WO2020257260A1 - Formulation de liposomes chargés de peptides et applications associées - Google Patents

Formulation de liposomes chargés de peptides et applications associées Download PDF

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Publication number
WO2020257260A1
WO2020257260A1 PCT/US2020/038101 US2020038101W WO2020257260A1 WO 2020257260 A1 WO2020257260 A1 WO 2020257260A1 US 2020038101 W US2020038101 W US 2020038101W WO 2020257260 A1 WO2020257260 A1 WO 2020257260A1
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liposome
mol
glycero
amount
palmitoyl
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PCT/US2020/038101
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English (en)
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Paula T. Hammond Cunningham
Betty GOURDON
Caroline CHEMIN
Jean-Manuel Pean
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Massachusetts Institute Of Technology
Les Laboratoires Servier
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Publication of WO2020257260A1 publication Critical patent/WO2020257260A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid

Definitions

  • the present invention is an anionic liposome, comprising: at least a first neutral lipid; at least one anionic lipid; and an active pharmaceutical ingredient (API), wherein: the API is encapsulated in the liposome; and the API comprises a polypeptide covalently attached to a fatty acid chain, optionally via a linker.
  • API active pharmaceutical ingredient
  • the present invention is a pharmaceutical composition, comprising any liposome described herein with respect to the first embodiment and various aspects thereof and a pharmaceutically acceptable carrier.
  • the present invention is a method of treating a condition, comprising: orally administering to a subject in need thereof an anionic liposome, wherein the liposome comprises: at least a first neutral lipid; at least one anionic lipid; and an active pharmaceutical ingredient (API), wherein: the API is encapsulated in the liposome; and the API comprises a polypeptide covalently attached to a fatty acid chain, optionally via a linker.
  • an anionic liposome wherein the liposome comprises: at least a first neutral lipid; at least one anionic lipid; and an active pharmaceutical ingredient (API), wherein: the API is encapsulated in the liposome; and the API comprises a polypeptide covalently attached to a fatty acid chain, optionally via a linker.
  • Figure 1 shows a schematic representation of the Biopharmaceuticals
  • Figure 2 shows a schematic representation of the gastrointestinal tract, demonstrating the modes of transport across the intestinal membrane.
  • FIG. 3 shows a schematic representation of the method of assembly of Layer-by-Layer (LbL) particle containing an anionic liposome core loaded with a peptide.
  • LbL Layer-by-Layer
  • Figure 4 shows the chemical structure and properties of liraglutide.
  • Figure 5 shows a flow chart representing the steps of an anionic liposome synthesis.
  • Figure 6 shows steps of liraglutide encapsulation and liposome formation, as well as tables listing drug loading, encapsulation efficiency, size, polydispersity and liraglutide concentration of the obtained liposomes prior to extrusion.
  • Figure 7 shows chemical structures of poly-L-lysine (PLK) and Valine (Val), and the coupling procedure between PLK and Val resulting in the formation of conjugated PLK-Val.
  • Figure 8 shows a schematic description of the four steps involved in the synthesis of LbL particles with a peptide-loaded liposome core.
  • Figure 9 shows a table listing size, Zeta potential, and polydispersity of free and liraglutide-loaded liposomes, free and liraglutide-loaded liposomes coated with PLK- Val, and free and liraglutide-loaded liposomes coated with PLR/DXS/PLK-Val.
  • Figure 9 also shows a table listing drug loading (DL) and encapsulation efficiency (EE) of liraglutide-loaded liposomes, liraglutide-loaded liposomes coated with PLK-Val, and liraglutide-loaded liposomes coated with PLR/DXS/PLK-Val.
  • DL drug loading
  • EE encapsulation efficiency
  • Figure 10 shows a chart demonstrating comparative rates of liraglutide release from liraglutide-loaded liposomes, liraglutide-loaded liposomes coated with PLK-Val, and liraglutide-loaded liposomes coated with PLR/DXS/PLK-Val.
  • Figure 11 shows TEM images of liraglutide-loaded liposomes and liraglutide- loaded liposomes coated with PLR/DXS/PLK-Val.
  • Figure 12 shows a chart demonstrating quantitative cellular uptake of liposomes, liposomes coated with PLK-Val, and liposomes coated with PLR/DXS/PLK- Val in Caco-2 cells (top); and competitive cellular uptake of liposomes or liposomes coated with PLR/DXS/PLK-Val in the presence of various concentrations of
  • Figure 13 shows confocal microscopy images of Caco-2 cells after incubation with liposomes, liposomes coated with PLK-Val, and liposomes coated with
  • Figure 14 shows bar graphs demonstrating toxicity of liraglutide, liposomes, liposomes coated with PLK-Val, and liposomes coated with PLR/DXS/PLK-Val in Caco- 2 cells.
  • Figure 15 shows a table listing size, Zeta potential, and polydispersity of liposomes and liposomes coated with PLR/DXS/PLK-Val, before and after
  • Figure 16 shows a bar graph demonstrating targeting Caco-2 cells by liposomes coated with PLR/DXS/PLK-Val before and after lyophilization; and bar graphs demonstrating toxicity of liposomes and liposomes coated with PLR/DXS/PLK-Val, in Caco-2 cells before and after lyophilization of the liposomes.
  • Figure 17 shows TEM images of free lyophilized liposomes and free lyophilized liposomes coated with PLR/DXS/PLK-Val.
  • Figure 18 shows a scheme demonstrating the setup of the peptide transport experiment; and a bar graph demonstrating apparent permeability of Caco-2 cells to liraglutide delivered as free peptide, or liraglutide encapsulated in liposomes, liposomes coated with PLK-Val, or liposomes coated with PLR/DXS/PLK-Val.
  • Figure 20 shows a bar graph demonstrating area under the curve (AUC) of blood glucose concentrations obtained from Figure 19.
  • Figure 22 shows a bar graph demonstrating AUC of blood glucose level reductions obtained from Figure 21.
  • Figure 24 shows a bar graph demonstrating AUC of blood glucose
  • Figure 25 shows a graph demonstrating blood glucose level reduction (%) as a function of time (h), after single oral administration of 2 mg/kg free liraglutide, liraglutide encapsulated in liposomes, and liraglutide encapsulated in liposomes coated with
  • Figure 26 shows a graph demonstrating AUC of blood glucose level reductions obtained from Figure 25.
  • Figure 28 shows a graph demonstrating blood glucose level reduction (%) as a function of time (h), after single oral administration of 0.02 mg/kg free liraglutide intravenously (TV), 0.2 mg/kg free liraglutide subcutaneously (SC), 2.5 mg/kg free liraglutide orally (PO), 2.5 mg/kg of liraglutide encapsulated in liposomes orally, and placebo liposomes orally as a control, to female 8 weeks old db/db mice.
  • Figure 29 shows a bar graph demonstrating AUC of blood glucose level reductions obtained from Figure 28.
  • One approach in solving the low bioavailability of the peptide- and protein- based therapeutic agents involves encapsulating the agents in liposomes, protecting them from enzymatic and acidic degradation, while enhancing transport through the intestinal membrane.
  • Liposomes are vesicles composed of concentric lipid bilayers which surround an inner lumen containing an aqeous solution.
  • a solute can be encapsulated in the liposome either within the inner lumen (polar solutes) or embedded in the lipid bilayers (lipophilic or amphiphilic solutes).
  • Liposomes are composed of lipid molecules, such as (phospho)lipid molecules, comprising a hydrophilic head group and a hydrophobic tail.
  • the lipid molecules assemble in an aqueous solu tion such that the hydrophobic parts get oriented toward each other to avoid contact with the aqueous phase, whereas the hydrophilic head groups are oriented such that they make maximal contact with the aqueous surrounding. This leads to spontaneous self-assembly into spherical structures that contain an inner aqueous compartment (lumen) surrounded by a lipid bilayer.
  • Liposomes thus contain an outer surface which is oriented towards the aqueous solution surrounding the liposomes and an inner surface lining the inner aqueous compartment.
  • the term“outer surface' of the liposome as used herein thus is meant to refer to the surface of the liposome which is oriented toward the aqueous phase surrounding the liposome.
  • liposomes and their subsequent applicability depend on the physical and physico-chemical characteristics of the liposomal membrane.
  • a neutral lipid is used as the basic lipid for the preparation of liposomes.
  • the net surface charge of liposomes can be modified by the incorporation of positively charged lipids (providing cationic liposomes), or negatively charged, such as phosphatidyl glycerols, phosphatidyl serines, or phosphatidic acids lipids (providing anionic liposomes).
  • Characteristics of a liposome loaded with a polypeptide also depend on the physico-chemical properties of the polypeptide.
  • Present disclosure demonstrates that introduction of a non-polar group, such as a fatty acid chain, into the structure of the polypeptide provides a liposome with a beneficial drug delivery profile.
  • the fatty acid chain attached to the polypeptide improves the embedment of the functionalized polypeptide into the liposomes.
  • polypeptide with a fatty acid chain can achieve higher drug loading in the liposomes compared to the
  • the fatty acid chain interacts with the lipids of the liposome bilayer, increasing encapsulation of the polypeptide not only in the core but also in the bilayer.
  • an anionic liposome is a liposome with a negative surface charge.
  • the surface charge of the liposome is evaluated through the measurement of Zeta potential.
  • Zeta potential of an anionic liposome is more negative than -20 mV, which corresponds to stable suspensions of anionic liposomes.
  • Anionic liposomes are capable of encapsulating of polypeptides that are soluble in solutions with a particular range of pH values. Additionally, anionic liposomes can facilitate transport of the liposome-contained cargo across the intestinal membrane.
  • Anionic liposomes can be prepared according to known methods, as disclosed, for example, in U.S. Patent Application Publication Nos. 2017/0056555, 2016/0228573, 2015/0284691, and 2003/0026831, each of which is incorporated herein by reference in its entirety.
  • Liraglutide is GLP-1 agonist used in the treatment of diabetes mellitus, and is administered once daily subcutaneously. Liraglutide is formed by adding a 16-carbon fatty acid at position 26 and replacing lysine 34 with arginine on GLP-1 ( Figure 4). As it is one of the latest and the most advanced drug for treating diabetes, liraglutide not only corrects the glucose metabolism disorder but also reduces the most common
  • diabetes is a chronic disease and even once-daily injection is still quite frequent for patients requiring life-long treatment.
  • development of a sustained-release drug-delivery system is needed for liraglutide.
  • the present disclosure relates to liraglutide-loaded liposomes formulated for oral administration.
  • Liraglutide-loaded liposomes provide the same level of blood glucose reduction when administered orally to diabetic mice as when liraglutide is administered subcutaneously.
  • Figures 27, 28 and 29 are demonstrate a similar decrease of blood glucose concentration after subcutaneous administration of 0.2 mg/kg of free liraglutide administered and oral administration of 2.5 mg/kg of liraglutide incapsulated in bare liposomes.
  • Formulations of peptide loaded layer-by-layer nanoparticles targeting intestinal transporters, such as PepTl, are provided herein, as well as methods of using and manufacturing such nanoparticles and populations of nanoparticles. These formulations and methods are useful for oral delivery of therapeutics, such as peptide- and/or protein-based therapeutics.
  • the disclosure provides formulations and methods for oral peptide delivery, namely a formulation that comprises peptide loaded layer-by-layer nanoparticles that target PepTl intestinal transporters.
  • the formulations provided herein include one or more peptide-loaded layer-by-layer nanoparticles that targets PepTl, wherein at least one nanoparticle comprises at least an initial layer and a second layer.
  • at least one nanoparticle includes a valine functionalization to target PepTl.
  • at least one nanoparticle comprises at least an initial layer, a second layer, and a third layer.
  • the initial layer comprises a negative liposome layer.
  • the initial layer comprises a negative liposome layer
  • the second layer comprises a polycation layer
  • the third layer comprises a polyanion layer.
  • the formulation includes a population of nanoparticles comprising two or more nanoparticles according to any one of the previous claims, wherein the initial layer comprises a negative liposome layer.
  • the disclosure also provides methods of using these formulations for oral delivery of a therapeutic.
  • the therapeutic is a peptide-based therapeutic, a protein- based-therapeutic, or a combination thereof.
  • the disclosure also provides methods of treating a disease or disorder in a subject by administering to the subject a therapeutically effective amount of a formulation described herein.
  • the nanoparticle or population of nanoparticles is administered orally to the subject.
  • the disclosure also provides methods of manufacturing the nanoparticle and/or the population of nanoparticles described herein using layer-by-layer assembly of the nanoparticle(s).
  • nanoparticles targeting intestinal transporters such as PepTl
  • methods of using and manufacturing such nanoparticles and populations of nanoparticles are also possible.
  • formulations and methods are useful for oral delivery of therapeutics, such as peptide- and/or protein-based therapeutics.
  • the disclosure provides formulations and methods for oral peptide delivery, namely a formulation that comprises peptide loaded layer-by-layer nanoparticles that target PepTl intestinal transporters.
  • Peptide- and protein-based therapeutics often have delivery challenges that have limited their clinical use. Although oral delivery is preferred, conventional formulation strategies cannot be applied due to limited oral bioavailability of peptide-and protein-based therapeutics. The low bioavailability of these compounds is directly linked to their poor ability to reach the systemic blood circulation because of limitations to cross the intestinal epithelium, degradation in the gastrointestinal tract and their large molecular size.
  • compositions and methods provided herein employ a current strategy to improve oral bioavailability of formulations by targeting intestinal transporters, such as PepTl.
  • the PepTl transporter is a desirable target for a number of reasons: (1) PepTl promotes the uptake of dipeptides, tripeptides, and peptidomimetics due to an inward proton gradient; and (2) PepTl has broad substrate specificity, which makes it an attractive transporter to target.
  • compositions and methods provided herein combine (i) a modified drug having a low oral bioavailability, where the modified drug is functionalized with a peptide-like ligand, thereby allowing the modified drug to be transported by PepTl into the bloodstream; and
  • a candidate peptide usually administered subcutaneously for the treatment of type 2 diabetes, was first encapsulated into negatively charged liposomes. These nanoparticles were then layered successively with three polyelectrolytes as shown in Figure 1 : a positively charged polymer selected for its controlled release properties, then a negatively charged polymer, and, finally, an outer positively charged polymer functionalized with valine in order to target intestinal transporter PepTl .
  • PepTl intestinal transporters
  • This transporter is well known to promote the uptake of dipeptides, tripeptides and peptidomimetics due to an inward proton gradient. Its broad substrate specificity makes it an attractive transporter to target.
  • modifying a drug having a low oral bioavailability with a peptide-like ligand it allows its intestinal transport by PepTl into the bloodstream.
  • Valacyclovir improved the oral bioavailability of the antiviral Acyclovir by 3- to 5- fold in humans
  • PKA-PEG valine functionalized polylactic acid-polyethylene glycol
  • NPs valine functionalized polylactic acid-polyethylene glycol
  • LbL assembly provides functional NPs with the ability to control peptide release while targeting intestinal PepTl transporter.
  • a candidate peptide usually administered subcutaneously for the treatment of type 2 diabetes, was first encapsulated into negatively charged liposomes. These NPs were then layered successively with three polyelectrolytes: a positively charged polymer selected for its controlled release properties, then a negatively charged polymer, and, finally, an outer positively charged polymer functionalized with valine in order to target intestinal transporter PepTl. NP synthesis is set up so as to get particles under 200 nm in diameter allowing them to cross the intestinal mucus barrier (L.M. Ensign et al., Oral drug delivery with polymeric nanoparticles: the gastrointestinal mucus barriers, Adv. Drug Deliv. Rev. 64 (2012) 557-570), and the surface charge was monitored at each layering step to maintain zeta potential >
  • NP uptake and intracellular location were assessed qualitatively and quantitatively in a Caco-2 cell culture model.
  • Peptide release was optimized such that 30 % of drug was released over 2 hours, allowing time for NPs to engage with PepTl and thus be processed by intestinal epithelial cells.
  • Drug transport experiments will evaluate peptide transport across a model of the intestinal membrane, and non-LbL particles and free compound will be compared, revealing the differential effects of LbL components and PepTl targeting on apparent permeability.
  • Oral administration of peptide- and protein-based therapeutics remains a challenging issue. Bringing together strategies of tissue-specific targeting and Layer-by- Layer assembly allows for the production of the formulations provided herein. These formulations can be used to identify candidates with translational potential.
  • the present invention is a liquid crystal [0068] in various embodiments.
  • a peptide-loaded layer-by-layer nanoparticle that targets PepT 1 wherein the nanoparticle comprises at least an initial layer and a second layer.
  • nanoparticle of claim 1 further comprising a valine functionalization to target PepTl.
  • nanoparticle of claim 1 or claim 2 wherein the nanoparticle comprises at least an initial layer, a second layer, and a third layer.
  • a population of nanoparticles comprising two or more nanoparticles according to any one of the previous claims, wherein the initial layer comprises a negative liposome layer.
  • a formulation comprising the nanoparticle of any one of claims 1 to 5 or the population of nanoparticles of claim 6.
  • the therapeutic is a peptide-based therapeutic, a protein- based-therapeutic, or a combination thereof.
  • a method of treating a disease or disorder in a subject comprising administering to the subject a therapeutically effective amount of the formulation of claim
  • nanoparticle or population of nanoparticles is administered orally to the subject.
  • the process for making a pharmaceutical composition comprising mixing one or more of the present liposomes and an optional
  • compositions resulting from such a process which process includes conventional pharmaceutical techniques.
  • Liposomes can be lyophilized prior to formulation of the pharmaceutical composition. Processes for lyophilization of liposomes are described, for example, in Chen et al., An overview of liposome lyophilization and its future potential, J. Cont. Release, 142(3): 299-311 (2010); Franze et al., Lyophilization of liposomal formulations: still necessary, still challenging, Pharmaceutics, 10(3): 139 (2016); and Arshinova et al., Pharm Chem J, Lyophilization of liposomal drug forms, 46: 228-233(2012).
  • compositions of the invention include ocular, oral, nasal, transdermal, topical with or without occlusion, intravenous (both bolus and infusion), inhalable, and injection (intraperitoneally, subcutaneously, intramuscularly, intratumorally, or parenterally) formulations.
  • the composition may be in a dosage unit such as a tablet, pill, capsule, powder, granule, liposome, ion exchange resin, sterile ocular solution, or ocular delivery device (such as a contact lens and the like facilitating immediate release, timed release, or sustained release), parenteral solution or suspension, metered aerosol or liquid spray, drop, ampoule, auto injector device, or suppository; for administration ocularly, orally, intranasally, sublingually, parenterally, or rectally, or by inhalation or insufflation.
  • a dosage unit such as a tablet, pill, capsule, powder, granule, liposome, ion exchange resin, sterile ocular solution, or ocular delivery device (such as a contact lens and the like facilitating immediate release, timed release, or sustained release), parenteral solution or suspension, metered aerosol or liquid spray, drop, ampoule, auto injector device, or suppository; for administration ocularly,
  • compositions of the invention suitable for oral administration include solid forms such as pills, tablets, caplets, capsules (each including immediate release, timed release, and sustained release formulations), granules and powders; and, liquid forms such as solutions, syrups, elixirs, emulsions, and suspensions.
  • forms useful for ocular administration include sterile solutions or ocular delivery devices.
  • forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.
  • the dosage form containing the composition of the invention contains an effective amount of the active ingredient necessary to provide a therapeutic effect.
  • the composition may be administered about 1 to about 5 times per day. Daily administration or post periodic dosing may be employed.
  • the composition is preferably in the form of a tablet or capsule containing, e.g., 500 to 0.5 milligrams of the active compound. Dosages will vary depending on factors associated with the particular patient being treated (e.g., age, weight, diet, and time of administration), the severity of the condition being treated, the compound being employed, the mode of administration, and the strength of the preparation.
  • the oral composition is preferably formulated as a homogeneous composition, wherein the liposome is dispersed evenly throughout the mixture, which may be readily subdivided into dosage units containing equal amounts of a liposome of the invention.
  • the compositions are prepared by mixing a liposome of the with one or more optionally present pharmaceutical carriers (such as a starch, sugar, diluent, granulating agent, lubricant, glidant, binding agent, and disintegrating agent), one or more optionally present inert pharmaceutical excipients (such as water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and syrup), one or more optionally present conventional tableting ingredients (such as com starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate, and any of a variety of gums), and an optional diluent (such as water).
  • pharmaceutical carriers such as a starch,
  • Binder agents include starch, gelatin, natural sugars (e.g., glucose and beta lactose), com sweeteners and natural and synthetic gums (e.g., acacia and tragacanth).
  • Disintegrating agents include starch, methyl cellulose, agar, and bentonite.
  • Tablets and capsules represent an advantageous oral dosage unit form. Tablets may be sugarcoated or filmcoated using standard techniques. Tablets may also be coated or otherwise compounded to provide a prolonged, control release therapeutic effect.
  • the dosage form may comprise an inner dosage and an outer dosage component, wherein the outer component is in the form of an envelope over the inner component.
  • the two components may further be separated by a layer which resists disintegration in the stomach (such as an enteric layer) and permits the inner component to pass intact into the duodenum or a layer which delays or sustains release.
  • enteric and nonenteric layer or coating materials such as polymeric acids, shellacs, acetyl alcohol, and cellulose acetate or combinations thereof may be used.
  • the liposome disclosed herein may be incorporated for administration orally or by injection in a liquid form such as aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil or peanut oil and the like, or in elixirs or similar pharmaceutical vehicles.
  • Suitable dispersing or suspending agents for aqueous suspensions include synthetic and natural gums such as tragacanth, acacia, alginate, dextran, sodium carboxymethylcellulose, methylcellulose, polyvinyl pyrrolidone, and gelatin.
  • the liquid forms in suitably flavored suspending or dispersing agents may also include synthetic and natural gums.
  • sterile suspensions and solutions are desired. Isotonic preparations, which generally contain suitable preservatives, are employed when intravenous administration is desired.
  • the liposomes may be administered parenterally via injection.
  • a parenteral formulation may consist of the active ingredient dissolved in or mixed with an
  • Acceptable liquid carriers usually comprise aqueous solvents and other optional ingredients for aiding solubility or preservation.
  • aqueous solvents include sterile water, Ringer's solution, or an isotonic aqueous saline solution.
  • Other optional ingredients include vegetable oils (such as peanut oil, cottonseed oil, and sesame oil), and organic solvents (such as solketal, glycerol, and formyl).
  • a sterile, non-volatile oil may be employed as a solvent or suspending agent.
  • the parenteral formulation is prepared by dissolving or suspending the active ingredient in the liquid carrier whereby the final dosage unit contains from 0.005 to 10% by weight of the active ingredient.
  • Other additives include preservatives, isotonizers, solubilizers, stabilizers, and pain soothing agents.
  • Injectable suspensions may also be prepared, in which case appropriate liquid carriers, suspending agents and the like may be employed.
  • Liposomes of the invention may be administered intranasally using a suitable intranasal vehicle.
  • Liposomes of the invention may also be administered topically or enhanced by using a suitable topical transdermal vehicle or a transdermal patch.
  • the composition is preferably in the form of an ophthalmic composition.
  • the ophthalmic compositions are preferably formulated as eye drop formulations and filled in appropriate containers to facilitate administration to the eye, for example a dropper fitted with a suitable pipette.
  • the compositions are sterile and aqueous based, using purified water.
  • an ophthalmic composition may contain one or more of: a) a surfactant such as a polyoxyethylene fatty acid ester; b) a thickening agents such as cellulose, cellulose derivatives, carboxyvinyl polymers, polyvinyl polymers, and polyvinylpyrrolidones, typically at a concentration in the range of about 0.05 to about 5.0% (wt/vol); c) (as an alternative to or in addition to storing the composition in a container containing nitrogen and optionally including a free oxygen absorber such as Fe), an anti-oxidant such as butylated hydroxy anisol, ascorbic acid, sodium thiosulfate, or butylated hydroxytoluene at a concentration of about 0.00005 to about 0.1% (wt/vol); d) ethanol at a concentration of about 0.01 to 0.5% (wt/vol); and e) other excipients such as an isotonic agent, buffer, pre
  • polypeptide refers to a linear polymer consisting of amino acid residues.
  • a polypeptide containing from 2 to 50 amino acid residues is also referred to as peptide, while a polypeptide containing over 100 amino acid residues is also referred to as protein.
  • the term polypeptide includes, for example, natural proteins, synthetic or recombinant polypeptides and peptides, epitopes, hybrid molecules, variants, homologs, analogs, peptoids, peptidomimetics, etc.
  • oral polypeptide refers to a polypeptide that can be efficiently delivered to a subject via oral administration, providing oral bioavailability of at least 2%, 5%, 10%, or 30%. Additionally,“oral polypeptide” can refer to a formulation comprising a polypeptide, wherein the formulation can be efficiently delivered to a subject via oral administration. Oral polypeptide exhibits improved bioavailability compared to the corresponding additive-free unmodified polypeptide (original polypeptide) due to higher acid stability, higher stability against enzymatic degradation, and higher intestinal membrane and basal membrane permeability.
  • oral polypeptide can be produced from the original polypeptide through chemical modification, use of appropriate formulation vehicles, or addition of enzyme inhibitors, absorption enhancers, and mucoadhesive polymers.
  • oral polypeptide can comprise the original polypeptide which has been chemically modified to improve encapsulation and stability of the polypeptide in the formulation vehicle.
  • oral polypeptide can be formulated to comprise agents that target intestinal transporters, thus increasing transport of the polypeptide from the small intestine lumen into the blood stream.
  • Orally available peptides, polypeptides, and proteins are disclosed, for example, in U.S. Patent Nos. 6,951,655, 8,088,734, 8,148,328, and 8,377,863, and U.S. Patent Application Publications 2013/0034597 and 2017/0304195, each of which is incorporated herein by reference in its entirety.
  • fatty acid chain refers to a monovalent radical obtained by removing the carboxylic group (COOH) from a fatty acid.
  • a fatty acid is a carboxylic acid with a straight or branched aliphatic chain, which is either saturated or unsaturated.
  • a fatty acid can be unsaturated, or it can have 1, 2, 3, 4, or more carbon- carbon double bonds. Most naturally occurring fatty acids have an unbranched chain of an even number of carbon atoms, from 4 to 28.
  • linker refers to a divalent chemical moiety covalently attached to two monovalent chemical moieties, thus connecting the two monovalent chemical moieties into a single molecule.
  • a linker can connect a polypeptide and a fatty acid chain.
  • the linker of the invention does not change the physico-chemical properties (e.g., solubility, thermal or chemical stability) or therapeutic properties of the monovalent chemical moieties to which is it attached.
  • the linker of the invention does not interfere with the therapeutic properties of the
  • Linkers of the present invention can comprise one or more of a C 1-12 alkylene, C 1-12 alkenylene, a C 6-10 arylene, a 5-12 membered heteroarylene, an amide, an ester, a thioester, a urea, a thiourea, an ether, a thioether, an amine, a sulfonamide, a sulfone, a sulfoxide, or a carbamate.
  • a linker can comprise one or more of a C 1-12 alkyl, a C 6-10 aryl,
  • the linker is selected from
  • alkyl means a saturated straight-chain or branched hydrocarbon.
  • An alkyl group is typically C 1-40 , more typically C 10-30 .
  • “C 10-30 alkyl” means a straight or branched saturated monovalent hydrocarbon radical having from 10 to 30 carbon atoms (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon atoms).
  • alkenyl means a straight-chain or branched hydrocarbon, which contains at least one carbon-carbon double bond.
  • an alkenyl can contain one, two, three, four, or more carbon-carbon double bond.
  • An alkenyl group is typically C 2-40 , more typically C 10-30 .
  • “C 10-30 alkyl” means a straight or branched monovalent hydrocarbon radical having from 10 to 30 carbon atoms (e.g., 10,
  • aromatic group used alone or as part of a larger moiety as in “aralkyl”,“aralkoxy”, or“aryloxy alkyl”, includes carbocyclic aromatic rings and heteroaryl rings.
  • aromatic group may be used interchangeably with the terms “aryl”,“aryl ring”“aromatic ring”,“aryl group” and“aromatic group”.
  • Carbocyclic aromatic ring groups have only carbon ring atoms (typically six to fourteen) and include monocyclic aromatic rings such as phenyl and fused polycyclic aromatic ring systems in which two or more carbocyclic aromatic rings are fused to one another. Examples include 1 -naphthyl, 2-naphthyl, 1-anthracyl and 2-anthracyl.
  • “carbocyclic aromatic ring” is a group in which an aromatic ring is fused to one or more non-aromatic rings (carbocyclic or heterocyclic), such as in an indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl, where the radical or point of attachment is on the aromatic ring.
  • non-aromatic rings such as in an indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl, where the radical or point of attachment is on the aromatic ring.
  • heteroaryl refers to heteroaromatic ring groups having five to fourteen members, including monocyclic heteroaromatic rings and polycyclic aromatic rings in which a monocyclic aromatic ring is fused to one or more other aromatic ring.
  • Heteroaryl groups have one or more ring heteroatoms.
  • heteroaryl groups include 2- furanyl, 3-furanyl, N-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-oxadiazolyl, 5-oxadiazolyl, 2-oxazolyl, 4-oxazolyl, 5- oxazolyl, 3-pyrazolyl, 4-pyrazolyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 3-pyridazinyl, 2-thiazolyl, 4- thiazolyl, 5-thiazolyl, 2-triazolyl, 5-triazolyl, tetrazolyl, 2-thienyl, 3-thienyl, caibazolyl, benzimidazolyl,
  • heteroaryl is a group in which an aromatic ring is fused to one or more nonaromatic rings (carbocyclic or heterocyclic), where the radical or point of attachment is on the aromatic ring.
  • An“alkylene group” is a bivalent alkyl group, where alkyl is defined above, for example, represented by -[CH 2 ]z-, wherein z is a positive integer, preferably from one to eight, more preferably from one to six, and one or both of the hydrogen atoms can be replaced by a suitable substituent as defined below.
  • arylene and“heteroarylene” refer to aryl or heteroaryl ring(s) in a molecule that are bonded to two other groups in the molecule through a single covalent from two of its ring atoms.
  • examples include phenylene [-(C 6 H 4 )-], thienylene [- (C 4 H 2 S)-], furanylene [-(C 4 H 2 O)-], 1,5-triazolylene, 1,4-triazolylene, pyrrolodinylene [- (C 4 H5N)-] and cyclohexylene [-(C 6 H 10 )-].
  • 1,4- phenylene, 2,5-thienylene, 1,4 cyclohexylene, 2,5-pyrrolodinylene, 1,5-triazolylene, and 1,4-triazolylene are shown below:
  • heteroatom means nitrogen, oxygen, or sulfur and includes any oxidized form of nitrogen and sulfur, and the quatemized form of any basic nitrogen.
  • nitrogen includes a substitutable nitrogen of a heteroaryl or non-aromatic heterocyclic group.
  • the nitrogen in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur or nitrogen, the nitrogen may be N (as in 3,4- dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR” (as in N-substituted pyrrolidinyl), wherein R” is a suitable substituent for the nitrogen atom in the ring of a non-aromatic nitrogen-containing heterocyclic group, as defined below.
  • R 10 represents a hydrogen, C 1-12 alkyl, C 1-12 alkenyl, a C 6-10 aryl, or a 5-12 membered heteroaryl group.
  • amine and“amino” are art-recognized and refer to both unsubstituted and substituted amines and salts thereof, e.g., a moiety that can be represented by
  • each R 10 independently represents a hydrogen, C 1-12 alkyl, C 1-12 alkenyl, a C 6-10 aryl, or a 5-12 membered heteroaryl group.
  • aralkyl refers to an alkyl group substituted with an aryl group.
  • R 10 represents a hydrogen, C 1-12 alkyl, C 1-12 alkenyl, a C 6-10 aryl, or a 5-12 membered heteroaryl group.
  • the term“ester”, as used herein, refers to -C(O)0- or -OC(O)-.
  • Hydrocarbyl groups include, but are not limited to aryl, heteroaryl, carbocycle, heterocyclyl, alkyl, alkenyl, alkynyl, and combinations thereof.
  • R 10 represents C 1-12 alkyl, C 1-12 alkenyl, a C 6-10 aryl, or a 5-12 membered heteroaryl group.
  • sulfoxide is art-recognized and refers to the group -S(O)-.
  • thioestef refers to a group -C(O)S- or -SC(O)-.
  • thioether is equivalent to an ether, wherein the oxygen is replaced with a sulfur.
  • R 9 and R 10 independently represent C 1-12 alkyl, C 1-12 alkenyl, a C 6-10 aryl, or a 5- 12 membered heteroaryl group, or either occurrence of R 9 taken together with R 10 and the intervening atom(s) complete a heterocycle having from 4 to 8 atoms in the ring structure.
  • R 9 and R 10 independently represent C 1-12 alkyl, C 1-12 alkenyl, a C 6-10 aryl, or a 5- 12 membered heteroaryl group, or either occurrence of R 9 taken together with R 10 and the intervening atom(s) complete a heterocycle having from 4 to 8 atoms in the ring structure.
  • heteroarylene groups disclosed herein can be unsubstituted or substituted.
  • substituted refers to moieties having substituents replacing a hydrogen on one or more carbons of the backbone. It will be understood that
  • substitution or“substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.
  • the term“substituted” is contemplated to include all permissible substituents of organic compounds.
  • the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds.
  • the permissible substituents can be one or more and the same or different for appropriate organic compounds.
  • the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms.
  • Substituents can include any substituents described herein, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxy carbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamo
  • API active pharmaceutical ingredient
  • API encapsulated in the liposome refers to an API disposed within the inner lumen of the liposome, within the lipid bilayer of the liposome, or partially within the inner lumen and partially within the lipid bilayer of the liposome.
  • An API encapsulated in the liposome is not disposed on the outer surface of the liposome.
  • Zero potential refers to the electrical potential at the interface between a particle surface and its liquid medium.
  • average size of the liposome refers to Z-average particle size as determined by Dynamic Light Scattering.
  • alkyl means a saturated straight-chain or branched hydrocarbon.
  • An alkyl group is typically Ci-4o, more typically C 10-30 .
  • “C 10-30 alkyl” means a straight or branched saturated monovalent hydrocarbon radical having from 10 to 30 carbon atoms (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon atoms).
  • alkenyl means a straight-chain or branched hydrocarbon, which contains at least one carbon-carbon double bond.
  • an alkenyl can contain one, two, three, four, or more carbon-carbon double bond.
  • An alkenyl group is typically C 2-40 , more typically C 10-30 .
  • “C 10-30 alkyl” means a straight or branched monovalent hydrocarbon radical having from 10 to 30 carbon atoms (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon atoms), which contains at least one carbon-carbon double bond.
  • “bare liposome”,“naked liposome” or“uncoated liposome” refers to a liposome that does not have a coating on its outer surface, e.g. does not have a polymer coating on its outer surface.
  • free liposome refers to a liposome that does not have an API, for example, a peptide, encapsulated within the liposome or disposed on the outer surface of the liposome.
  • the term“subject” means a mammal in need of treatment or prevention, e.g., companion animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, pigs, horses, sheep, goats and the like) and laboratory animals (e.g., rats, mice, guinea pigs and the like).
  • the subject is a human in need of the specified treatment.
  • the term“treating” or‘treatment” refers to obtaining desired pharmacological and/or physiological effect.
  • the effect can include achieving, partially or substantially, one or more of the following results: partially or totally reducing the extent of the disease, disorder or syndrome; ameliorating or improving a clinical symptom or indicator associated with the disorder; delaying, inhibiting or decreasing the likelihood of the progression of the disease, disorder or syndrome; or preventing the disease, disorder or syndrome.
  • “preventing” or“prevention” refers to reducing the likelihood of the onset or development of disease, disorder or syndrome.
  • pharmaceutically acceptable refers to a substance that is acceptable for use in pharmaceutical applications from a toxicological perspective and does not adversely interact with the active ingredient. Accordingly, pharmaceutically acceptable carriers are those that are compatible with the other ingredients in the formulation and are biologically acceptable. Supplementary active ingredients can also be incorporated into the pharmaceutical compositions.
  • the term“about” refers to a ⁇ 10% variation from the nominal value unless otherwise indicated or inferred.
  • the term“about” can refer to a ⁇ 5%, or a ⁇ 2.5%, or a ⁇ 1% variation from the nominal value or a fixed variation from the nominal value, for example, ⁇ 0.1 mV or ⁇ 0.2 mV.
  • the present invention is an anionic liposome, comprising: at least a first neutral lipid; at least one anionic lipid; and an active pharmaceutical ingredient (API), wherein: the API is encapsulated in the liposome; and the API comprises a polypeptide covalently attached to a fatty acid chain, optionally via a linker.
  • API active pharmaceutical ingredient
  • the first neutral lipid is selected from one or more of l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC), l-palmitoyl-2-oleoyl-sn-glycero-3- phosphoethanolamine (POPE), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) 9 9 l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), egg phosphatidylcholine (EPC), soy phosphatidylcholine (SPC), dilauryloylphosphatidylcholine (DLPC),
  • DOPC 1,2-dimyristoyl- sn-glycero-3-phosphocholine
  • POPE l-palmitoyl-2-oleoyl-sn-glycero
  • DMPC dimyristoylphosphatidylcholine
  • MPPC l-myristoyl-2-palmitoyl phosphatidylcholine
  • PMPC l-palmitoyl-2-myristoyl phosphatidylcholine
  • PSPC l-palmitoyl-2-stearoyl phosphatidylcholine
  • DBPC l,2-diarachidoyl-sn-glycero-3-phosphocholine
  • SPPC dimyristoylphosphatidylcholine
  • DEPC l-dieicosenoyl-sn-glycero-3- phosphocholine
  • POPC N- palmitoyl-D-erythro-sphingosylphosphorylcholine
  • PLPC phosphatidyl choline
  • the liposome further comprises a second neutral lipid.
  • the second neutral lipid is one or both of cholesterol and sitosterol.
  • the remainder of the values and example values of the variables of the liposome are as described above with respect to the first aspect of the first embodiment.
  • the anionic lipid is selected from one or more of l,2-dioleoyl-sn-glycero-3-[phospho-rac-(l-gylcerol)] (DOPG); 1,2- dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS); l,2-dioleoyl-sn-glycero-3 -phosphate (DOPA); dimyristoyl-phosphatidyl glycerol (DMPG), l,2-distearoyl-sn-glycero-3- phosphoglycerol (DSPG), dipalmitoyl-phosphatidyl glycerol (DPPG), diethylenetriamine pentaacetic acid (DPTA), l,4-dipalmitoyl-tartarate-2,3-diglutaric acid (DPTGA), 1,4- disteroyl-tartarate-2,3-disucc
  • the anionic lipid is Li + , Na + , K + ,
  • dipalmitoylphosphatidylserine DPPS
  • palmitoyl-oleoylphosphatidylserine POPS
  • dioleoylphosphatidylglycerol DOPG
  • palmitoyl-oleoylphosphatidylglycerol POPG
  • dimyristoylphosphatidic acid DMPA
  • dipalmitoylphosphatidic acid DPP A
  • POP A palmitoyl-oleoylphosphatidic acid
  • the first neutral lipid is present in an amount from about 20 mol. % to about 80 mol. %, e.g., about 30 mol. % to about 60 mol. % or about 30 mol. % to about 40 mol. % of the total amount of lipids in the liposome.
  • the first neutral lipid is present in an amount of about 30 mol. %, about 40 mol. %, about 50 mol. %, about 60 mol. %, about 70 mol. %, or about 80 mol.
  • the second neutral lipid is present in an amount from about 0 mol. % to about 50 mol. %, e.g., about 10 mol. % to about 40 mol. % or about 20 mol. % to about 40 mol. % of the total amount of lipids in the liposome.
  • the second neutral lipid is absent or present in an amount of about 10 mol. %, about 20 mol. %, about 30 mol. %, about 40 mol. %, or about 50 mol. %, such as about 33.3 mol. % of the total amount of lipids in the liposome.
  • the remainder of the values and example values of the variables of the liposome are as described above with respect to the first through the fifth aspects of the first embodiment.
  • the anionic lipid is present in an amount from about 5 mol. % to about 40 mol. %, e.g., about 10 mol. % to about 40 mol. % or about 20 mol. % to about 40 mol. % of the total amount of lipids in the liposome.
  • the anionic lipid is present in an amount of about 5 mol. %, about 10 mol. %, about 15 mol. %, about 20 mol. %, about 25 mol. %, about 30 mol. %, about 35 mol. %, or about 40 mol. %, such as about 33.3 mol. % of the total amount of lipids in the liposome.
  • the remainder of the values and example values of the variables of the liposome are as described above with respect to the first through the sixth aspects of the first embodiment.
  • the ratio of mol. % amounts of the first neutral lipid, the second neutral lipid, and the anionic lipid is about 1:1:1.
  • the remainder of the values and example values of the variables of the liposome are as described above with respect to the first through the seventh aspects of the first embodiment.
  • the liposome comprises DSPC, DSPG, and cholesterol.
  • the remainder of the values and example values of the variables of the liposome are as described above with respect to the first through the eighth aspects of the first embodiment.
  • the liposome further comprises a buffer, e.g., HEPES buffer, a PBS buffer, Tris buffer, citrate buffer, lactate buffer, acetate buffer, MES buffer, or maleate buffer.
  • a buffer e.g., HEPES buffer, a PBS buffer, Tris buffer, citrate buffer, lactate buffer, acetate buffer, MES buffer, or maleate buffer.
  • the buffer is HEPES buffer.
  • the fatty acid chain is selected from C 10-30 alkyl or C 10-30 alkenyl, e.g., fatty acid chain is selected from straight-chain C 10-30 alkyl or straight-chain C 10-30 alkenyl.
  • the fatty acid chain is a C 16 alkyl or a Cie alkenyl.
  • the remainder of the values and example values of the variables of the liposome are as described above with respect to the first through the tenth aspects of the first embodiment.
  • the fatty acid chain is covalently attached to the polypeptide via a linker.
  • the linker is selected from
  • the linker is selected from
  • the polypeptide is selected from liraglutide, semaglutide, albiglutide, bivalirudin, blisibimod, buserelin, carfilzomib, cosyntropin, cilengitide, dulaglutide, enfuvirtide, eptifibatide, exenatide, glatiramer, goserelin, gramicidin D, leuprolide, linaclotide, lixisenatide, nesiritide, oxytocin, octreotide, oritavancin, pramlintide, pasireotide, salmon calcitonin, teduglutide, somatostatin, or teriparatide.
  • the polypeptide is selected from liraglutide, semaglutide, albiglutide, dulaglutide, exenatide, lixisenatide, or pramlintide.
  • the remainder of the values and example values of the variables of the liposome are as described above with respect to the first through the twelfth aspects of the first embodiment.
  • the API is liraglutide or semaglutide.
  • the API is liraglutide.
  • the remainder of the values and example values of the variables of the liposome are as described above with respect to the first through the thirteenth aspects of the first embodiment.
  • the amount of the API in the liposome is from about 1 wt. % to about 30 wt. %., such as from about 5 wt. % to about 20 wt. %.
  • the amount of the API in the liposome is about 10.5 wt. % .
  • the amount of the API in the liposome is about 12.5 wt. %.
  • the remainder of the values and example values of the variables of the liposome are as described above with respect to the first through the fourteenth aspects of the first embodiment.
  • the average size of the liposome is from about 30 nm to about 800 nm, e.g., from about 100 nm to about 300 nm.
  • the average size of the liposome is less than 200 nm, such as less than 100 nm.
  • the average size of the liposome is about 231 nm.
  • the average size of the liposome is about 140 nm. The remainder of the values and example values of the variables of the liposome are as described above with respect to the first through the fifteenth aspects of the first embodiment.
  • the encapsulation efficiency of the API in the liposome is from about 20 % to about 100 %, e.g. about 20 %, about 30 %, about 40 %, about 50 %, about 60 %, about 70 %, about 80 %, about 90 %, about 100 %.
  • the encapsulation efficiency of the API in the liposome is about 42 %.
  • the encapsulation efficiency of the API in the liposome is about 50 %.
  • the remainder of the values and example values of the variables of the liposome are as described above with respect to the first through the sixteenth aspects of the first embodiment.
  • the Zeta potential of the liposome is from about -20 mV to about -90m V, e.g., about -20 mV about -30 mV, about -40 mV, about -50 mV, about -60 mV, about -70 mV, about -80 mV, or about -90 mV.
  • the Zeta potential of the liposome is about -59 mV.
  • the Zeta potential of the liposome is about -76 mV.
  • the remainder of the values and example values of the variables of the liposome are as described above with respect to the first through the seventeenth aspects of the first embodiment.
  • the liposome comprises an inner lumen, a lipid bilayer, and an outer surface; and the API is disposed within the inner lumen, within the lipid bilayer, or partially within the inner lumen and partially within the lipid bilayer.
  • the remainder of the values and example values of the variables of the liposome are as described above with respect to the first through the eighteenth aspects of the first embodiment.
  • the API is not disposed on the outer surface of the liposome.
  • the remainder of the values and example values of the variables of the liposome are as described above with respect to the first through the nineteenth aspects of the first embodiment.
  • the present invention is a pharmaceutical composition, comprising any liposome described herein with respect to the first embodiment and various aspects thereof and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further comprises a polyol.
  • the pharmaceutical composition further comprises glucose, mannitol, sucrose, trehalose, or sorbitol.
  • the liposome further comprises a cyclodextrin.
  • the liposome further comprises SBE-b- cyclodextrin or HR-b-cyclodextrin.
  • the remainder of the values and example values of the variables of the liposome are as described above with respect to the first aspect of the second embodiment.
  • the present invention is a method of treating a condition, comprising: orally administering to a subject in need thereof an anionic liposome, wherein the liposome comprises: at least a first neutral lipid; at least one anionic lipid; and an active pharmaceutical ingredient (API), wherein: the API is encapsulated in the liposome; and the API comprises a polypeptide covalently attached to a fatty acid chain, optionally via a linker.
  • an anionic liposome wherein the liposome comprises: at least a first neutral lipid; at least one anionic lipid; and an active pharmaceutical ingredient (API), wherein: the API is encapsulated in the liposome; and the API comprises a polypeptide covalently attached to a fatty acid chain, optionally via a linker.
  • the condition is selected from a cancer, an infectious disease, a neurological disorder, or a metabolic disorder.
  • the condition is cancer, such as malignant hemopathy, e.g., lymphoma, leukemia, myeloma, or myelodysplastic syndrome, bladder cancer, brain cancer, breast cancer, uterine cancer, ovarian cancer, melanoma, colorectal cancer, liver cancer, or esophageal cancer.
  • the malignant hemopathy is Hodgkin’s B-cell lymphoma, diffuse large B-cell lymphoma, chronic lymphoid leukemia, acute myeloid leukemia, chronic lymphoid leukemia, lymphoblastic leukemia, or multiple myeloma.
  • the condition is infectious disease, such as a viral infectious disease, a bacterial infectious disease, or a fungal infectious disease.
  • the condition is metabolic disorder, such as type I diabetes, type P diabetes, obesity, nonalcoholic fatty liver disease (NAFLD), or Nonalcoholic steatohepatitis (NASH).
  • NAFLD nonalcoholic fatty liver disease
  • NASH Nonalcoholic steatohepatitis
  • the anionic liposome is administered once daily or twice daily. The remainder of the values and example values of the variables of the method are as described above with respect to the first aspect of the third embodiment.
  • the method further comprises administering sulfonylurea, an SGLT2 inhibitor, or metformin.
  • the method further comprises administering the sulfonylurea selected from glipizide, glimepiride, gliclazide, glibomuride, glibenclamide, or carbutamide.
  • the method further comprises administering the SGLT2 inhibitor selected from glipizide, canagliflozin, dapaglifozine, empagliflozin, ertugliflozin, ipragliflozin, luseogliflozin, remogliflozin etabonate, or tofogliflozin.
  • the SGLT2 inhibitor selected from glipizide, canagliflozin, dapaglifozine, empagliflozin, ertugliflozin, ipragliflozin, luseogliflozin, remogliflozin etabonate, or tofogliflozin.
  • the first neutral lipid is selected from one or more of l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC), l-palmitoyl-2-oleoyl-sn-glycero-3- phosphoethanolamine (POPE), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) 9 9 l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), egg phosphatidylcholine (EPC), soy phosphatidylcholine (SPC), dilauryloylphosphatidylcholine (DLPC),
  • DOPC 1,2-dimyristoyl- sn-glycero-3-phosphocholine
  • POPE l-palmitoyl-2-oleoyl-sn-glycero
  • DMPC dimyristoylphosphatidylcholine
  • MPPC l-myristoyl-2-palmitoyl phosphatidylcholine
  • PMPC l-palmitoyl-2-myristoyl phosphatidylcholine
  • PSPC l-palmitoyl-2-stearoyl phosphatidylcholine
  • DBPC l,2-diarachidoyl-sn-glycero-3-phosphocholine
  • SPPC dimyristoylphosphatidylcholine
  • DEPC l-dieicosenoyl-sn-glycero-3- phosphocholine
  • POPC N- palmitoyl-D-erythro-sphingosylphosphorylcholine
  • PLPC phosphatidyl choline
  • the liposome comprises a second neutral lipid.
  • the second neutral lipid is one or both of cholesterol and sitosterol.
  • the anionic lipid is selected from one or more of l,2-dioleoyl-sn-glycero-3-[phospho-rac-(l-gylcerol)] (DOPG); 1,2- dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS); l,2-dioleoyl-sn-glycero-3 -phosphate (DOPA); dimyristoyl-phosphatidyl glycerol (DMPG), l,2-distearoyl-sn-glycero-3- phosphoglycerol (DSPG), dipalmitoyl-phosphatidyl glycerol (DPPG), diethylenetriamine pentaacetic acid (DPTA), l,4-dipalmitoyl-tartarate-2,3-diglutaric acid (DPTGA), 1,4- disteroyl-tartarate-2,3-disucc
  • DPP A dipalmitoylphosphatidic acid
  • POP A palmitoyl-oleoylphosphatidic acid
  • the anionic lipid is Li + , Na + , K + ,
  • dipalmitoylphosphatidylserine DPPS
  • palmitoyl-oleoylphosphatidylserine POPS
  • dioleoylphosphatidylglycerol DOPG
  • palmitoyl-oleoylphosphatidylglycerol POPG
  • dimyristoylphosphatidic acid DMPA
  • dipalmitoylphosphatidic acid DPP A
  • POP A palmitoyl-oleoylphosphatidic acid
  • the first neutral lipid is present in an amount from about 20 mol. % to about 80 mol. %, e.g., about 30 mol. % to about 60 mol. % or about 30 mol. % to about 40 mol. % of the total amount of lipids in the liposome.
  • the first neutral lipid is present in an amount of about 30 mol. %, about 40 mol. %, about 50 mol. %, about 60 mol. %, about 70 mol. %, or about 80 mol.
  • the second neutral lipid is present in an amount from about 0 mol. % to about 50 mol. %, e.g., about 10 mol. % to about 40 mol. % or about 20 mol. % to about 40 mol. % of the total amount of lipids in the liposome.
  • the second neutral lipid is absent or present in an amount of about 10 mol. %, about 20 mol. %, about 30 mol. %, about 40 mol. %, or about 50 mol. %, such as about 33.3 mol. % of the total amount of lipids in the liposome.
  • the remainder of the values and example values of the variables of the method are as described above with respect to the first through the eighth aspects of the third embodiment.
  • the anionic lipid is present in an amount from about 5 mol. % to about 40 mol. %, e.g., about 10 mol. % to about 40 mol. % or about 20 mol. % to about 40 mol. % of the total amount of lipids in the liposome.
  • the anionic lipid is present in an amount of about 5 mol. %, about 10 mol.
  • the ratio of mol. % amounts of the first neutral lipid, the second neutral lipid, and the anionic lipid is about 1:1:1.
  • the remainder of the values and example values of the variables of the method are as described above with respect to the first through the tenth aspects of the third
  • the liposome comprises DSPC, DSPG, and cholesterol.
  • the remainder of the values and example values of the variables of the method are as described above with respect to the first through the eleventh aspects of the third embodiment.
  • the liposome further comprises a buffer, e.g., HEPES buffer, a PBS buffer, Tris buffer, citrate buffer, lactate buffer, acetate buffer, MES buffer, or maleate buffer.
  • a buffer e.g., HEPES buffer, a PBS buffer, Tris buffer, citrate buffer, lactate buffer, acetate buffer, MES buffer, or maleate buffer.
  • the buffer is HEPES buffer.
  • the fatty acid chain is selected from C 10-30 alkyl or C 10-30 alkenyl, e.g., fatty acid chain is selected from straight- chain C 10-30 alkyl or straight-chain C 10-30 alkenyl.
  • the fatty acid chain is a C 16 alkyl or a C 16 alkenyl.
  • the fatty acid chain is covalently attached to the polypeptide via a linker.
  • the linker is selected
  • the linker indicates a point of attachment of the linker to the polypeptide, or to the fatty acid chain.
  • the linker is selected from
  • the polypeptide is selected from liraglutide, semaglutide, albiglutide, bivalirudin, blisibimod, buserelin, carfilzomib, cosyntropin, cilengitide, dulaglutide, enfuvirtide, eptifibatide, exenatide, glatiramer, goserelin, gramicidin D, leuprolide, linaclotide, lixisenatide, nesiritide, oxytocin, octreotide, oritavancin, pramlintide, pasireotide, salmon calcitonin, teduglutide, somatostatin, or teriparatide.
  • the polypeptide is selected from liraglutide, semaglutide, albiglutide, dulaglutide, exenatide, lixisenatide, or pramlintide.
  • the remainder of the values and example values of the variables of the method are as described above with respect to the first through the fifteenth aspects of the third embodiment.
  • the API is liraglutide or semaglutide.
  • the API is liraglutide.
  • the remainder of the values and example values of the variables of the method are as described above with respect to the first through the sixteenth aspects of the third embodiment.
  • the amount of the API in the liposome is from about 1 wt. % to about 30 wt. %., such as from about 5 wt. % to about 20 wt. %.
  • the amount of the API in the liposome is about 10.5 wt. %.
  • the amount of the API in the liposome is about 12.5 wt. %.
  • the remainder of the values and example values of the variables of the method are as described above with respect to the first through the seventeenth aspects of the third embodiment.
  • the average size of the liposome is from about 30 nm to about 800 nm, e.g., from about 100 nm to about 300 nm.
  • the average size of the liposome is less than 200 nm, such as less than 100 nm.
  • the average size of the liposome is about 231 nm.
  • the average size of the liposome is about 140 nm.
  • the encapsulation efficiency of the API in the liposome is from about 20 % to about 100 %, e.g. about 20 %, about 30 %, about 40 %, about 50 %, about 60 %, about 70 %, about 80 %, about 90 %, about 100 %.
  • the encapsulation efficiency of the API in the liposome is about 42 %.
  • the encapsulation efficiency of the API in the liposome is about 50 %.
  • the remainder of the values and example values of the variables of the method are as described above with respect to the first through the nineteenth aspects of the third embodiment.
  • the Zeta potential of the liposome is from about -20 mV to about -90m V, e.g., about -20 mV about -30 mV, about -40 mV, about -50 mV, about -60 mV, about -70 mV, about -80 mV, or about -90 mV.
  • the Zeta potential of the liposome is about -59 mV.
  • the Zeta potential of the liposome is about -76 mV.
  • the remainder of the values and example values of the variables of the method are as described above with respect to the first through the twentieth aspects of the third embodiment.
  • the liposome comprises an inner lumen, a lipid bilayer, and an outer surface; and the API is disposed within the inner lumen, within the lipid bilayer, or partially within the inner lumen and partially within the lipid bilayer.
  • the remainder of the values and example values of the variables of the liposome are as described above with respect to the first through the twenty-first aspects of the third embodiment.
  • the API is not disposed on the outer surface of the liposome.
  • the remainder of the values and example values of the variables of the liposome are as described above with respect to the first through the twenty-second aspects of the third embodiment.
  • ChemicTrypsine was procured from Life Technologies.
  • Glycylsarcosine was purchased from Moravek.
  • DIEA N,N-diisopropylethylamine
  • NEAA non-essential amino acids
  • Dulbecco’s Phosphate Buffer Saline DPBS
  • Dulbecco modified eagle medium DMEM
  • Lucifer Yellow LY
  • HPES 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid
  • MES 2-(N- morpholino)ethanesulfonic acid
  • DMSO Dimethylsulfoxide
  • Caco-2 cells were obtained from Sender Biology Research Department (Orleans, France). Cells were grown in plastic culture flasks (Coming) in culture medium containing DMEM, 10% FBS (heat-inactivated), 1% NEAA, 1% L- Glutamine 200 mM and 1% P/S (10,000 UI/mL) and used between passages 77 and 90. When confluency reached 80%, these cells were detached by treating them with trypsin/EDTA, and then plated at a density of 30,000 cells/unit on 24-well plates
  • Example 1 Formulation of free or liraglutide-loaded lyposomes.
  • DSPC and cholesterol were dissolved in chloroform at a concentration of 25 and 50 mg/mL, respectively, and DSPG was dissolved in a mixture of chloroform, methanol and deionized water (65:35:8, v/v/v) at a concentration of 25 mg/mL.
  • Lipid mixture composed of DSPC/DSPG/cholesterol (1:1:1, w/w/w) was prepared in a 10 mL round bottom flask, for a final amount of lipids of 10 mg. Solvents were evaporated using a rotary evaporator under heat (60 °C, water bath) until completely dry ( ⁇ 15 mbar) and lipid film was formed. In a sonicator bath at 65 °C, 2 mL of 25 mM HEPES pH 9
  • the suspension was extruded two times through successively smaller nucleopores membranes (400 nm, 200 nm, 100 nm). The extrusion process was optimized in order to have small liposomes with a minimal loss of drug encapsulation.
  • Non-encapsulated liraglutide was removed using the tangential flow filtration (TFF) method.
  • Liposome suspension was connected to a Spectrum Laboratories KrosFlo P system using masterflex, Teflon-coated tubing. D02-E100-05-N (liposome amount > 2.5 mg) or C02-E100-05-N (liposome amount ⁇ 2.5 mg) 100 kDa filters were used to purify the particles until 8 volume- equivalents were collected in the permeate.
  • Exchange buffer was 25 mM HEPES at pH 9, which is the pH of aqueous solutions in which liraglutide is the most soluble. Finally, exchange buffer was switched for 2 volume-equivalents with 25 mM HEPES at pH 4.9, which is the pH matching isoelectric point of liraglutide. Suspensions were run at 75 mL/min (size 16 tubing, used with D02-E100-05-N column) or 13 mL/min (size 13 tubing, used with C02-E100-05-N column). Once washed, liposomes were concentrated and recovered by reversing the direction of the peristaltic pump. For complete recovery, 1 to 3 mL of the appropriate buffer was run backward through the tubing to recover any remaining particles.
  • Example 2 Formulation of LbL particles.
  • Nanoparticles were layered by adding a volume of the liposome core suspension at 1 mg/mL in 25 mM HEPES at pH 4.9, to an equal volume of a poly electrolyte solution in 50 mM MES, 40 mM NaCl at pH 4.9, under sonication at room temperature. The mixture was sonicated for 10 seconds and incubated for 30 min under agitation. The optimal mass ratio between each layer and the liposome core was determined prior to the deposition step via polyelectrolyte titration using 50 mL samples of the liposome suspension for each tested mass ratio. The test ratios were mixed as described previously but only incubated for 5 min.
  • the test ratio was chosen as the optimal ratio.
  • optimal ratio core/polyelectrolyte was determined as 1:0.5 (w/w).
  • Interlayer purification was made using the tangential flow filtration method described above. Excess of polyelectrolyte was removed after 5 volume-equivalents were collected in the permeate.
  • Exchange buffer was 25 mM HEPES at pH 4.9. Schematic representation of the LbL particle formulation process is shown in Figure 8.
  • Poly-L-lysine-Valine was obtained from the conjugation of poly-L- lysine (PLK) and Boc-protected valine ligand. Covalent linkage of the ligand on the polymer was obtained by combining 1 equivalent ofPLK, 3 equivalents of Boc- Valine, 4 equivalents of (2-(lH-benzotriazol- 1 -yl)- 1 , 1 ,3,3 -tetramethy luronium hexafluorophosphate (HBTU) and 5 equivalents of DIE A, in dimethylformamide (DMF). The reaction was continued for 2 hours under constant stirring at room temperature.
  • Nanoparticles size and PDI were characterized by dynamic light scattering (DLS) using a Malvern ZS90 Particle Analyzer. Zeta potential was measured using Doppler electrophoresis on the Malvern ZS90. Samples were diluted in water or all measurements. Results from the Malvern are reported using the SD of three measurements. Shape and size of nanoparticles were also observed by negative stained-electron microscopy. 7 uL of the suspension was dropped on a 200 meshes copper grid coated with a continuous carbon film and allowed to rest for 60 seconds. Excess of suspension was removed. 10 uL of negative staining solution (phosphotungstic acid, 1% aqueous solution) were dropped on the TEM grid and immediately removed.
  • DLS dynamic light scattering
  • Total amount of drug and total amount of liposomes were determined by HPLC dosage of liraglutide and cholesterol after addition of 500 uL of MeOH to 500 uL of nanoparticle (NP) suspension in order to destruct the NPs.
  • the drug loading was described as the percentage of encapsulated drug compared to the total amount of NPs.
  • the entrapment efficiency was defined as the percentage of encapsulated drug compared to the total amount of drug initially in the solution used for lipid film rehydration.
  • Liraglutide loading and entrapment efficiency were calculated using the following equations:
  • Liposome suspension was diluted in 10 mM MES pH 6 in order to have 20 ug/mL of liraglutide corresponding to the targeted dose in vivo.
  • the suspension was kept under stirring for 4 hours. 1.75 mL were sampled at 20, 60, 120, 240 min. Desalting columns (Disposable PD- 10 Desalting Columns, GE Healthcare) were used in order to separate liposomes from free released liraglutide.
  • the top filter was previously removed and the column was equilibrated with 20 mL of buffer. 5 mL of buffer were added and the column was spun down at 1000 x g for 2 min to pack the bed. Samples were added to the top of the column and eluted by centrifugation at 1000 x g for 2 min.
  • Liraglutide encapsulation in liposomes was assayed using HPLC.
  • the system is composed of an Agilent separation module coupled with an Agilent UV dual l absorbance UV. Detector was set at 280 nm.
  • the column used was a Sunfire C8 (15 cm x 14 mm, 5 um).
  • the mobile phase consists in a gradient of water, 1% TFA (v/v) and Acetonitrile, 1% TFA (v/v), 65:10:25.
  • the flow rate was 1.2 mL/min and the injected volume 50 uL.
  • detector was set at 210 nm.
  • the column used was the same as described previously.
  • the mobile phase consists in an isocratic gradient of isopropanol / water, 1% TFA (v/v) / acetonitrile, 1% TFA (v/v), 65:10:25.
  • the flow rate was 1.5 mL/min and the injected volume 50 uL.
  • Samples were analyzed against a set of calibration standards prepared in the mobile phase. The determination of drug concentration was carried out over a calibration range obtained from a control sample and a 10% and 1% dilution of the control sample in mobile phase.
  • An analytical run consisted of control sample, a set of calibration standard, and blank samples interspersed among the study samples. Analyte concentrations were evaluated using the internal standard method. The standard curves were generated from the peak area ratios of analyte/intemal standard and the nominal analyte concentration using linear regression analysis with 1/x 2 weighting.
  • Example 5 In Vitro PepTl targeting.
  • Liposomes were made as described in Example 1 with addition of 1% NBD- ethylenediamine dye (mol/mol lipids) in the formulation.
  • the 24-wells plate was seeded with 20,000 Caco-2 cells (heterogeneous human epithelial colorectal adenocarcinoma cells) and allowed to attach and grow for 7 days.
  • the old media was removed from wells and replaced by 450 uL of fresh cell media at pH 6 with or without 10 mM of
  • glycylsarcosine 50 uL of the tested formulation was added to each well in order to have a final NPs concentration of 35 ug/mL. NPs were incubated with cells for 90 minutes at 37 °C and then removed from wells. Cells were washed 2 times with 500 uL warmed DPBS and 100 uL of tryspin EDTA were added to each well and incubated for 5 minutes at 37 °C. 400 uL of warmed media were added to each well and were pipetted up and down rigorously, then transferred in clear FACS tubes. Cells were kept in ice until analysis in FACS (FACS LSR P HTS-2) using software BD FACS Diva.
  • lipid mixture composed of
  • DSPC/DSPG/cholesterol/DOPE 31:31:31 :7, % w/w/w was prepared in a 50 mL round bottom flask, for a final amount of lipids of 50 mg. Solvents were evaporated until completely dry ( ⁇ 15 mbar) and lipid film was formed. In a sonicator bath at 65 °C, 25 mL of water, pre-heated at 65°C, were added under sonication in order to resuspend the lipid film. The liposome suspension was sonicated for 5 min and then transferred to a liposome extruder maintained at a temperature >65 °C.
  • the suspension was extruded two times through 400 nm and 200 nm nucleopore membranes, two times through 100 nm and two times through 50 nm.
  • 1 mL at a time of NaHCO 3 0.1M, pH 8.4 was added to 5 mg of the Sulfo-Cyanine5 NHS ester dye and transferred to liposomes as long as there is dye in the falcon.
  • the volume added was no more than 10 mL for a final volume of 35 mL.
  • the mixture was allowed to stir overnight and covered with foil.
  • the resulting liposomes were washed by TFF in 5 volumes of lxPBS and 15 volumes of water. Once washed, the liposomes were concentrated and recovered by reversing the direction of the peristaltic pump. For complete recovery, 1 to 3 mL of the appropriate buffer was run backward through the tubing to recover any remaining particles.
  • Cells were plated at a density of 30,000 cells/unit on 24-well Transwells® (polyester membranes, 0.4 um pore size, 6.5 mm diameter wells) and were grown in culture medium for 21-30 days.
  • Cell media was removed from both apical and basolateral sides and replaced by 135 uL of culture media at pH 6 to the apical side and 500 uL of media at pH7.4 to the basolateral side.
  • Apical and basolateral conventional transport media were composed of culture medium at pH 6 and pH 7.4, respectively.
  • Apical and basolateral conventional transport media are disclosed, for example, in Gourdon, B.
  • Formaldehyde solution was removed and cells were washed 3 times with 4 °C HBBS for 5 minutes. Each polycarbonate membrane loading the cells was cut out from the filter and disposed into a well of a Coming® 24-well multi-dishes. 300 uL of WGA AF555 were added to each well and incubated for 10 minutes at room temperature. During incubation with WGA, a 1 ug/mL Hoescht solution in PBS was prepared. WGA was removed and membranes were washed 3 times with PBS for 5 minutes. 500 uL of 4% of formaldehyde solution was added to each well for a second fixation of the cells for 15 min at room temperature.
  • Caco-2 cells were obtained from the High Throughput Sciences Facility of The Koch Institute for Integrative Cancer Research at Massachusetts Institute of Technology. Cells were grown in plastic culture flasks (Falcon) in culture medium containing DMEM complemented with L -Glutamine, 10% FBS (heat- inactivated), 1% NEAA and 1% P/S (10,000 UI/mL) and used between passages 10 and 50. When confluency reached 80%, Caco-2 cells were detached by treating them with trypsin/EDTA and seeded at a density of 10,000 cells/wells on 96-well plate. Cells were then grown for 7 days (37°C, 5% C02).
  • Example 7 In Vitro peptide transport.
  • Caco-2 cells were plated at a density of 30,000 cells/unit on 24-well Transwells® (polyester membranes, 0.4 um pore size, 6.5 mm diameter wells). Cells were then grown in culture medium for 21-30 days. Lucifer yellow (100 uM) transport was used as marker of cell monolayer integrity. Apical and basolateral conventional transport media were composed of culture medium at pH6 and ph7.4, respectively. Finally, 75 nM doses of free liraglutide and liraglutide loaded in bare liposomes or layered and valine-functionalized liposomes were incubated in apical chambers for 4 hours. Liraglutide concentrations were determined by liraglutide Elisa test and apparent permeability was calculated for each group. Results of these experiments are shown in Figure 18.
  • Example 8 In Vivo efficacy of orally administered liraglutide encapsulated in liposomes, liposomes coated with PLK-Val, and liposomes coated with PLR/DXS/PLK- Val.
  • Free Liraglutide SC 0.2 mg/kg, solution in saline;
  • Liraglutide liposomes PO, 2 mg/kg, suspension in water;
  • Liposomes placebo, PO, suspension in water.
  • Example 9 Formulation of a liposome functionalized with Val.
  • a liposome cores containing phospholipid-alkyne will be prepared. It will be conjugated with Val-N 3 at the surface using the following the protocol:

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Abstract

L'invention concerne des formulations de liposomes chargés de peptides, ainsi que des méthodes d'utilisation et des procédés de fabrication de liposomes. Ces formulations et méthodes sont utiles pour l'administration par voie orale d'agents thérapeutiques, tels que des agents thérapeutiques à base de peptides et/ou de protéines.
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CN114452258A (zh) * 2022-03-09 2022-05-10 成都大学 利拉鲁肽脂质体制剂及其制备方法和应用
CN116211827A (zh) * 2023-03-17 2023-06-06 浙江大学 一种特立帕肽固体脂质纳米粒及其制备方法和应用

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Cited By (5)

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Publication number Priority date Publication date Assignee Title
CN114081963A (zh) * 2021-11-16 2022-02-25 上海理工大学 一种提高活性肽生物利用度的纳米载体及其制备与应用
CN114081963B (zh) * 2021-11-16 2023-09-26 上海理工大学 一种提高活性肽生物利用度的纳米载体及其制备与应用
CN114452258A (zh) * 2022-03-09 2022-05-10 成都大学 利拉鲁肽脂质体制剂及其制备方法和应用
CN116211827A (zh) * 2023-03-17 2023-06-06 浙江大学 一种特立帕肽固体脂质纳米粒及其制备方法和应用
CN116211827B (zh) * 2023-03-17 2024-04-05 浙江大学 一种特立帕肽固体脂质纳米粒及其制备方法和应用

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