pressed orange oil, medium chain triglyceride oil, ibuprofen and water.

[0201] A crude emulsion was prepared by heating a mixture of oil phase components consisting of 6.2% laureth-23, 4.8% PEG40 stearate, 3.1% sorbitan oleate, 5.5% soy lecithin (Alcolec XTRA-A, a product of American Lecithin), 1.8% lauric acid, 14.1% isopropyl myristate, 5.2% cold-pressed orange oil, 10.1% medium chain triglyceride oil, 5.7% ibuprofen, 0.8% Jeecide CAP-4, 0.2% sodium citrate dihydrate, and 34.5% deionized water. In a separate beaker, 0.072% nonapeptide-1 (available from Active Peptide Company) was dissolved in 10.8% 0.1N HCl. To the resulting clear nonapeptide-1 solution was added a 30% aqueous solution of 0.04% sodium lauryl sulfate under stirring to give a white suspension, and an oil phase was added to give a coarse emulsion mill base. The ratio of water-immiscible oil to nonapeptide-1 was 4:12 to 1. The coarse extrudable emulsion mill base was placed in a 60 mL polyethylene syringe and heated in a microwave oven for 7 seconds to raise the temperature to 31°C and pumped at a rate of 12.5 mL / min through a heat exchanger maintained at a temperature between 86°C and 94°C into the inlet of a modified Thermo Fisher Process 11 extruder with a screw rotating at 300 rpm to give a translucent milky white liquid with a viscosity similar to honey. The volume average particle size, number average particle size, and polydispersity index were 67.6 nm, 29.5 nm, and 0.0979, respectively.

[0202] Example 5. Nonapeptide-1 D NLF dispersion for topical application, comprising nonapeptide-1, ceteareth-20, sorbitan oleate, soy lecithin, isopropyl myristate, niacinamide, d-panthenol, kojic acid and water.

[0203] A coarse emulsion was prepared by heating a mixture of oil phase components consisting of 8.6% ceteareth-20, 8.6% sorbitan oleate, 0.5% soy lecithin, and 25.9% isopropyl myristate. In a separate beaker, 0.06% nonapeptide-1 was dissolved in 3.5% 0.1N HCl and 0.30% sodium lauryl sulfate was added to produce a white precipitate. The oil phase and the nonapeptide-1 hydrophobic ion pair suspension were mixed and a coarse emulsion millbase was prepared by adding 0.52% Jeecide CAP-4, 51.1% water, 0.19% niacinamide, 0.6% panthenol, 0.5% kojic acid, and 0.15% sodium chloride. The ratio of water-immiscible oil to nonapeptide-1 was 452 to 1. The coarse extrudable emulsion mill base (1000 g) was placed in a 2 L Pyrex measuring cup and heated to 95° C. in a microwave and poured into the inlet of a modified Leistritz ZSE 27 / GL-36D extruder with a screw rotating at 200 rpm to obtain a translucent milky white liquid with a viscosity similar to honey. The volume average particle size, number average particle size, and polydispersity index were 46.7 nm, 38.0 nm, and 0.1186, respectively.

[0204] The sample (60.4 g) was placed in a 100 mL beaker and gently heated to 40°C in a microwave oven using the thaw setting to reduce the viscosity, and then heated and stirred on a hot plate while measuring the conductivity and temperature. The graph of d (normalized conductivity) / dT showed a 0.51°C drop at 89.4°C. -1 The positive peak of magnitude 1.5 indicates that the DNLF dispersion has a potential lamellar structure. The phase inversion start and end temperatures are 84°C and 92°C, respectively.

[0205] Example 6. Copper tripeptide DNLF dispersion for topical application, comprising copper tripeptide, ceteareth-20, sorbitan oleate, soy lecithin, isopropyl myristate, niacinamide, d-panthenol, kojic acid, and water.

[0206] A crude emulsion was prepared by adding an oil phase consisting of 9.5% ceteareth-20, 9.5% sorbitan oleate, 0.6% soy lecithin, 28.6% isopropyl myristate, and 0.6% Jeecide CAP-4 to an aqueous phase consisting of 0.16% copper tripeptide, 0.17% sodium chloride, and 51.0% water. The ratio of water-immiscible oil to copper tripeptide was 183 to 1. The crude extrudable emulsion mill base (1000 g) was placed in a 2 L Pyrex measuring cup, heated to 95° C. in a microwave, and poured into the inlet of a modified Leistritz ZSE 27 / GL-36D extruder with a screw rotating at 200 rpm, resulting in a translucent milky white liquid with a viscosity similar to honey. The volume average and number average particle sizes and the polydispersity index were 46.9 nm, 40.2 nm, and 0.0600, respectively.

[0207] The sample (56.3 g) was placed in a 100 mL beaker and gently heated to 60°C in a microwave oven using the thaw setting to reduce the viscosity, and then heated and stirred on a hot plate while measuring the conductivity and temperature. The graph of d (normalized conductivity) / dT showed a 0.12°C drop at 87°C. -1 The positive peak of magnitude 1.8 indicates that the DNLF dispersion has a potential lamellar structure. The phase inversion start and end temperatures are 79°C and 87°C, respectively.

[0208] Example 7. Palmitoyl Tripeptide-1DNLF dispersion for topical application, comprising hydrolyzed collagen, palmitoyl tripeptide-1, palmitoyl tetrapeptide-7, spermidine, ceteareth-20, ceteareth-30, sorbitan oleate, cetyl alcohol, isopropyl myristate, medium chain triglyceride oil, mineral oil and water.

[0209] A coarse emulsion was prepared by adding an oil phase consisting of 550 ppm palmitoyl tripeptide-1, 540 ppm palmitoyl tetrapeptide-7, 900 ppm spermidine, 6.2% ceteareth-20, 3.1% ceteareth-30, 9.3% sorbitan oleate, 0.5% cetyl alcohol, 9.8% isopropyl myristate, 8.9% medium chain triglyceride oil, 9.4% light mineral oil, and 0.6% Jeecide CAP-4 to an aqueous phase consisting of 0.76% hydrolyzed collagen, 0.2% sodium chloride, and 51.1% water. The ratio of water-immiscible oil to hydrolyzed collagen, palmitoyl tripeptide-1, and palmitoyl tetrapeptide-7 was 32 to 1. The coarse extrudable emulsion mill base (1000 g) was placed in a 2 L Pyrex measuring cup and heated to 95° C. in a microwave and poured into the inlet of a modified Leistritz ZSE 27 / GL-36D extruder with a screw rotating at 200 rpm to obtain a translucent milky white liquid with a viscosity similar to honey. The volume average particle size, number average particle size and polydispersity index were 55.8 nm, 43.4 nm and 0.0866, respectively.

[0210] The sample (56.6 g) was placed in a 100 mL beaker and gently heated to 60°C in a microwave oven using the thaw setting to reduce the viscosity, and then heated and stirred on a hot plate while measuring the conductivity and temperature. The graph of d (normalized conductivity) / dT showed a value of 0.32°C at 94°C. -1 The positive peak of magnitude 1.5 indicates that the DNLF dispersion has a potential lamellar structure. The phase inversion start and end temperatures are 92°C and 99°C, respectively.

[0211] Example 8. DNLF dispersion for topical administration comprising undecylenoyl phenylalanine, glutathione, arbutin, nicotinamide mononucleotide, ceteareth-20, ceteareth-30, sorbitan oleate, cetyl alcohol, isopropyl myristate, medium chain triglyceride oil, mineral oil, and water.

[0212] A coarse emulsion was prepared by adding an oil phase consisting of 0.55% undecylenoyl phenylalanine, 5.9% ceteareth-20, 3.0% ceteareth-30, 9.4% sorbitan oleate, 0.5% cetyl alcohol, 9.4% isopropyl myristate, 8.5% medium chain triglyceride oil, 9.0% light mineral oil, and 0.5% Jeecide CAP-4 to an aqueous phase consisting of 1.1% glutathione, 0.5% arbutin, 0.5% nicotinamide mononucleotide, 0.2% sodium chloride, and 51.4% water. The ratio of water-immiscible oil to undecylenoyl phenylalanine plus glutathione was 16 to 1. The coarse extrudable emulsion mill base (1000 g) was placed in a 2 L Pyrex measuring cup and heated to 95° C. in a microwave and poured into the inlet of a modified Leistritz ZSE 27 / GL-36D extruder with a screw rotating at 200 rpm to obtain a translucent milky white liquid with a viscosity similar to honey. The volume average particle size, number average particle size, and polydispersity index were 54.9 nm, 43.9 nm, and 0.0724, respectively.

[0213] The sample (63.3 g) was placed in a 100 mL beaker and gently heated to 50°C in a microwave oven using the thaw setting to reduce the viscosity, and then heated and stirred on a hot plate while measuring the conductivity and temperature. The graph of d (normalized conductivity) / dT showed a 1.1°C drop at 90°C. -1 The positive peak of magnitude 1.8 indicates that the DNLF dispersion has a potential lamellar structure. The phase inversion start and end temperatures are 87°C and 93°C, respectively.

[0214] Example 9. DNLF dispersion for topical application comprising acetaminophen, ceteareth-20, sorbitan oleate, isopropyl myristate, medium chain triglyceride oil, and water.

[0215] A crude emulsion was prepared by heating a mixture of the oil phase components consisting of 8.9% ceteareth-20, 13.8% sorbitan oleate, 7.9% isopropyl myristate, and 15.7% medium chain triglyceride oil to obtain a clear solution. 0.16% sodium chloride, 1.5% acetaminophen, and 52.1% water were added to obtain a crude emulsion mill base. This was the same mill base as Example 1 except for the addition of acetaminophen. The crude extrudable emulsion mill base (1000 g) was placed in a 2 L Pyrex measuring cup and heated to 95° C. in a microwave and poured into the inlet of a modified Leistritz ZSE 27 / GL-36D extruder with a screw rotating at 200 rpm to obtain a translucent milky white liquid with a viscosity similar to that of vegetable oil. The volume average particle size, number average particle size, and polydispersity index were 58.7 nm, 43.2 nm, and 0.1421, respectively.

[0216] A sample (23.4 g) was placed in a 30 mL beaker and heated on a hot plate while measuring conductivity and temperature. When heated to 87°C, the sample became transparent and birefringent, indicating that the DNLF dispersion possesses a latent lamellar structure. The phase inversion onset and end temperatures were 88°C and 95°C, respectively.

[0217] After 5 days of storage between 18°C ​​and 21°C, no acetaminophen crystals were visible in the DNLF dispersion. In a separate experiment, 1.50 g of acetaminophen was heated in 53.0 g of deionized water (the same composition as the extruded DNLF, except that the lipid was omitted), resulting in a clear solution. After standing at room temperature for 3 hours, acetaminophen formed crystals.

[0218] This example demonstrates that the addition of acetaminophen and a hydrophilic drug to a mill-base composition of ceteareth-20, sorbitan oleate, isopropyl myristate, medium-chain triglyceride oil, and water effectively extrudes the mill-base to yield a DNLF dispersion having a volume average particle size of less than 100 nm, wherein the DNLF dispersion has a latent lamellar structure. This example also demonstrates that the lipid nanoparticles effectively solubilize acetaminophen in an aqueous solution relative to a solution lacking the lipid nanoparticles.

[0219] Example 10. DNLF dispersion for topical application comprising caffeine, ceteareth-20, sorbitan oleate, isopropyl myristate, medium chain triglyceride oil, and water.

[0220] A crude emulsion was prepared by heating a mixture of the oil phase components consisting of 8.9% ceteareth-20, 13.8% sorbitan oleate, 7.9% isopropyl myristate, and 15.7% medium chain triglyceride oil to obtain a clear solution. 0.16% sodium chloride, 1.5% caffeine, and 52.1% water were added to obtain a crude emulsion mill base. This was the same mill base as in Example 1, except for the addition of caffeine. The crude extrudable emulsion mill base (1000 g) was placed in a 2 L Pyrex measuring cup and heated to 95° C. in a microwave and poured into the inlet of a modified Leistritz ZSE27 / GL-36D extruder with a screw rotating at 200 rpm to obtain a translucent milky white liquid with a viscosity similar to that of vegetable oil. The volume average and number average particle sizes and the polydispersity index were 51.2 nm, 40.3 nm, and 0.0789, respectively.

[0221] A 25g sample was placed in a 30mL beaker and heated on a hot plate while measuring conductivity and temperature. When heated to 87°C, the sample became transparent and birefringent, indicating that the DNLF dispersion possesses a latent lamellar structure. The phase inversion onset and end temperatures were 88°C and 95°C, respectively.

[0222] In a separate experiment, 1.50 g of caffeine was heated in 53.0 g of deionized water (the same composition as the extruded DNLF, except that the lipids were omitted), resulting in a clear solution. After standing overnight at room temperature, the caffeine crystallized into long needle-like crystals.

[0223] This example demonstrates that the addition of caffeine, a hydrophilic drug, to a mill-base composition of ceteareth-20, sorbitan oleate, isopropyl myristate, medium chain triglyceride oil, and water effectively extrudes the mill-base to yield a DNLF dispersion having a volume average particle size of less than 100 nm, wherein the DNLF dispersion has a latent lamellar structure. This example also demonstrates that the lipid nanoparticles effectively solubilize caffeine in an aqueous solution relative to a solution lacking the lipid nanoparticles.

[0224] In a separate experiment, the conductivity versus temperature was measured for a base composition consisting of 9.0% ceteareth-20, 14.0% sorbitan oleate, 15.9% medium chain triglyceride oil, 10% isopropyl myristate, 0.2% sodium chloride, and 50.9% water. 1.0% caffeine was then added to the composition, and the conductivity versus temperature was measured again. The conductivity versus temperature graph is shown below. Figure 1 As shown in Figure 2, caffeine addition resulted in a 5°C increase in the phase inversion temperature and a reduction in the negative peak in conductivity attributed to a weakened lamellar structure. The change in phase inversion temperature and the weakening of the lamellar structure provide evidence that caffeine is encapsulated in or otherwise associated with the lipid phase (water-immiscible oil plus surfactant).

[0225] This example demonstrates that the addition of caffeine, a hydrophilic drug, to a mill-base composition of ceteareth-20, sorbitan oleate, isopropyl myristate, medium-chain triglyceride oil, and water effectively extrudes the mill-base to yield a DNLF dispersion having a volume average particle size of less than 100 nm, wherein the DNLF dispersion has a latent lamellar structure. This example also demonstrates that the lipid nanoparticles effectively solubilize caffeine in an aqueous solution relative to a solution lacking the lipid nanoparticles, and provides additional evidence that caffeine is encapsulated within or otherwise associated with lipids in the DNLF dispersion.

[0226] Example 11. DNLF dispersion for topical application comprising 5-fluorouracil, ceteareth-20, sorbitan oleate, isopropyl myristate, medium chain triglyceride oil, and water.

[0227] A crude emulsion was prepared by heating a mixture of the oil phase components consisting of 8.7% ceteareth-20, 13.4% sorbitan oleate, 11.5% isopropyl myristate, and 15.3% medium chain triglyceride oil to obtain a clear solution. To this was added 0.15% sodium chloride, 0.5% 5-fluorouracil, and 50.6% water to obtain a crude emulsion mill base. This was the same mill base as in Example 1 except that 5-fluorouracil and isopropyl myristate were added. The crude extrudable emulsion mill base (1000 g) was placed in a 2 L Pyrex measuring cup and heated to 95° C. in a microwave and poured into the inlet of a modified Leistritz ZSE 27 / GL-36D extruder with a screw rotating at 200 rpm to obtain a translucent milky white liquid with a viscosity similar to that of vegetable oil. The volume average particle size, number average particle size and polydispersity index were 53.2 nm, 38.4 nm and 0.0821, respectively.

[0228] The sample (25 g) was placed in a 30 mL beaker and heated on a hot plate while measuring conductivity and temperature. The graph of d (normalized conductivity) / dT showed a 1°C drop at 88°C. -1 The positive peak of magnitude 1.5 indicates that the DNLF dispersion has a potential lamellar structure. The phase inversion start and end temperatures are 85℃ and 91℃, respectively.

[0229] This example shows the preparation of an extruded DNLF dispersion containing 5-fluorouracil, ceteareth-20, sorbitan oleate, isopropyl myristate, medium chain triglyceride oil, and water, with a volume average particle size of less than 100 nm.

[0230] Example 12. DNLF dispersion for topical administration comprising gemcitabine, ceteareth-20, sorbitan oleate, isopropyl myristate, medium chain triglyceride oil, and water.

[0231] A crude emulsion was prepared by heating a mixture of the oil phase components consisting of 8.5% ceteareth-20, 13.3% sorbitan oleate, 11.5% isopropyl myristate, and 15.4% medium chain triglyceride oil to obtain a clear solution. 0.17% sodium chloride, 0.5% gemcitabine, and 50.6% water were added thereto to obtain a crude emulsion mill base. This was the same mill base as in Example 1 except that gemcitabine and isopropyl myristate were added. The crude extrudable emulsion mill base (500 g) was placed in a 2 L Pyrex measuring cup and heated to 95° C. in a microwave and poured into the inlet of a modified Leistritz ZSE 27 / GL-36D extruder with a screw rotating at 200 rpm to obtain a translucent, milky white liquid with a viscosity similar to that of vegetable oil. The volume average particle size, number average particle size and polydispersity index were 53.2 nm, 38.4 nm and 0.0821, respectively.

[0232] The sample (25 g) was placed in a 30 mL beaker and heated on a hot plate while measuring conductivity and temperature. The graph of d (normalized conductivity) / dT showed a 1°C drop at 88°C. -1 The positive peak of magnitude 1.5 indicates that the DNLF dispersion has a potential lamellar structure. The phase inversion start and end temperatures are 85℃ and 91℃, respectively.

[0233] This example shows the preparation of an extruded DNLF dispersion comprising gemcitabine, ceteareth-20, sorbitan oleate, isopropyl myristate, medium chain triglyceride oil, and water, with a volume average particle size of less than 100 nm.

[0234] Examples 13 to 15. Preparation of extruded DNLF dispersions containing hydrophilic and / or amphiphilic bioactive agents for oral administration.

[0235] Surprisingly, as shown in Examples 13 to 15, DNLF dispersions can also be prepared into formulations comprising hydrophilic and / or amphiphilic bioactive agents and suitable for oral administration.

[0236] Example 13. Insulin DNLF for oral administration.

[0237] Prepare thick emulsion by heating the mixture of the oil phase component consisting of 12.9% polysorbate 80, 3.1% sorbitan stearate, 2.5% soy lecithin, 2.6% stearic acid, 27.8% ethyl oleate and 11.5% medium chain triglyceride oil and adding 0.2% sodium chloride and 39.4% deionized water. In an independent beaker, 93.9mg recombinant human insulin (can be obtained from Sigma Aldrich, catalog number (Cat. No.) 91077C) is dissolved among the 19.7g 0.1N HCl. Under agitation, 0.68g 10% sodium lauryl sulfate is added to the clear insulin solution of gained, obtaining a white suspension. In this white suspension, add 180.0g thick emulsion and 2.27g 10% sodium citrate dihydrate, obtaining a thick extrudable emulsion premix. The crude extrudable emulsion premix contains 0.0463% insulin, 11.4% polysorbate 80, 0.033% sodium lauryl sulfate, 2.7% sorbitan stearate, 2.2% soy lecithin, 2.3% stearic acid, 24.6% ethyl oleate, 10.2% medium chain triglyceride oil, 0.036% HCl, 0.11% sodium citrate dihydrate, 0.16% sodium chloride, and 46.2% water. The ratio of water-immiscible oil to insulin is 753 to 1. The crude emulsion (150 g) in a 250 mL pyrex beaker is heated in a microwave oven for 60 seconds to a temperature of approximately 65° C. It is then heated with a handheld torch for approximately 2 minutes while stirring with a stick thermometer until it reaches a temperature of 85° C. The hot coarse emulsion was poured into the inlet of a modified Process 11 extruder with zone temperatures of Zone 2 = approximately 50° C., Zone 3 = 35° C., Zone 4 = 9° C., Zone 5 = 9° C., Zone 6 = 7° C., Zone 7 = 9° C., Zone 8 = 8° C. and screw speed = 200 rpm. The processing rate was approximately 40 g / min, and the entire batch was processed in less than 4 minutes to give an insulin DNLF dispersion as a translucent beige viscous liquid.

[0238] The volume average particle size, number average particle size and polydispersity index were 68.7 nm, 54.5 nm and 0.0698, respectively, and the lipid concentration was 53.5%. The pH was 4.9.

[0239] The sample (22.2 g) was placed in a 30 mL beaker and heated on a hot plate while measuring conductivity and temperature. At 66°C and 77°C, the sample was transparent and birefringent, and a plot of d (normalized conductivity) / dT showed a 0.19°C drop at 76.5°C. -1 The positive peak of magnitude 1.8 indicates that the DNLF dispersion has a potential lamellar structure. The phase inversion start and end temperatures are 66°C and 78°C, respectively.

[0240] DNLF dispersion sample (0.41g) is diluted with 3.60g 4% bovine serum albumin PBS buffer, and the sample of dilution is diluted twice with 1 to 100 using 4% bovine serum albumin PBS buffer, and total dilution is 1 to 100,000.Insulin concentration based on formulation data is 4.7 parts per billion by weight.Use the human insulin ELISA kit from Crystal Chem (catalog number (Cat. No.) 90095), use the standard curve measurement insulin concentration prepared by 1.0, 2.0, 3.0, 4.0 and 5.0ppb standard substance, and this standard substance is prepared by identical Sigma Aldrich (catalog number (Cat. No.) 91077C) human recombinant insulin.According to this measurement, the concentration of insulin is 1.2ppb, is equivalent to 26% insulin recovery.

[0241] In a separate measurement, a sample (1.00 g) of the DNLF dispersion was diluted with 10.0 g of 10% Triton X-100 (4-tert-octylphenol ether with 9 moles of poly(ethylene glycol), a product of Union Carbide) to obtain a translucent dispersion. The diluted dispersion was diluted twice 1 to 100 using 4% bovine serum albumin PBS buffer, for a total dilution of 1 to 110,000. The insulin concentration based on the formulation data was 4.2 ppb. When measured using the human insulin ELISA test described above, the insulin concentration was 1.1 ppb, equivalent to an insulin recovery of 26%.

[0242] Prepare empty extrudable emulsion premix as mentioned above, but omit insulin.This empty extrudable emulsion premix contains 11.4% polysorbate 80, 0.031% sodium lauryl sulfate, 2.7% sorbitan stearate, 2.2% soy lecithin, 2.3% stearic acid, 24.6% ethyl oleate, 11.4% medium chain triglyceride oil, 0.033% HCl, 0.10% sodium citrate dihydrate, 0.16% sodium chloride and 45.0% water.The thick emulsion (150g) in the 250mL pyrex beaker is heated to a temperature of about 65 ℃ for 60 seconds in a microwave oven.Then heat with a handheld torch for about 2 minutes, stir with a stick thermometer simultaneously, until it reaches a temperature of 85 ℃. The hot coarse emulsion was poured into the inlet of a modified Process 11 extruder with zone temperatures of approximately 50°C (Zone 2), 35°C (Zone 3), 9°C (Zone 4), 9°C (Zone 5), 7°C (Zone 6), 9°C (Zone 7), and 8°C (Zone 8) at a screw speed of 200 rpm. The processing rate was approximately 40 g / min, and the entire batch was processed in less than 4 minutes to yield a DNLF dispersion as a translucent, beige, viscous liquid.

[0243] The volume average particle size, number average particle size and polydispersity index were 67.6 nm, 53.7 nm and 0.1038, respectively, and the lipid concentration was 54.7%. The pH was 4.8.

[0244] A DNLF dispersion sample (0.39 g) was diluted with 3.58 g of 4% bovine serum albumin (PBS) buffer, and the diluted sample was diluted twice with 4% bovine serum albumin (PBS) buffer at a 1:100 ratio, for a total dilution of 1:100,000. Insulin concentration was measured using a human insulin ELISA kit from Crystal Chem (Cat. No. 90095) using a standard curve prepared from 1.0, 2.0, 3.0, 4.0, and 5.0 ppb standards, prepared from the same Sigma Aldrich (Cat. No. 91077C) human recombinant insulin. Based on this measurement, the concentration of insulin in the DNLF dispersion was 0.26 ppb (baseline value - empty lipid nanoparticles).

[0245] Example 14. Insulin DNLF for oral administration

[0246] By heating the mixture of the oil phase component composed of 11.7% polysorbate 80, 2.8% sorbitan stearate, 2.2% soy lecithin, 2.3% stearic acid, 14.0% ethyl oleate, 10.9% isopropyl myristate and 10.3% medium chain triglyceride oil and adding 0.16% sodium citrate dihydrate and 35.4% deionized water to prepare a crude emulsion. The ratio of water-immiscible oil to insulin is 682 to 1. In a separate beaker, 520ppm recombinant human insulin (available from SigmaAldrich, catalog number (Cat. No.) 91077C) is dissolved in 10.1%g 0.1N HCl. Under stirring, 300ppm sodium lauryl sulfate is added to the clear insulin solution of gained to obtain a white suspension. The oil phase component is added to the suspension of insulin to obtain a thick extrudable emulsion. The mill base was pumped at a rate of 12.5 mL / min through a heat exchanger maintained at a temperature between 85°C and 90°C into the inlet of a modified Thermo Fisher Process 11 extruder having a screw rotating at 300 rpm to obtain a translucent milky white liquid with a viscosity similar to that of vegetable oil. The volume average particle size and number average particle size and polydispersity index were 77.6 nm, 55.9 nm and 0.0714, respectively. At a flow rate of 12.5 mL / min, the mill base was heated in a heat exchanger (path length = 50.0 cm, cross-sectional area = 0.071 cm) and heated to 400 rpm for 10 minutes. 2 The residence time in the flask (volume = 3.5 mL) was 17 seconds. The pH of the extruded sample was 5.5.

[0247] DNLF dispersion sample (0.56g) was diluted with 5.67g 4% bovine serum albumin PBS buffer, and the diluted sample was diluted twice with 1 to 100 using 4% bovine serum albumin PBS buffer, with a total dilution of 1 to 100,000. The insulin concentration based on the formulation data was 5.2 parts per billion by weight. Using a human insulin ELISA kit from Crystal Chem (catalog number 90095), the standard curve prepared by 1.0, 2.0, 3.0, 4.0 and 5.0ppb standards was used to measure insulin concentration, which was prepared by identical Sigma Aldrich (catalog number 91077C) human recombinant insulin. According to this measurement, the concentration of insulin was 3.6ppb, equivalent to 70% insulin recovery. In this example, the crude emulsion was heated to 85°C to 90°C in 17 seconds to form an intermediate structure that could be processed in a twin-screw extruder, resulting in a DNLF dispersion that only lost 30% of its initial insulin concentration.

[0248] Example 15. Cyclosporin A DNLF for oral administration.

[0249] A crude emulsion was prepared by heating a mixture of oil phase components consisting of 0.5% cyclosporin A, 11.5% polysorbate 80, 2.7% sorbitan stearate, 2.2% soy lecithin, 2.2% stearic acid, 18.6% isopropyl myristate, 8.7% acetate esters of mono- and diglycerides, and 7.4% medium-chain triglyceride oil, and adding 0.2% sodium chloride and 46.0% deionized water. The ratio of water-immiscible oil to cyclosporin A was 68 to 1. The millbase was pumped at a rate of 12.5 mL / min through a heat exchanger maintained at a temperature between 87°C and 89°C into the inlet of a modified Thermo Fisher Process 11 extruder with a screw rotating at 300 rpm, resulting in a translucent, milky white liquid with a viscosity similar to that of vegetable oil. The volume average particle size, number average particle size and polydispersity index were 67.1 nm, 46.4 nm and 0.0852, respectively.

[0250] Examples 16 to 21. Improvement of oral bioavailability of DNLF formulations.

[0251] With the understanding that DNLF dispersions containing hydrophilic and amphiphilic compounds may be suitable for oral administration (e.g., insulin, as demonstrated in Examples 13 to 14), the oral bioavailability of DNLF was examined for suspensions. As shown in Examples 16 to 21 below, quite surprisingly, DNLF containing dasatinib was able to deliver the greatest concentration of active compound in the bloodstream (T max ), which is on the order of minutes rather than more than an hour. The maximum dasatinib concentration in the bloodstream (Cmax ) was also relatively high, which is attributed to the rapid absorption from the GI system. Notably, Examples 20 to 21 showed further improvement in bioavailability by conferring lipolytic resistance to DNLF.

[0252] Comparative Example 16. Oral administration of Dasatinib suspension.

[0253] A suspension of dasatinib powder (1.4%, a product of ChemScene, Monmouth Junction, NJ) in a solution of methylcellulose HV (0.75%, a product of Modernist Pantry, Eliot ME) and phosphate buffered saline (pH 7.4) was prepared. The suspension was diluted to 0.125% dasatinib (1.25 mg / mL) using 0.75% methylcellulose HV in phosphate buffered saline (pH 7.4). The suspension was administered to three Jackson Labs C57BL / 6J mice at a dose of 10 mg / kg, and blood samples were drawn regularly. The blood samples of the three mice were combined, the combined plasma was collected by centrifugation, and dasatinib was quantified by HPLC-MS. The relationship between plasma concentration and time is shown in Table 2.

[0254] Example 17. Oral administration of a readily lipolytic DNLF dispersion comprising polysorbate 80, sorbitan stearate, soy lecithin, oleic acid, isopropyl myristate, medium chain triglyceride oil, dasatinib, and water.

[0255] A crude emulsion was prepared containing 14.6% polysorbate 80, 1.5% sorbitan stearate, 1.8% soy lecithin, 5.3% oleic acid, 26.6% isopropyl myristate, 6.5% medium-chain triglyceride oil, 0.3% sodium benzoate, 0.1% citric acid, 42.2% water, and 1.2% dasatinib. The crude emulsion was processed in a Process 11 extruder, which had been modified with a heat exchanger for barrel cooling and a heat exchanger for heating the crude emulsion before introduction into the barrel. This yielded a DNLF dispersion with a volume-average particle size of 54.7 nm, a number-average particle size of 35.0 nm, and a zeta potential of 5.6 mV. In this DNLF, polysorbate 80, sorbitan stearate, isopropyl myristate, and medium-chain triglyceride oil contain ester bonds and are susceptible to lipolysis.

[0256] DNLF was diluted with deionized water to a concentration of 0.125% dasatinib (1.25 mg / mL) and administered to three Jackson Labs C57BL / 6J mice at a dose of 10 mg / kg. Blood samples were drawn regularly, pooled from the three mice, and the combined plasma was collected by centrifugation. Dasatinib was quantified by HPLC-MS. The relationship between plasma concentrations and time is shown in Table 2.

[0257] Example 18. Oral Administration of a Readily Lipolysable DNLF Dispersion Comprising Polysorbate 80, PEG40 Stearate, Sorbitan Stearate, Soy Lecithin, Oleic Acid, Isopropyl Myristate, Medium Chain Triglyceride Oil, Trilinolein, Dasatinib, and Water.

[0258] A coarse emulsion was prepared containing 6.7% polysorbate 80, 6.8% PEG 40 stearate, 2.0% sorbitan stearate, 0.9% soy lecithin, 6.2% oleic acid, 21.4% isopropyl myristate, 7.9% medium chain triglyceride oil, 5.3% trilinolein (Maisine CC, a product of Gattefosse), 0.3% sodium benzoate, 0.1% citric acid, 41.0% water, and 1.4% dasatinib. The coarse emulsion was processed in a Process 11 extruder that had been modified with a heat exchanger for cooling the barrel and a heat exchanger for heating the coarse emulsion before introduction into the barrel, resulting in a DNLF dispersion having a volume average particle size of 62.1 nm, a number average particle size of 46.9 nm, and a zeta potential of -0.8 mV. In this DNLF, polysorbate 80, PEG40 stearate, sorbitan stearate, isopropyl myristate, medium chain triglyceride oil and trilinolein contain ester bonds and are easy to lipolytic. DNLF is diluted to 0.125% dasatinib (1.25mg / mL) with deionized water and administered to three Jackson Labs C57BL / 6J mice at a dosage of 10mg / kg. Blood samples were drawn regularly, and the blood samples of the three mice were combined. The combined plasma was collected by centrifugation, and dasatinib was quantitatively measured by HPLC-MS. The relationship between plasma concentration and time is shown in Table 2.

[0259] Example 19. Oral administration of a readily lipolytic DNLF dispersion comprising polysorbate 80, PEG 40 stearate, sorbitan stearate, soy lecithin, oleic acid, isopropyl myristate, medium chain triglyceride oil, trilinolein, sesame oil, sodium benzoate, citric acid, dasatinib, and water.

[0260] A crude emulsion was prepared containing 6.6% polysorbate 80, 6.6% PEG40 stearate, 2.0% sorbitan stearate, 0.8% soy lecithin, 6.1% oleic acid, 13.7% isopropyl myristate, 5.9% medium chain triglyceride oil, 7.9% trilinolein (Maisine CC, a product of Gattefosse), 7.9% sesame oil, 0.2% sodium benzoate, 0.1% citric acid, 40.8% water, and 1.4% dasatinib.

[0261] The coarse emulsion was processed in a Process 11 extruder that had been modified with a heat exchanger for cooling the barrel and a heat exchanger for heating the coarse emulsion before introduction into the barrel, resulting in a DNLF dispersion with a volume average particle size of 66.6 nm, a number average particle size of 42.5 nm, and a zeta potential of 7.4 mV.

[0262] In this DNLF, polysorbate 80, PEG40 stearate, sorbitan stearate, isopropyl myristate, medium chain triglyceride oil, trilinolein, and sesame oil contain ester bonds and are susceptible to lipolysis.

[0263] DNLF was diluted with deionized water to a concentration of 0.125% dasatinib (1.25 mg / mL) and administered to three Jackson Labs C57BL / 6J mice at a dose of 10 mg / kg. Blood samples were drawn regularly, pooled from the three mice, and the combined plasma was collected by centrifugation. Dasatinib was quantified by HPLC-MS. The relationship between plasma concentrations and time is shown in Table 2.

[0264] Example 20. Oral administration of a non-lipolytic DNLF dispersion comprising polysorbate 80, sorbitan stearate, soy lecithin, oleic acid, isopropyl myristate, medium chain triglyceride oil, orlistat, dasatinib, and water

[0265] A coarse emulsion was prepared containing 14.4% polysorbate 80, 1.5% sorbitan stearate, 1.8% soy lecithin, 5.2% oleic acid, 26.4% isopropyl myristate, 6.4% medium-chain triglyceride oil, 1.0% orlistat, 0.2% sodium benzoate, 0.1% citric acid, 41.8% water, and 1.2% dasatinib. The coarse emulsion was processed in a Process 11 extruder modified with a heat exchanger for barrel cooling and a heat exchanger for heating the coarse emulsion before introduction into the barrel, resulting in a DNLF dispersion with a volume average particle size of 110.3 nm, a number average particle size of 46.4 nm, and a zeta potential of -0.1 mV.

[0266] In this DNLF, polysorbate 80, sorbitan stearate, isopropyl myristate, and medium-chain triglyceride oil contain ester bonds and are susceptible to lipolysis, and orlistat is an inhibitor of lipase.

[0267] DNLF was diluted with deionized water to a concentration of 0.125% dasatinib (1.25 mg / mL) and administered to three Jackson Labs C57BL / 6J mice at a dose of 10 mg / kg. Blood samples were drawn regularly, pooled from the three mice, and the combined plasma was collected by centrifugation. Dasatinib was quantified by HPLC-MS. The relationship between plasma concentrations and time is shown in Table 2.

[0268] Example 21. Oral administration of a non-lipolytic DNLF dispersion comprising ceteareth-30, sorbitan stearate, oleic acid, isopropyl myristate, medium chain triglyceride oil, mineral oil, dasatinib, and water.

[0269] A coarse emulsion was prepared containing 9.5% ceteareth-30, 3.8% sorbitan stearate, 8.3% oleic acid, 9.8% isopropyl myristate, 8.2% medium chain triglyceride oil, 8.9% mineral oil, 0.3% sodium benzoate, 0.1% citric acid, 49.7% water, and 1.5% dasatinib. The coarse emulsion was processed in a Process 11 extruder modified with a heat exchanger for barrel cooling and a heat exchanger for heating the coarse emulsion prior to introduction into the barrel, resulting in a DNLF dispersion having a volume average particle size of 66.0 nm, a number average particle size of 47.1 nm, and a zeta potential of 3.9 mV.

[0270] In this DNLF, sorbitan stearate, isopropyl myristate, and medium chain triglyceride oil contain ester bonds and are easily lipolyzed, and ceteareth-30 and mineral oil do not have ester bonds and are not easily lipolyzed.

[0271] DNLF was diluted with deionized water to a concentration of 0.125% dasatinib (1.25 mg / mL) and administered to three Jackson Labs C57BL / 6J mice at a dose of 10 mg / kg. Blood samples were drawn regularly, pooled from the three mice, and the combined plasma was collected by centrifugation. Dasatinib was quantified by HPLC-MS. The plasma concentration versus time and the AUC4 are shown in Table 2. Local maxima in the concentration versus time plot are underlined.

[0272] Table 2. Relationship between plasma concentration and time

[0273]

[0274] Discussion of Examples 16 to 21

[0275] Three dasatinib DNLFs based on readily lipolyzed polysorbate 80 were prepared and tested for oral bioavailability (Examples 2 to 4) and compared with a dasatinib suspension (Example 1). DNLFs showed enhanced absorption compared to the suspension powder ( Figure 2 They also showed that, in addition to a local maximum at 2 hours, a local maximum in the dasatinib plasma concentration versus time curve occurred at the earliest sampling time point. The early local maximum was attributed to rapid absorption of already formed nanoemulsion droplets, while the peak at 2 hours was attributed to absorption of micelles formed from bile salts and lipolytic degradation products of the nanoemulsion droplets.

[0276] The conclusion that the second local maximum in the plasma concentration versus time curve can be attributed to micellization and absorption of lipolytic degradation products from the lipid nanoemulsion droplets is supported by the observation that inclusion of orlistat (a lipase inhibitor) in DNLF resulted in the disappearance of the second peak ( Figure 3 (Example 20 in

[15] ). The truly striking feature of this graph is the monotonic decrease in plasma concentration. While improvements in AUC of up to 4-fold or even greater are not uncommon and are actually quite typical in the academic literature, a Cmax at the first time point has never been observed in oral dosage forms. In fact, this is a characteristic of intravenously administered drugs. Compared to DNLF without orlistat, the AUC is not improved because the diameter of the nanoemulsion droplets is doubled due to the inclusion of orlistat.

[0277] The attribution of the second local maximum to the micellization and absorption of lipolytic degradation products is further supported by the observation that replacement of the lipolytically susceptible polysorbate 80 with ceteareth-30 (insensitive to lipolysis) also resulted in the disappearance of the second peak ( Figure 4 In this case, the AUC is greatly improved compared to DNLF containing polysorbate 80 in the absence of orlistat, because the drug is not lost by precipitation when the lipid and surfactant are hydrolyzed.

[0278] Examples 22 to 24. DNLF dispersions maintain particle size distribution under lipolytic conditions.

[0279] The performance of DNLF under lipolysis conditions was also examined, and it was shown that the DNLF formulated as disclosed herein can retain its properties (e.g., particle size less than 100 nm) for a considerable period of time. Considering that the time to pass through the GI tract under lipolysis conditions is in the range of 1 hour, the unexpected ability of DNLF to largely maintain a particle size suitable for absorption over this time range suggests that the DNLF of Examples 1 to 15 may be capable of being administered in certain aspects without further consideration of lipase inhibitors, encapsulation, etc.

[0280] Comparative Example 22. Preparation of a digestible DNLF dispersion comprising polysorbate 80, sorbitan stearate, soy lecithin, isopropyl myristate, medium chain triglyceride oil, glycerol, sodium chloride, and water.

[0281] A DNLF dispersion was prepared consisting of 14.6% polysorbate 80, 7.2% sorbitan stearate, 0.9% soy lecithin, 25.5% isopropyl myristate, 10.9% medium-chain triglyceride oil, 2.3% glycerol, 0.17% sodium chloride, and 40.2% water. The dispersion was a turbid, transparent, light yellow liquid with a viscosity similar to that of a light syrup. The volume-average and number-average particle sizes, as well as the polydispersity index, were 70.5 nm, 57.5 nm, and 0.0733, respectively. This sample contained 1.86 mmol of ester bonds per gram.

[0282] A pH static experiment of susceptibility to ester bond hydrolysis by lipase was conducted at 40°C using 1.52 g of DNLF dispersion in 60.06 g of lipolytic medium. The experiment was started by adding 0.25 g of Carolina Biologicals laboratory grade lipase (Cat. No. 872500). The particle size distribution was measured at the beginning of the experiment and one hour later using a NanoFlex dynamic light scattering (DLS) instrument (Microtrac Instruments, York PA). The particle size distribution was as follows: Figure 5 As shown, and the relationship between the cumulative volume percentage of particles with a diameter smaller than (% passing the diameter sieve) and the diameter is as follows Figure 6 Carolina Biologicals laboratory-grade lipase is useful for determining particle size versus time using DLS because it dissolves in water, yielding a clear, homogeneous solution that does not interfere with particle size measurements.

[0283] This example shows that after 60 minutes of lipolysis using Carolina Biologicals laboratory grade lipase, no particles had a diameter less than 100 nm.

[0284] Example 23. Preparation of a DNLF dispersion wherein greater than 80% by volume of the nanoparticles have a diameter less than 100 nm after 1 hour of lipolysis, the dispersion comprising ceteareth-20, sorbitan stearate, isopropyl myristate, medium chain triglyceride oil, mineral oil, quinine, sodium chloride, and water.

[0285] A DNLF dispersion was prepared consisting of 9.9% ceteareth-20, 9.9% sorbitan stearate, 9.8% isopropyl myristate, 9.8% medium chain triglyceride oil, 9.9% mineral oil, 1.1% quinine, 0.2% sodium chloride, and 49.3% water. This sample had 1.17 mmol of ester bonds per gram.

[0286] A pH static experiment of susceptibility to ester bond hydrolysis by lipase was performed at 40°C using 1.83g of DNLF dispersion in 60.11g of lipolytic medium. Once the pH stabilized at 6.80, the experiment was started by adding 0.24g of Carolina Biologicals laboratory grade lipase (Cat. No. 872500) and recording the pH. After the addition of lipase, the pH dropped to 6.90 and then remained constant at 6.90 for 40 minutes. A graph of pH and % ester bond hydrolysis over time is shown in Figure 2. Figure 7 The particle size distribution was measured at the beginning of the experiment and one hour later using a NanoFlex dynamic light scattering (DLS) instrument (Microtrac Instruments, York PA). Figure 8 As shown, and the relationship between the cumulative volume percentage of particles with a diameter smaller than (% passing the diameter sieve) and the diameter is as follows Figure 9 shown.

[0287] This example shows that after 60 minutes of lipolysis using Carolina Biologicals laboratory grade lipase, 84% by volume of the particles had a diameter less than 100 nm, even though more than 20% of the ester groups had been cleaved by lipase hydrolysis.

[0288] Example 24. Preparation of a DNLF dispersion with zwitterionic particles, the dispersion comprising ceteareth-20, sorbitan stearate, medium chain triglyceride oil, isopropyl myristate, mineral oil, cocamidopropyl betaine, palmitoyl tripeptide 1, palmitoyl tetrapeptide 7, sodium chloride, and water.

[0289] A DNLF dispersion was prepared consisting of 9.8% ceteareth-20, 9.7% sorbitan stearate, 9.7% medium chain triglyceride oil, 9.8% isopropyl myristate, 10.0% mineral oil, 1.5% cocamidopropyl betaine, 0.13% palmitoyl tripeptide 1, 0.12% palmitoyl tetrapeptide 7, 0.19% sodium chloride, and 49.1% water. The DNLF dispersion had a volume average particle size of 54.7 nm and a number average particle size of 37.1 nm.

[0290] Examples 25 to 27. Preparation of Encapsulated DNLF Dispersions.

[0291] In addition to the improvements resulting from resistance to lipolysis and maintenance of particle size distribution, it is contemplated herein that DNLF containing a bioactive agent can be formulated within capsules for delivery to specific regions of the GI tract where absorption conditions are most favorable without destroying the bioactive agent. For example, delivering DNLF to the small intestine via capsules can avoid exposure to digestion by lipases present elsewhere in the GI tract. Examples 25 to 27 provide encapsulated DNLF in stable forms for storage and oral administration.

[0292] Example 25. An oral DNLF dispersion comprising polysorbate 80, sorbitan stearate, soy lecithin, isopropyl myristate, light mineral oil, clove essential oil, and water was encapsulated in capsules composed of Eudragit FL 30D-55 polymer.

[0293] A DNLF dispersion was prepared containing 14.7% polysorbate 80, 5.2% sorbitan stearate, 0.7% soy lecithin, 26.0% isopropyl myristate, 11.2% light mineral oil, 0.3% clove essential oil, 0.1% sodium benzoate, 0.3% sodium citrate dihydrate, 0.4% citric acid, 2.3% glycerol, and 39.0% water. The volume-average and number-average particle sizes and polydispersity index were 58.7 nm, 44.7 nm, and 0.0766, respectively, and the lipid concentration was 58.0%. The pH was 4.1.

[0294] Eudragit FL 30D-55 polymer films were prepared by drying the liquid pool on a silicone baking tray. The film weight per unit area was 0.048 g / cm 2. The film was wrapped around a rod with a diameter of 7 mm and heat sealed to give a tube weighing 0.89 g. One end of the tube was heat sealed, partially filled with 1.46 g of DNLF dispersion, and the other end of the tube was heat sealed. After 4 days of storage at room temperature, the weight of the capsule and contents decreased from 2.36 g to 2.16 g, the capsule remained intact and firm, and did not soften significantly compared to before filling. The capsule was cut open and the DNLF contents squeezed out, leaving an empty capsule weighing 0.95 g (an increase of 6%) and a content weighing 1.21 g (a decrease of 17%). The volume average particle size and number average particle size and polydispersity index of the stored DNLF dispersion were 62.4 nm, 50.1 nm and 0.0549, respectively. This example shows that when a DNLF dispersion with a pH of 4.1 is stored in Eudragit FL30D-55 capsules, both the DNLF dispersion and the capsule are stable and about 35% of the water initially present in the DNLF dispersion is lost due to evaporation. In a separate experiment, when two similar capsules were stored at 40°C for four days, they became stuck together, indicating that filled Eudragit FL 30D-55 capsules become sticky under such storage conditions.

[0295] Example 26. An oral DNLF dispersion comprising polysorbate 80, sorbitan stearate, soy lecithin, isopropyl myristate, light mineral oil, clove essential oil, and water was encapsulated in capsules composed of Eudragit FL 30D-55 polymer having an outer surface layer comprising carnauba wax, polydimethylsiloxane, and an organomodified silicone.

[0296] The polymer film was prepared by drying a slurry of 7.5 g of Eudragit FL 30D-55 polymer contained in a 6 cm x 8 cm rectangular silicone soap mold. The weight per unit area of ​​the film was 0.047 g / cm 2. While still in the mold, the film was sprayed with 0.20 g of Meguar brand Gold Class Carnauba Plus premium fast wax spray (available from Meguiar, Irvine CA). The applied beaded spray wet film was manually flattened to obtain a continuous film and dried at 50°C for 18 hours. The film was wrapped around a rod with a diameter of 7 mm with the coated side facing outward and heat sealed to obtain a tube weighing 0.99 g. One end of the tube was heat sealed, partially filled with 1.90 g of the DNLF dispersion from the example, and the other end of the tube was heat sealed. This capsule plus a second similar capsule was stored at 40°C for two days. The capsules remained intact and firm and were not noticeably softer than before filling, and stuck together very slightly and separated easily. This example shows that Eudragit FL 30D-55 capsules coated with carnauba wax, polydimethylsiloxane and organically modified silicone do not become sticky when filled with DNLF dispersion at pH 4.1 and stored at 40°C.

[0297] Example 27. An oral DNLF dispersion comprising polysorbate 80, sorbitan stearate, soy lecithin, isopropyl myristate, light mineral oil, clove essential oil, and water was encapsulated in capsules composed of Eudragit FL 30D-55 polymer having an outer surface layer comprising carnauba wax.

[0298] The polymer film was prepared by drying a 5.0 g slurry of Eudragit FL 30D-55 polymer contained in a 6 cm x 8 cm rectangular silicone soap mold. The weight per unit area of ​​the film was 0.032 g / cm 2. While still in the mold, 0.5 g of a 1.0 wt % solution of carnauba wax (food grade flakes, available from Better Shea Butter Company, Cedar Park TX) in trichloroethylene was spread on top of the film and dried at 50°C for 4 hours. The film was wrapped around a 7 mm diameter rod with the coated side facing outward and heat sealed to give a tube weighing 0.55 g. One end of the tube was heat sealed, partially filled with 1.08 g of the DNLF dispersion from the example, and the other end of the tube was heat sealed. The capsule plus a second similar capsule was stored at 40°C overnight, whereby the weight of the capsule and contents decreased from 1.63 g to 1.45 g. The capsules remained intact and firm, not noticeably softer than before filling, and did not stick together. When placed in 500 mL of slowly stirred PBS 7.4 buffer at 40°C, the capsules became opaque within 5 minutes and had the consistency of melted cheese after 90 minutes. They could be broken by gently squeezing. This example shows that Eudragit FL 30D-55 capsules coated with carnauba wax do not become sticky when filled with a DNLF dispersion at pH 4.1 and stored at 40°C, but become very soft and physically brittle when stirred in pH 4 buffer at 40°C for 90 minutes.

[0299] Examples 28 to 30. Preparation of extruded DNLF dispersions containing insulin for oral administration with high bioavailability.

[0300] In view of the previous examples, it is contemplated herein that bioactive compounds that could not previously be obtained by oral administration can be provided as oral formulations with high bioavailability. Preparations for oral administration of insulin have long been sought, but without success. Examples 28 to 29 present anti-lipolytic DNLF comprising insulin that, according to the results of Examples 20 to 21, are able to deliver insulin to the bloodstream via oral administration with acceptable Tmax and Cmax, similar to less desirable alternative administration routes (e.g., injection).

[0301] Example 28. Insulin. Preparation of an oral DNLF dispersion that is resistant to lipolysis, the dispersion comprising insulin, polysorbate 80, sodium lauryl sulfate, sorbitan stearate, soy lecithin, stearic acid, ethyl oleate, isopropyl myristate, medium chain triglyceride oil, orlistat, and water.

[0302] A DNFL dispersion was prepared containing an oil phase consisting of 540 ppm insulin as a hydrophobic ion pair with 310 ppm sodium lauryl sulfate, 11.7% polysorbate 80, 2.8% sorbitan stearate, 2.1% soy lecithin, 2.3% stearic acid, 13.9% ethyl oleate, 10.7% isopropyl myristate, and 10.2% medium-chain triglyceride oil, plus 0.23% orlistat as a lipase inhibitor and an aqueous phase consisting of 0.2% sodium citrate dihydrate, 410 ppm HCl, and 45.7% water. The dispersion was a cloudy, transparent yellow liquid with a viscosity similar to that of a light syrup. The volume average particle size, number average particle size, and polydispersity index were 61.7 nm, 31.7 nm, and 0.0652, respectively, and the pH was 4.93. This sample had 1.66 mmol of ester bonds per gram. This was the same formulation as Example 14, except that it contained orlistat.

[0303] A pH static experiment to investigate the sensitivity of ester bond hydrolysis to lipase was performed using 1.61 g of DNLF dispersion in 59.68 g of lipolysis medium at 40°C. Once the pH stabilized at 6.93, 0.28 g of Sigma-Aldrich catalog number L-3126 porcine pancreatic lipase (≥125 units / mg protein, using olive oil and incubating for 30 minutes) was added as a solid to obtain a lipolysis mixture of ≥560 units / mL, and the pH was recorded. After the addition of lipase, the pH dropped to 6.90 and then remained stable at 6.90 for 40 minutes. No NaOH was added to neutralize the acid produced by lipolysis. In contrast, for the same DNLF dispersion without orlistat, 35% of the ester bonds were hydrolyzed within 25 minutes. Figure 10 and 11 Graphs of pH versus time and % hydrolysis versus time are shown, respectively. This example demonstrates that lipid nanoparticles comprising insulin, polysorbate 80, sodium lauryl sulfate, sorbitan stearate, soy lecithin, stearic acid, ethyl oleate, isopropyl myristate, medium chain triglyceride oil, and orlistat are not degraded by pancreatic lipase at pH 6.93.

[0304] Example 29. Insulin. Preparation of a non-digestible oral DNLF dispersion with lipid nanoparticles containing insulin, polysorbate 80, PEG 100 stearate, sodium lauryl sulfate, sorbitan stearate, soy lecithin, stearic acid, isopropyl myristate, mineral oil, sodium citrate dihydrate, and water.

[0305] A DNFL dispersion was prepared comprising an oil phase consisting of 485 ppm of insulin as a hydrophobic ion pair with 360 ppm of sodium lauryl sulfate, 11.5% polysorbate 80, 3.3% PEG 100 stearate, 3.9% sorbitan stearate, 0.4% soy lecithin, 7.2% stearic acid, 28.4% isopropyl myristate, and 3.7% light mineral oil, and an aqueous phase consisting of 0.2% sodium citrate dihydrate, 329 ppm of HCl, and 41.4% water. The dispersion was a turbid, transparent, light yellow liquid with a viscosity similar to honey. The volume average particle size, number average particle size, and polydispersity index were 87.3 nm, 63.3 nm, and 0.0834, respectively. This sample had 1.25 mmol of ester bonds per gram.

[0306] At 40 ° C, a pH static experiment was conducted to test the sensitivity of ester bond hydrolysis by lipase using 1.58g DNLF dispersion in 59.68g lipolysis medium. Once the pH value stabilized at 6.71, 0.26g Sigma-Aldrich catalog number L-3126 porcine pancreatic lipase was added as a solid to obtain a lipolysis mixture of ≥540 units / mL, and the pH was recorded. After adding the lipase, the pH rose to 6.74 and then slowly increased to 6.78 within 13 minutes, maintaining this value during the 60-minute experiment. It was not necessary to add NaOH to neutralize the acid produced by lipolysis. This example shows that lipid nanoparticles containing insulin, polysorbate 80, PEG 100 stearate, sodium lauryl sulfate, sorbitan stearate, soy lecithin, stearic acid, isopropyl myristate, and mineral oil were not degraded by pancreatic lipase at pH 6.78.

[0307] Example 30. Cyclosporin A. Preparation of a non-digestible oral DNLF dispersion with cationic lipid nanoparticles comprising cyclosporin A, polysorbate 80, sorbitan stearate, soy lecithin, acetates of mono- and diglycerides, orlistat, dimethyllaurylamine, sodium chloride, and water.

[0308] A DNLF dispersion was prepared consisting of 0.43% cyclosporin A, 12.0% polysorbate 80, 3.1% sorbitan stearate, 2.2% soy lecithin, 4.0% dimethyl laurylamine, 9.1% mono- and diglyceride acetates (Kerry Myvacet 9-45K), 20.3% isopropyl myristate, 0.25% sodium chloride, 0.12% orlistat, and 48.5% water. The dispersion was a cloudy, transparent yellow liquid with a viscosity similar to that of a light syrup. The volume and number average particle sizes and polydispersity index were 59.5 nm, 43.3 nm, and 0.0703, respectively, and the pH was 8.59. This sample had 1.61 mmol of ester bonds per gram.

[0309] A pH static experiment was conducted at 40°C using 1.59 g of DNLF dispersion in 59.42 g of lipolysis medium to assess susceptibility to ester hydrolysis by lipase. Once the pH stabilized at 6.93, 0.28 g of Sigma-Aldrich catalog number L-3126 porcine pancreatic lipase was added as a solid to obtain a lipolysis mixture of ≥560 units / mL, and the pH was recorded. After the addition of lipase, the pH dropped to 6.90 and then remained constant at 6.90 for 40 minutes. No NaOH was added to neutralize the acids produced by lipolysis. Figure 11 Graphs of pH and % hydrolysis versus time are shown.

[0310] The zeta potential of the sample was measured using a Malvern Zetasizer. DNLF dispersion (0.82 g) was dispersed in 17.9 g of 0.1 M potassium dihydrogen phosphate / dipotassium hydrogen phosphate buffer. At pH 6.4, the zeta potential was +42.4.

[0311] This sample illustrates the preparation of a cationic small particle size DNLF dispersion that is stable to degradation by pancreatic lipase.

[0312] aspect

[0313] Aspect 1. A nanoparticle dispersion comprising:

[0314] 0.01% to 20% by weight of one or more biologically active agents;

[0315] 0.1 to 20 wt. % of one or more high hydrophilic-lipophilic balance (HLB) surfactants;

[0316] 0.1 to 20 wt. % of one or more low hydrophilic-lipophilic balance (HLB) surfactants;

[0317] 10% to 45% by weight of one or more water-immiscible oils; and

[0318] 25% to 75% by weight water;

[0319] wherein the bioactive agent is hydrophilic or amphiphilic (eg, the bioactive agent is not hydrophobic, and / or the bioactive agent has a logP of less than 1).

[0320] Aspect 2. A nanoparticle dispersion comprising:

[0321] 0.01% to 20% by weight of one or more biologically active agents;

[0322] 0.1 to 20 wt. % of one or more high hydrophilic-lipophilic balance (HLB) surfactants;

[0323] 0.1 to 20 wt. % of one or more low hydrophilic-lipophilic balance (HLB) surfactants;

[0324] 10% to 45% by weight of one or more water-immiscible oils; and

[0325] 25% to 75% by weight water;

[0326] wherein the lipophilic phase (e.g., a surfactant, a water-immiscible oil, a hydrophobic therapeutic agent, etc.) of the nanoparticle dispersion is resistant to lipolysis (e.g., less than 25% of the ester bonds are hydrolyzed within one hour in the presence of 500 units / mL porcine pancreatic lipase; alternatively, is non-digestible).

[0327] Aspect 3. The nanoparticle dispersion according to aspect 1 or 2, wherein:

[0328] The one or more high hydrophilic-lipophilic balance (HLB) surfactants include ester-type polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactants; and

[0329] The one or more low hydrophilic-lipophilic balance (HLB) surfactants include phospholipid low hydrophilic-lipophilic balance (HLB) surfactants.

[0330] Aspect 4. The nanoparticle dispersion according to any one of aspects 1 to 3, wherein:

[0331] The ester-type polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactant is selected from the group consisting of PEG100 stearate, PEG20 stearate, PEG30 cocoglyceryl, PEG32 stearate, polysorbate 20, and polysorbate 80; and

[0332] The phospholipid low hydrophilic-lipophilic balance (HLB) surfactant is selected from the group consisting of phosphatidylcholine and lecithin.

[0333] Aspect 5. The nanoparticle dispersion according to any one of aspects 1 to 4, wherein:

[0334] The one or more high hydrophilic-lipophilic balance (HLB) surfactants include one or more ether-type polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactants.

[0335] Aspect 6. The nanoparticle dispersion according to aspect 5, further comprising one or more ester-type polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactants (eg, ester-type polyethoxylated surfactants comprising at least 40 ethoxylated groups per molecule).

[0336] Aspect 7. The nanoparticle dispersion according to aspect 6, wherein the weight ratio of the ether-type polyethoxylated high hydrophilic-lipophilic balance HLB surfactant to the ester-type polyethoxylated high hydrophilic-lipophilic balance HLB surfactant is greater than 1:1.

[0337] Aspect 8. The nanoparticle dispersion according to aspect 6, wherein:

[0338] the ether-type polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactant selected from the group consisting of Laureth-23, Laureth-30, Steareth-100, Steareth-20, Steareth-40, Ceteareth-20, and Ceteareth-30; and

[0339] The ester type polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactant is selected from the group consisting of PEG100 stearate, PEG20 stearate, PEG30 glyceryl cocoate, PEG32 stearate and polysorbate 20.

[0340] Aspect 9. The nanoparticle dispersion according to any one of aspects 1 to 4, further comprising one or more non-polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactants.

[0341] Aspect 10. The nanoparticle dispersion of aspect 9, wherein the non-polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactant is sodium lauryl sulfate.

[0342] Aspect 11. The nanoparticle dispersion according to any one of aspects 1 to 10, wherein the bioactive agent is a skin care agent.

[0343] Aspect 12. The nanoparticle dispersion according to any one of aspects 1 to 11, wherein the bioactive agent is a tyrosinase inhibitor.

[0344] Aspect 13. The nanoparticle dispersion according to any one of aspects 1 to 12, wherein the bioactive agent is a peptide.

[0345] Aspect 14. The nanoparticle dispersion according to aspect 13, wherein the peptide has a molar mass in the range of 1 kDa to 1,000 kDa.

[0346] Aspect 15. The nanoparticle dispersion according to aspect 13 or 14, wherein the peptide is a signaling peptide, an AA peptide, a hormone, or a derivative thereof.

[0347] Aspect 16. The nanoparticle dispersion according to aspect 13, wherein the peptide is insulin.

[0348] Aspect 17. The nanoparticle dispersion according to aspect 13, wherein the peptide is bovine serum albumin.

[0349] Aspect 18. The nanoparticle dispersion according to any one of aspects 1 to 12, wherein the bioactive agent is a hydrophilic bioactive agent.

[0350] Aspect 19. The nanoparticle dispersion according to aspect 18, wherein the hydrophilic bioactive agent is a nucleotide or a nucleic acid.

[0351] Aspect 20. The nanoparticle dispersion of aspect 18 or 19, wherein the hydrophilic bioactive agent has a MW of less than 1,000 g / mol.

[0352] Aspect 21. The nanoparticle dispersion of aspect 18, wherein the hydrophilic bioactive agent is selected from the group consisting of spermidine, caffeine, acetaminophen, and nicotinamide.

[0353] Aspect 22. The nanoparticle dispersion according to any one of aspects 1 to 21, comprising a latent lamellar structure, characterized in that the nanoparticle dispersion exhibits a lamellar structure when heated to a temperature in the range of 40°C to 95°C.

[0354] Aspect 23. The nanoparticle dispersion according to any one of aspects 1 to 22, comprising a latent lamellar structure, characterized in that the dispersion exhibits a positive peak between about 45°C and 80°C in a first derivative plot of normalized conductivity versus temperature, the positive peak having a relative humidity greater than about 0.1°C. -1 The peak amplitude of .

[0355] Aspect 24. The nanoparticle dispersion of any one of aspects 1 to 23, comprising a latent lamellar structure, characterized in that when heated to a temperature in the range of 60°C to 95°C, the dispersion exhibits optical birefringence when the dispersion is viewed through crossed polarizers.

[0356] Aspect 25. The nanoparticle dispersion of any one of Aspects 1 to 24, wherein the bioactive agent does not form crystals within the nanoparticle dispersion at a temperature of about 18°C ​​to about 22°C (e.g., for more than about 1 month, more than about 2 months, more than about 3 months, more than about 8 months).

[0357] Aspect 26. The nanoparticle dispersion according to any one of aspects 1 to 25, wherein:

[0358] The nanoparticle dispersion is free of crystals and the concentration of the bioactive agent is greater than the solubility limit in the aqueous phase of the dispersion outside the environment of the nanoparticles; and

[0359] The bioactive agent has a logP of less than 1.

[0360] Aspect 27. The nanoparticle dispersion according to any one of aspects 1 to 26, further comprising a structure-promoting additive.

[0361] Aspect 28. The nanoparticle dispersion according to any one of aspects 1 to 27, wherein the nanoparticle dispersion has a pH less than the isoelectric point of the peptide.

[0362] Aspect 29. The nanoparticle dispersion of any one of aspects 1 to 28, comprising zwitterionic nanoparticles.

[0363] Aspect 30. The nanoparticle dispersion according to any one of aspects 1 to 29, wherein the bioactive agent is present as a component of a hydrophobic ion pair further comprising an anionic surfactant.

[0364] Aspect 31. The nanoparticle dispersion of any one of aspects 1 to 30, wherein the volume average particle size of the dispersion is less than 80 nm (eg, in the range of 30 nm to 80 nm).

[0365] Aspect 32. The nanoparticle dispersion according to any one of aspects 1 to 31, wherein the oleophilic phase comprises greater than 25% by weight of the nanoparticle dispersion.

[0366] Aspect 33. The nanoparticle dispersion of aspect 2, wherein the high HLB surfactant is inert to lipolysis (eg, non-digestible).

[0367] Aspect 34. The nanoparticle dispersion of aspect 33, wherein the high HLB surfactant lacks an ester group.

[0368] Aspect 35. The nanoparticle dispersion according to aspect 32 or 33, wherein the high HLB surfactant is an ether-type high HLB polyethoxylated surfactant.

[0369] Aspect 36. The nanoparticle dispersion according to any one of aspects 1 to 35, further comprising a lipase inhibitor.

[0370] Aspect 37. The nanoparticle dispersion of aspect 36, wherein the lipase inhibitor comprises orlistat.

[0371] Aspect 38. The nanoparticle dispersion of any one of aspects 1 to 37, wherein the nanoparticles within the nanoparticle dispersion have a net positive charge.

[0372] Aspect 39. The nanoparticle dispersion of any one of aspects 1 to 38, wherein the nanoparticles within the nanoparticle dispersion comprise a zeta potential greater than 1.0 millivolt.

[0373] Aspect 40. The nanoparticle dispersion of any one of aspects 2 and 33 to 39, wherein oral administration of the nanoparticle dispersion to Jackson Labs C57BL / 6J mice produces a peak plasma concentration less than one hour after administration.

[0374] Aspect 41. The nanoparticle dispersion of aspect 40, wherein in a graph of plasma concentration versus time, d(plasma concentration) / dt is a negative value at each time greater than one hour (e.g., 1 to 24 hours) after administration to Jackson Labs C57BL / 6J mice by oral gavage.

[0375] Aspect 42. The nanoparticle dispersion of any one of aspects 1 to 41, wherein after 60 minutes in a lipolysis solution containing calcium, one or more bile salts, and 0.4% lipase at a pH of about 6.8 containing 2.6% DNLF dispersion, more than 80% by volume of the particles in the DNLF have a diameter of less than 100 nm.

[0376] Aspect 43. The nanoparticle dispersion of any one of aspects 1 to 42, wherein after more than 30% of the lipid nanoparticle ester bonds are hydrolyzed by lipase, more than 80% of the particles by volume have a diameter less than 100 nm.

[0377] Aspect 44. The nanoparticle dispersion of any one of aspects 1 to 43, wherein less than 10% of the lipid ester bonds are lipolyzed after 60 minutes in a lipolysis solution containing calcium, one or more bile salts, and 0.4% lipase at a pH of about 6.8.

[0378] Aspect 45. The nanoparticle dispersion according to any one of aspects 1 to 44, wherein the rate of drug absorption in the gastrointestinal tract is greater than the rate of drug release from the lipid phase of the nanoparticle dispersion.

[0379] Aspect 46. The nanoparticle dispersion according to any one of aspects 1 to 45, wherein in the gastrointestinal tract, the rate of drug absorption is greater than the rate of hydrolysis of ester bonds in the lipid phase of the nanoparticle dispersion.

[0380] Aspect 47. A capsule comprising the nanoparticle dispersion according to any one of aspects 1 to 46.

[0381] Aspect 48. The capsule of aspect 47, comprising an encapsulating polymer surrounding the nanoparticle dispersion.

[0382] Aspect 49. The capsule of aspect 48, wherein the encapsulating polymer comprises a carboxylic acid.

[0383] Aspect 50. The capsule of aspect 48 or 49, wherein the pH of the nanoparticle dispersion is less than the pKa of the carboxylic acid groups of the encapsulating polymer (eg, at least one pH unit less).

[0384] Aspect 51. The capsule of any one of aspects 48 to 50, wherein the encapsulating polymer comprises a plurality of acrylate monomer units, a plurality of methacrylate monomer units, a plurality of methacrylate monomer units, a plurality of vinyl acetate monomer units, a plurality of acrylic acid monomer units, a plurality of methacrylic acid monomer units, a plurality of vinyl 4-hydroxyphthalate monomer units, a vinyl polymer, a modified cellulose, or a combination thereof.

[0385] Aspect 52. The capsule according to any one of aspects 48 to 51, wherein the encapsulating polymer comprises a modified cellulose comprising cellulose esterified with acetic acid and / or phthalic acid, or cellulose esterified with acetic acid and / or succinic acid.

[0386] Aspect 53. The capsule according to any one of aspects 48 to 52, wherein the encapsulating polymer is selected from the group consisting of methyl acrylate-methacrylic acid copolymer, methyl methacrylate-methacrylic acid copolymer, ethyl acrylate-methacrylic acid copolymer, vinyl acetate-4-hydroxyvinyl phthalate copolymer, hydroxypropyl methylcellulose acetate succinate, cellulose acetate phthalate, Eudragit L 30D-55, or a combination thereof;

[0387] Aspect 54. The capsule of any one of aspects 48 to 53, wherein the capsule further comprises a surface layer located external to the encapsulating polymer.

[0388] Aspect 55. The capsule of aspects 48 to 54, wherein the surface layer comprises carnauba wax, polydimethylsiloxane, organomodified silicone, or any combination thereof.

[0389] Aspect 56. The capsule of any one of aspects 48 to 55, wherein the capsule further comprises an intermediate layer located between the encapsulating polymer and the surface layer.

[0390] Aspect 57. The capsule of aspect 56, wherein the capsule comprises an innermost layer composed of the encapsulating polymer, a middle layer comprising gelatin, and a surface layer comprising carnauba wax.

[0391] Aspect 58. The capsule according to any one of aspects 47 to 57, wherein the capsule is not digested by gastric acid (eg, HCl) having a pH of less than 2 within 30 minutes.

[0392] Aspect 59. A method for treating a condition selected from the group consisting of type 1 diabetes and type 2 diabetes, the method comprising: orally administering insulin, the method comprising orally administering to a patient in need thereof:

[0393] The nanoparticle dispersion according to any one of aspects 1 to 46; or

[0394] The capsule according to any one of aspects 47 to 58.

[0395] Aspect 60. The method of aspect 59, wherein oral administration of the nanoparticle dispersion produces a peak plasma concentration in the patient in less than one hour (or 10 minutes to 1 hour, or less than 30 minutes, or less than 15 minutes, or 5 to 30 minutes) after administration of the nanoparticles or capsules to the patient.

[0396] Aspect 61. The method of aspect 59 or 60, wherein in a graph of plasma concentration versus time, d(plasma concentration) / dt is a negative value at each time point more than one hour (e.g., 1 to 24 hours) after administration of the nanoparticle or capsule.

Claims

1. A nanoparticle dispersion comprising: 0.01% to 20% by weight of one or more biologically active agents; 0.1 to 20 wt. % of one or more high hydrophilic-lipophilic balance (HLB) surfactants; 0.1 to 20 wt. % of one or more low hydrophilic-lipophilic balance (HLB) surfactants; 10% to 45% by weight of one or more water-immiscible oils; and 25% to 75% by weight water; The bioactive agent is hydrophilic or amphiphilic.

2. The nanoparticle dispersion according to claim 1, wherein: The one or more high hydrophilic-lipophilic balance (HLB) surfactants include ester-type polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactants; and The one or more low hydrophilic-lipophilic balance (HLB) surfactants include phospholipid low hydrophilic-lipophilic balance (HLB) surfactants.

3. The nanoparticle dispersion according to claim 1, wherein: The ester-type polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactant is selected from the group consisting of PEG100 stearate, PEG20 stearate, PEG30 cocoglyceryl, PEG32 stearate, polysorbate 20, and polysorbate 80; and The phospholipid low hydrophilic-lipophilic balance (HLB) surfactant is selected from the group consisting of phosphatidylcholine and lecithin.

4. The nanoparticle dispersion according to claim 1, wherein: The one or more high hydrophilic-lipophilic balance (HLB) surfactants include one or more ether-type polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactants.

5. The nanoparticle dispersion according to claim 4, further comprising one or more ester-type polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactants. 6 . The nanoparticle dispersion according to claim 5 , wherein the weight ratio of the ether-type polyethoxylated high hydrophilic-lipophilic balance HLB surfactant to the ester-type polyethoxylated high hydrophilic-lipophilic balance HLB surfactant is greater than 1:

1.

7. The nanoparticle dispersion according to claim 5, wherein: The ether-type polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactant is selected from the group consisting of Laureth-23, Laureth-30, Steareth-100, Steareth-20, Steareth-40, Ceteareth-20, and Ceteareth-30; and The ester type polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactant is selected from the group consisting of PEG100 stearate, PEG20 stearate, PEG30 glyceryl cocoate, PEG32 stearate and polysorbate 20.

8. The nanoparticle dispersion of claim 4, further comprising one or more non-polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactants.

9. The nanoparticle dispersion of claim 8, wherein the non-polyethoxylated high hydrophilic-lipophilic balance (HLB) surfactant is sodium lauryl sulfate.

10. The nanoparticle dispersion of claim 1, wherein the hydrophilic bioactive agent is selected from the group consisting of spermidine, caffeine, acetaminophen, and niacinamide.

11. The nanoparticle dispersion of claim 1, wherein the bioactive agent is a peptide.

12. The nanoparticle dispersion of claim 11, wherein the peptide is insulin.

13. The nanoparticle dispersion of claim 11, wherein the peptide is bovine serum albumin.

14. The nanoparticle dispersion of claim 1 comprising a latent lamellar structure.

15. The nanoparticle dispersion of claim 1, wherein the bioactive agent does not form crystals within the nanoparticle dispersion at a temperature of about 18°C ​​to about 22°C for more than about 8 months.

16. The nanoparticle dispersion of claim 1, wherein the nanoparticle dispersion has a pH less than the isoelectric point of the peptide.

17. An anti-lipolytic nanoparticle dispersion comprising: 0.01% to 20% by weight of one or more biologically active agents; 0.1 to 20 wt. % of one or more high hydrophilic-lipophilic balance (HLB) surfactants; 0.1 to 20 wt. % of one or more low hydrophilic-lipophilic balance (HLB) surfactants; 10% to 45% by weight of one or more water-immiscible oils; and 25% to 75% by weight water; in: The one or more high HLB polyethoxylated surfactants consist of ether-type high HLB polyethoxylated surfactants; The nanoparticle dispersion further comprises a lipase inhibitor; The nanoparticle dispersion comprises a zeta potential greater than 1.0 millivolt; or any combination thereof.

18. A capsule comprising: The nanoparticle dispersion according to claim 1 or the nanoparticle dispersion according to claim 17, an encapsulating polymer comprising a carboxylic acid and selected from the group consisting of methyl acrylate-methacrylic acid copolymer, methyl methacrylate-methacrylic acid copolymer, ethyl acrylate-methacrylic acid copolymer, vinyl acetate-4-hydroxyvinyl phthalate copolymer, hydroxypropyl methylcellulose acetate succinate, cellulose acetate phthalate, Eudragit L 30D-55, or a combination thereof; Optionally, a surface layer located external to the encapsulating polymer, the surface layer comprising carnauba wax, polydimethylsiloxane, organomodified silicone, or any combination thereof; and Optionally, an intermediate layer located between the encapsulating polymer and the surface layer; in: The pH of the nanoparticle dispersion is less than the pKa of the carboxylic acid groups of the encapsulating polymer; and The capsules remained undigested after 30 minutes in an aqueous mixture with a pH of less than 2.

19. The capsule of claim 18, wherein the capsule comprises an innermost layer composed of the encapsulating polymer, a middle layer comprising gelatin, and a surface layer comprising carnauba wax.

20. A method for treating a condition selected from the group consisting of type 1 diabetes and type 2 diabetes, the method comprising orally administering to a patient in need thereof: The nanoparticle dispersion according to claim 1; The nanoparticle dispersion according to claim 17; or The capsule according to claim 18; in: The time to peak plasma concentration (Tmax) in the patient is in the range of 2 minutes to 1 hour; and In a plot of plasma concentration versus time at each time point in the range of 1 hour to 24 hours after oral administration, d(plasma concentration) / dt is a negative value.

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