The invention relates to oral pharmaceutical compositions of an active agent and a lipid conju gate.

Prior art

Oral drug delivery is considered as the most advantageous route of drug application, in particu lar for the treatment of chronic diseases, which demand long-term and repeated drug admin istration. The oral route offers high drug safety and is widely accepted among patients due to its convenience. Additionally, as sterility is not required for oral drug forms, costs in production, storage and distribution are reduced, which may contribute to health care improvement in third world countries. It is estimated, that 90% of all marketed drug formulations are for oral use.

However, the number of drugs with low oral bioavailability, such as macromolecular drugs like peptides, proteins, and antibodies is steadily increasing. Because of their sizes, macromolecular drugs have particularly low oral bioavailability. In particular, many macromolecular drugs show poor absorption across the gastrointestinal barrier. Thus, such drugs have to be administered subcutaneously or intravenously which increases necessary medical efforts and causes in creased costs, decreased patient compliance, and increased risk of complications.

To overcome this problem, different approaches to improve the oral bioavailability have been tested in the past years including nanoparticles, and liposomes. However, conventional liposo mal formulations have not been very convincing due to their instability in the gastrointestinal tract.

Nanoparticle and liposome preparation involves multi-step processes requiring special expertise and expensive equipment. Achieving pure, consistent and stable products requires expert level personnel and experience.

There is a need for pharmaceutical compositions that improve bioavailability of active agents with poor mucosal absorption. Manufacture of the compositions, in particular manufacturing un der GMP rules, should be as simple as possible, preferably not involving production of nanopar ticles or liposomes. Description of the invention

In an aspect, the invention relates to a pharmaceutical composition for oral administration com prising

- a conjugate comprising a cell penetrating peptide conjugated to a lipid, and

- an active agent, optionally selected from the group consisting of peptides, polypeptides and proteins, wherein the composition is essentially free of liposomes.

In another aspect, the invention relates to a pharmaceutical composition comprising at least one conjugated lipid comprising a cell penetrating peptide conjugated to a lipid, such as a phospholipid or fatty acid, and at least one active agent, wherein the amount of conjugated lipid is from 0.1 to 100 mol% relative to the total amount of oily components in the composition.

Optionally, the amount of lipid conjugate may be at least 1.25 mol%, at least 3.0 mol%, greater than 5.0 mol%, at least 10.0 mol%, at least 15.0 mol%, at least 20.0 mol% or at least 25.0 mol% relative to the total amount of oily component in the composition.

In an optional embodiment, the composition is a self-emulsifying drug delivery system (SEDDS), such as a self-microemulsifying or self-nanoemulsifying drug delivery system. SEDDS form droplets in the Gl tract. The droplets may protect active agents from enzymatic degradation. SEDDS may comprise considerable amounts of oily components so that the amount of lipid conjugate relative to the total amount of oily components in the composition may be low. Op tionally, the amount of lipid conjugate in the pharmaceutical composition may be up to 10.0 mol%, up to 8.0 mol%, up to 6.0 mol%, up to 5.0 mol%, up to 4.0 mol% or up to 3.0 mol% rela tive to the total amount of oily components. The lipid conjugate may serve two functions in SEDDS, e.g. it may enhance resorption of the active agents, and it may additionally serve as surfactant so that no additional surfactant is needed, or the amount of surfactant may be re duced. Optionally, the amount of certain co-solvents may be reduced, e.g. propylene glycol. Op tional compositions comprise at least 50 mol%, at least 75 mol%, at least 85 mol% or at least 90 mol% of lipid conjugate relative to the total amount of oily components in the composition. The peptide may be conjugated to the lipid directly or indirectly via a covalent bond. In this con text, indirect conjugation means that one or more linkers may be positioned between lipid and peptide.

It was found that the conjugated lipid acts as a permeation enhancer for the active agent in the composition. In other words, oral bioavailability of the active agent increases when co-adminis- tered together with the lipid conjugate described herein. Without wishing to be bound by this theory, the inventors believe that the lipid conjugate may form micelles, droplets, vesicles or particles (hereinafter collectively referred to as “particle”) that enhance resorption of the active agent in a patient’s gastrointestinal tract. It appears that the particles form spontaneously with out any need for specialized equipment or expertise. The particles may be present in a liquid pharmaceutical composition or they may form after ingestion of a dosage form by a patient.

The term “particle” means a micelle, vesicle, droplet or particle of colloidal dimensions that ex ists in equilibrium with the molecules or ions in solution from which it is formed. The particles may form spontaneously, i.e. simply by combining the components. In the context of this de scription, “colloidal dimension” means a particle size of less than 100 nm, preferably less than 75 nm or less than 50 nm. A particle may form spontaneously. The term „particle“ does not in clude „liposome“. As opposed to liposomes, the pharmaceutical compositions of disclosed herein typically do not contain cholesterol in amounts of more than 1.0 mol% relative to the total amount of the oily component. The amount of cholesterol may even be limited to less than 0.5 mol% or less than 0.1 mol% relative to the total amount of oily component. In certain embodi ments, the particles may be larger, e.g. in the case of SEDDS larger particles may be formed spontaneously. Optionally, the particles may have a particle size of larger than 100 nm, e.g. from 100 nm to 200 nm, preferably from 100 nm to 300 nm, from 120 nm to 200 nm or from 120 nm to 180 nm. In an embodiment, the particle size ranges from 20 to 500 nm, from 50 to 400 nm, from 100 to 300 nm or from 120 to 200 nm.

The term "liposome" refers to artificially prepared vesicles composed of lipid bilayers. Lipo somes can be used for delivery of APIs due to their unique property of encapsulating a portion of an aqueous solution inside a lipophilic bilayer membrane. Lipophilic compounds can be dis solved in the lipid bilayer, and in this way liposomes can carry both lipophilic and hydrophilic compounds. To deliver the molecules to sites of action, the lipid bilayer can fuse with other bi layers such as cell membranes, thus delivering the liposome contents. In an embodiment, the compositions of this disclosure are essentially free of liposomes, particularly of liposomes hav ing an average particle size of at least 100 nm. “Essentially free of liposomes” means that the number of liposomes within the composition is less than 10% or less than 1% relative to the to tal number of particles in the composition. The number of particles may be determined by cryo- electron microscopy and counting the particles. In some embodiments, e.g. if the composition is in the form of SEDDS, the composition may be dissolved in simulated gastric fluid using the paddle apparatus (Ph. Eur.) before conducting the cryo-electron microscopy. Preferably, the number of liposomes is less than 5%, less than 3% or less than 1% of the total number of parti cles in the composition. In an embodiment, the composition contains less than 0.1% by weight of liposomes, preferably less than 0.05% by weight of liposomes relative to the total weight of the composition.

In an embodiment, oral bioavailability of the active agent is increased by at least 50%, at least 100%, at least 150%, at least 200% or at least 250% compared to oral bioavailability of the ac tive agent in the same composition administered to a human without the lipid conjugate. Option ally, the absolute oral bioavailability of the active agent in the composition comprising the conju gate may be at least 0.01%, at least 0.05%, at least 0.1%, at least 0.5%, at least 1.0%, at least 2.5% or at least 3.0%.

The absolute oral bioavailability of the active agent may be less than 10.0%, less than 2.5%, less than 1.0%, less than 0.5%, or less than 0.2% when administered to a human in the compo sition described herein without the conjugate. In other words, the active agent may be an agent with low absolute oral bioavailability. In the absence of any other indication, references to ad ministration or bioavailability refer to administration to a human and bioavailability after admin istration to a human. Oral bioavailability can be assessed by oral administration to a human in a fasting state. Fasting state means more than 8 hours after food ingestion, e.g. in the morning before breakfast. Subjects should not ingest any food within 2 hours after administration.

Conjugated lipids

The expressions “conjugated lipid” and “lipid conjugate” are used interchangeably in this de scription. The lipid conjugates of this invention may have the following general structure:

L -AG - CPP (Formula I)

In this formula, L represents the lipid, AG represents an optional activating group and/or linker and CPP represents the cell penetrating peptide. The hyphens represent covalent bonds. In other words, the lipid conjugate may comprise the lipid covalently attached to the CPP, wherein the covalent bond may optionally be achieved via an activating group and/or linker, or directly without any activating group or other intermediate group in between.

Preferred lipid conjugates have molecular weights in the range of 1.000 to 10.000 g/mol, prefer ably from 1.200 to 5.000 g/mol, or from 1.500 to 3.500 g/mol. Optionally, the lipid conjugate may carry a positive charge and/or the net charge of the lipid con jugate may be positive. A conjugate has a positive net charge, if the number of positive charges is larger than the number of negative charges in the conjugate.

Preferred lipid conjugates are shown in Figures 11 and 12. An optional lipid conjugate is the fol lowing compound A:

RQIKIWFQNRRMKWKK

An optional lipid conjugate is the following compound B:

An optional lipid conjugate is the following compound C:

An optional lipid conjugate is the following compound D:

An optional lipid conjugate is the following compound E:

An optional lipid conjugate is the following compound F: An optional lipid conjugate is the following compound G:

An optional lipid conjugate is the following compound H:

An optional lipid conjugate is the following compound I, wherein R represents arginine and n may be an integer of > 3:

Pharmaceutical composition

The pharmaceutical composition may be liquid, solid, or semi-solid. The pharmaceutical compo sition may be in the form of a solution, emulsion, suspension, powder, lyophilisate, granules, pellets, gel, tablet, pill, capsule, effervescent formulation, paste, lozenge, chewing gum, or spray. Liquid compositions may include solvents, such as water, in amounts of at least 50.0 wt%, at least 75.0 wt%, or at least 90.0 wt%. Solid compositions may comprise only limited amounts of solvent, such as less than 50.0 wt%, less than 30.0 wt%, less than 15.0 wt% or less than 5.0 wt%. Optional compositions are free of solvents.

The relative weight amount of lipid conjugates in the pharmaceutical compositions may be in the area of 1 to 25 wt.-% relative to the total weight of the composition. Preferred lower limits in clude 2 wt.-%, 3 wt.-% or 5 wt.-%. Preferred upper limits include 20 wt.-%, 15 wt.-% or 10 wt.-%.

The weight amount of lipid conjugate in the pharmaceutical composition may be at least as high as the amount of active agent. In an embodiment, the weight amount of lipid conjugate exceeds the amount of active agent by a factor of at least 1.5, at least 2.0, at least 2.5 or at least 3.0. Op tionally, the weight amount of lipid conjugate may be limited to about 100 times the amount of active agent, or up to about 50 times, or up to about 25 times or up to about 15 times the amount of active agent.

In an embodiment, the amount of lipid conjugate in the composition is at least 0.1 mg/g relative to the composition. Optionally, the amount may be at least 0.3 mg/g, at least 0.5 mg/g or at least 0.7 mg/g. The amount may be limited to up to 700 mg/g, up to 500 mg/g, up to 300 mg/g, up to 200 mg/g or up to 100 mg/g. In embodiments, the amount of lipid conjugate is up to 70 mg/g, up to 50 mg/g or up to 35 mg/g of the composition. In an exemplary embodiment, the amount is from 0.1 to 700 mg/g, from 0.3 to 300 mg/g, from 0.5 to 100 mg/g or from 0.7 to 35 mg/g. The amount may range up to 1000 mg/g.

In an embodiment of a liquid composition, the amount of lipid in the composition may be se lected in a range of from 0.1 to 2.0 mg/ml, or from 0.3 to 1.5 mg/ml or from 0.5 to 1.0 mg/ml. Op tionally, the amount is at least 0.7 mg/ml. It was found that these concentrations are best suited for particle formation.

Optionally, the composition comprises an aqueous solution with particles therein, wherein the particles comprise the lipid conjugate. It was found that the lipid conjugates described herein have the capability of forming particles upon contact with aqueous media, such as buffers. The aqueous solution may be a buffer, such as a citrate buffer or a phosphate buffer or a mixture thereof. Because the particles form spontaneously upon mixing the lipid conjugates with aque ous media (self-emulsifying), the difficult preparation of liposomes is not necessary. Optionally, the particles have an average particle size of less than 100 nm, or less than 75 nm, or less than 50 nm, or less than 40 nm or less than 30 nm. The particles may be much smaller than lipo- somes. Typically, liposomes of the lipids used herein are much larger than 100 nm. It is surpris ing that the liposomes are not needed to achieve the effect of increased bioavailability. The zeta potential of the particles may be positive, such as at least 1.0 mV, preferably at least 2.0 mV, at least 3.0 mV or at least 4.0 mV. Conventional liposomes typically have negative zeta potentials because of the negative charges of the lipids in their bilayers.

Pharmaceutical compositions of this invention may or may not contain the particles described above. Optional embodiments are in the form of solid compositions without any particles. The inventors hypothesize that upon resuspension of the solid composition in the stomach particles may form after administration. In one embodiment, the pharmaceutical composition is a solid dosage form comprising active agent and lipid conjugate in lyophilized form.

In an embodiment, the invention includes pharmaceutical compositions comprising one or more oily components in an amount of not more than 10.0 wt% relative to the pharmaceutical compo sition, one or more lipid conjugates in a total amount of at least 25.0 mol% relative to the total amount of oily components, and one or more active agents. The active agent may be a peptide. The pharmaceutical composition may be a solid composition, such as a tablet, or a capsule.

In another embodiment, the invention includes pharmaceutical compositions comprising one or more oily components in a total amount of at least 50.0 wt% relative to the pharmaceutical com position, one or more lipid conjugates in a total amount of at least 1.0 mol% relative to the total amount of oily components, and one or more active agents. The active agent may be a peptide. The amount of cholesterol in the pharmaceutical composition may be limited to not more than 1.0 mol% relative to the total amount of oily component. The pharmaceutical composition may be a liquid composition, such as an emulsion and/or a SEDDS. Optionally, the pharmaceutical composition may be essentially free of nanoparticles and/or essentially free of any particles of more than 100 nm particle size. In this context, “essentially free” means that the content of the mentioned constituents is less than 0.5 wt.%, less than 0.1 wt.% or even less than 0.01 wt.% of the composition.

The pharmaceutical compositions may comprise at least one pharmaceutically acceptable ex cipient, and/or at least one protease inhibitor, and/or at least one lipase inhibitor. These sub stances can be incorporated into the dosage form. Preferably, said at least one pharmaceuti cally acceptable excipient is selected from the group consisting of sorbitan monostearate, tripal- mitin, cetyl palmitate, alginate, ethyl oleate, C8 triglycerides, C10 triglycerides, cellulose, disac charides, monosaccharides, oligosaccharides, magnesium stearate, corn starch, citric acid, tar taric acid, acid salts of amino acids, and combinations thereof. Some of these excipients may form part of or constitute the oily components fraction of the composition. Furthermore, said at least one protease inhibitor is preferably selected from the group consisting of aprotinin, soy bean trypsin inhibitor, bacitracin, sodium glycocholate, bestatin, leupeptin, cystatin, camostat mesilate, and combinations thereof. Furthermore, said at least one lipase inhibitor is preferably selected from the group, consisting of orlistat, lipstatin, chitin, chitosan, saponin, flavonoid gly coside, polyphenole, ebelacton A and B, esterastin, valilactone, panclicine, proanthocyanidin, vibralactone, and combinations thereof.

Optionally, the lipid conjugate is not part of a liposome’s lipid double layer. Particularly, the cell penetrating peptide may be attached to a compound that is not part of a liposome’s lipid double layer. Optionally, the pharmaceutical compositions are free of tetraetherlipids.

Lipids

The lipid may be selected from the group consisting of steroids, fatty acids, fatty alcohols, fatty amines, hydrocarbons with carbon chain lengths of at least eight carbon atoms (e.g. liquid par affin), phospholipids, sphingolipids, ceramides, glycolipids, etherlipids, polyethers, carotenoids, and glycerides (mono-, di- and/or triglycerides) and combinations thereof. Steroids include com pounds having a sterane structure. Steroids include cholesterol and its derivatives. Triglycerides include medium chain triglycerides (e.g. C6 to C12 fatty acids). Mono-, di- or triglycerides and/or fatty acids may be modified, such as PEGylated, ethoxylated, esterified (e.g. with propylene gly col, sorbitol or sorbitan) and/or in salt form. Mono-, di- or triglycerides include vegetable oils.

The lipids in the conjugated lipid may be selected from amphiphilic compounds/surfactants. Op tional lipids with surfactant properties may be selected from the group consisting of mono- and/or diglycerides, ethoxylated mono-, di- or triglycerides (e.g. Kolliphor® EL), medium chain mono- and/or diglycerides (e.g. C6 to C12 fatty acids), ethoxylated plant oil (e.g. ethoxylated castor oil), monoglycerides of C12 to C20 saturated or unsaturated fatty acids (e.g. C14 to C18), esters of saturated or unsaturated fatty acids with diols (such as C8 to C20 fatty acids; e.g. 2-hydroxypropyl octanoate; propylene glycol monolaurate), mono- and di-esters esters of polyethylene glycol with medium chain fatty acids (e.g. C6 to C10 fatty acids), sorbitol or sorbi tan esters, esters of sorbitol or sorbitan with polyethylene glycol and/or fatty acids (e.g. poly- sorbates), transesterified ethoxylated vegetable oils (e.g. Labrafil® M1944CS), polyethers (e.g. copolymers of polyalkylene glycols, such as Poloxamer®), salts of fatty acids, cetrimonium bro mide, bis(2-ethylhexyl) sulfosuccinate and mixtures thereof.

Examples of lipids suitable for conjugate formation within the concept of this invention are listed in Nardin et al., Successful development of oral SEDDS: screening of excipients from the indus trial point of view, Advanced Drug Delivery Reviews 142 (2019) 128-140. The disclosure of Nardin et al. is incorporated by reference as if fully set forth herein. Lipids may include the lipids disclosed in Nardin et al. in Tables 1 and 2; and/or the amphiphilic substances listed in Table 3.

The hydrocarbons preferably comprise at least one activating group for chemical coupling, such as maleimide active ester, amine alcohol, halogenide, thiol, ketone or aldehyde, triple and/or double carbon bonds.

In an embodiment the fatty acids, fatty amines, fatty alcohols and/or hydrocarbons may have carbon chain lengths of from 8 to 24 carbon atoms, preferably from 12 to 20 carbon atoms, or from 16 to 20 carbon atoms. The fatty acids, fatty amines, fatty alcohols and/or hydrocarbons may be saturated or unsaturated. Saturated compounds have the advantage of being chemi cally more stable than the unsaturated ones.

Preferred lipids include phospholipids and fatty acids.

In an embodiment, the phospholipids may be synthetic, semi-synthetic or natural phospholipids, or combinations thereof. Preferred phospholipids include phosphatidylcholines, phosphatidyleth- anolamines, phosphatidylinosites, phosphatidylserines, cephalines, phosphatidylglycerols, lyso- phospholipids, and combinations thereof. Preferred lipids may be selected from 1,2-dioleoyl-sn- glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cycloh exane-carboxamide] (sodium salt), 1,2-Dipalmitoyl-sn-Glycero-3-Phosphothioethanol (Sodium Salt), 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine-N-(succinyl) (sodium salt), 1,2-distearoyl-sn-glycero-3-phosphoethanola- mine-N-[maleimide(polyethylene glycol)-2000] (ammonium salt), and combinations thereof.

In an embodiment, the lipid is activated. Activation of the lipid may facilitate reaction of the CPP with the first lipid during CPP-lipid conjugate formation. An activated lipid includes lipids that comprise an activating group and/or linker. An activating group may be a group that has a greater reactivity towards the CPP than the lipid without the activating group. Suitable activating groups include activated polymeric groups (molecular weight more than 1.000 g/mol) and small molecule activating groups (molecular weight up to 1.000 g/mol). The reaction product of acti vated lipid and CPP is a lipid conjugate wherein the lipid and CPP are indirectly, covalently linked. The activating group forms a covalent bond to the CPP.

Activated polymeric groups may be selected from the group consisting of a polymeric part, e.g. polyethylene glycol (PEG), covalently linked to one or more small molecule activating groups selected from a maleimide group (Mai), active esters, such as N-hydroxy succinimide (NHS), tetra fluorophenol (Tfp), and para-nitrophenol esters; amines, alcohols, ketones, aldehydes, thi ols, halides, triple and double carbon bonds, and combinations thereof. Thus, an activating group may comprise a polymeric part and a reactive group covalently coupled to each other so that the polymeric part links the reactive group to the lipid. Exemplary activated polymeric groups include SM(PEG)24 (PEGylated, long-chain SMCC crosslinker), SMCC (succinimidyl-4- (N-maleimidomethyl)cyclohexane-1-carboxylate)-linker, 6-maleimido hexanoic acid linker, and combinations thereof. The length of the polymeric part may influence the composition’s proper ties. Accordingly, a preferred PEG polymeric part of an activated polymeric group may have a length of 8 to 50 individual PEG units.

Preferred activated lipids may be selected from the group consisting of (2R)-3-((((4,46-dioxo-46- (2,3,5,6-tetrafluorophenoxy)-7,10,13,16,19,22,25,28,31,34,37 ,40,43-tridecaoxa-3-azahex- atetracontyl)oxy) (hydroxy) phosphoryl)oxy)propane-1,2-diyl distearate (PEG(13)-dis- tearoylphosphatidylethanolamine-tetrafluorophenyl ester); 1 ,2-dipalmitoyl-sn-glycero-3-phos- phoethanolamine-N-[4-(p-maleimidomethyl)cyclo-hexane-carboxa mide] (sodium salt), 1,2-di- oleoyl-sn-glycero-3-phosphoethanol-amine-N-[4-(p-maleimidome thyl)cyclohexane-carboxamide] (sodium salt), DSPE-PEG(2000) Maleimide (1,2-distearoyl-sn-glycero-3-phosphoethanolamine- N-[maleimide(polyethylene glycol)-2000] (ammonium salt)), Tfp-PEG -DSPE, Mal-PEGi2-DSPE and combinations thereof.

Oily components

Oily components are lipid and amphiphilic substances comprised in the composition that are not conjugated to a cell penetrating peptide. Oily components may be used in pharmaceutical com positions, e.g. in emulsions, SEDDS or other compositions.

In an embodiment, the oily components include all ingredients of the pharmaceutical composi tion, except the conjugated lipids, having an n-octanol/water partition coefficient of at least 1.0, at least 2.0 or at least 3.0 at 25°C. Oily components may be the components of the composition, except the conjugated lipids, that are immiscible with water at 25°C. Oily components may be components having saturated or unsaturated carbon chain lengths of more than 6, more than 8 or more than 10 carbon atoms. Optional oily components are modified or unmodified fatty acids.

Oily components may be selected from the group consisting of steroids, fatty acids, fatty alco hols, fatty amines, hydrocarbons with carbon chain lengths of at least eight carbon atoms (e.g. liquid paraffin), phospholipids, sphingolipids, ceramides, glycolipids, etherlipids, polyethers, ca rotenoids, and glycerides (mono-, di- and/or triglycerides) and combinations thereof. Steroids may include compounds having a sterane structure. Steroids may include cholesterol and its derivatives. Triglycerides include medium chain triglycerides (e.g. C6 to C12 fatty acids). Mono-, di- or triglycerides and/or fatty acids may be modified, such as PEGylated, ethoxylated, esteri- fied (e.g. with propylene glycol, sorbitol or sorbitan) and/or in salt form. Mono-, di- or triglycer ides include vegetable oils.

Oily components may include amphiphilic compounds/surfactants. Optional oily components with surfactant properties may include mono- and/or diglycerides, ethoxylated mono-, di- or tri glycerides (e.g. Kolliphor® EL), medium chain mono- and/or diglycerides (e.g. C6 to C12 fatty acids), ethoxylated plant oil (e.g. ethoxylated castor oil), monoglycerides of C12 to C20 satu rated or unsaturated fatty acids (e.g. C14 to C18), esters of saturated or unsaturated fatty acids with diols (such as C8 to C20 fatty acids; e.g. 2-hydroxypropyl octanoate; propylene glycol monolaurate), mono- and di-esters esters of polyethylene glycol with medium chain fatty acids (e.g. C6 to C10 fatty acids), sorbitol or sorbitan esters, esters of sorbitol or sorbitan with polyeth ylene glycol and/or fatty acids (e.g. polysorbates), transesterified ethoxylated vegetable oils (e.g. Labrafil® M1944CS), polyethers (e.g. copolymers of polyalkylene glycols, such as Polox- amer®), salts of fatty acids, cetrimonium bromide, bis(2-ethylhexyl) sulfosuccinate and mixtures thereof.

Examples of oily components suitable within the concept of this invention are listed in Nardin et al., Successful development of oral SEDDS: screening of excipients from the industrial point of view, Advanced Drug Delivery Reviews 142 (2019) 128-140. The disclosure of Nardin et al. is incorporated by reference as if fully set forth herein. Oily components may include the lipids dis closed in Nardin et al. in Tables 1 and 2; and/or the amphiphilic substances listed in Table 3.

The total amount of oily components in the composition comprises the cumulative amounts of the oily components listed above, in particular of steroids (including cholesterol and its deriva tives), fatty acids, fatty alcohols, fatty amines, hydrocarbons with carbon chain lengths of at least eight carbon atoms, phospholipids, sphingolipids, ceramides, glycolipids, etherlipids, poly ethers, carotenoids, and glycerides (mono-, di- and/or triglycerides) and combinations thereof, including modified mono-, di- or triglycerides and/or modified fatty acids, such as PEGylated, ethoxylated, esterified (e.g. with propylene glycol, sorbitol or sorbitan) and/or in salt form. Mono- , di- or triglycerides include vegetable oils.

The pharmaceutical composition may include oily components in amounts of at least 1.0 wt%, at least 5.0 wt%, at least 10.0 wt%, at least 20.0 wt%, at least 30.0 wt%, at least 40.0 wt% or at least 50.0 wt%. Optionally, the amount of oily components in the composition may be limited to up to 99.0 wt%, up to 95.0 wt%, up to 90.0 wt%, or up to 85.0 wt%. In certain embodiments with higher amounts of oily components, such as SEDDS, the total amount of oily components in the pharmaceutical composition may be at least 60.0 wt%, at least 70.0 wt% or at least 80.0 wt%.

Alternative embodiments of pharmaceutical compositions with lower amounts of oily compo nents, such as optional types of solid dosage forms, include up to 10.0 wt%, up to 8.0 wt%, up to 6.0 wt% or up to 4.0 wt% of oily components.

Optional embodiments include oily components in amounts of from 1.0 wt% to 99.0 wt%, from 5.0 wt% to 95.0 wt%, or from 10.0 wt% to 90.0 wt%.

In embodiments, the total amount of oily components in the composition is from 0.1 to 100 mol% relative to the total amount of lipid conjugate in the composition. Optionally, the total amount of oily components in the composition is from 5.0 to 100 mol% relative to the total amount of lipid conjugate in the composition. Optionally, the total amount of oily components may be at least 1.25 mol%, at least 3.0 mol%, greater than 5.0 mol%, at least 10.0 mol%, at least 15.0 mol%, at least 20.0 mol% or at least 25.0 mol% relative to the amount of lipid conju gate in the composition. Optional compositions comprise at least 50 mol%, at least 75 mol%, at least 85 mol% or at least 90 mol% of oily components relative to the amount of lipid conjugate in the composition.

The “total lipid content” of the composition is the sum of the proportions of lipid conjugate and oily components.

In certain embodiments, the amount of conjugated lipid is from 0.1 to 100 mol% relative to the total lipid content in the composition. Optionally, the amount of lipid conjugate may be at least 1.25 mol%, at least 3.0 mol%, greater than 5.0 mol%, at least 10.0 mol%, at least 15.0 mol%, at least 20.0 mol% or at least 25.0 mol% relative to the total lipid content in the composition. Op tionally, the amount of lipid conjugate in the pharmaceutical composition may be up to 10.0 mol%, up to 8.0 mol%, up to 6.0 mol%, up to 5.0 mol%, up to 4.0 mol% or up to 3.0 mol% rela tive to the total lipid content. Optional compositions comprise at least 50 mol%, at least 75 mol%, at least 85 mol% or at least 90 mol% of lipid conjugate relative to the total lipid content in the composition.

Further excipients

The pharmaceutical composition may contain excipients, including solubilizers and solvents. Solubilizers may include 2-(2-Ethoxyethoxy)ethanol, propylene glycol, glycerol, tetraglycol and combinations thereof. Generally, solubilizers may be present in the pharmaceutical composition in amounts of up to 20.0 wt%, up to 15.0 wt%, or up to 10.0 wt%. Optionally, the amount may be at least 1.0 wt%, or at least 3.0 wt%.

Solvents may include water, dimethyl sulfoxide, ethanol, isopropanole and combinations thereof. The amount of solvent in the pharmaceutical composition may range from 1.0 wt% to 99.0 wt%. Liquid compositions may contain more solvent than semi-solid and solid composi tions.

Cell penetrating peptides

The cell penetrating peptides (CPPs) in the conjugate may be selected from penetratin, TAT (transactivator of transcription), MAP (model amphiphatic peptide), polyarginines (including R3, R4, R5, R6, R7, R8, R9, R10, R11 and R12), pVEC, transportan, MPG, and combinations thereof. The CPPs may be cyclized or linear, dimerized or un-dimerized. The CPPs may consist of the following sequences, or comprise the following sequences. Penetratin may include SEQ ID NO: 1; TAT may include SEQ ID NO: 2; MAP may include SEQ ID NO: 3; R9 may include SEQ ID NO: 4; pVEC may include SEQ ID NO: 5; transportan may include SEQ ID NO: 6; and/or MPG may include SEQ ID NO: 7. The listed CPPs include functional derivatives and peptide-mimetics of the mentioned sequences. Functional derivatives include CPPs that consist of or comprise the above-mentioned sequences, or sequences having at least 90%, or at least 95% sequence identity therewith. Optional functional derivatives have the sequences disclosed above with up to one, up to two or up to three amino acids replaced by other amino acids. Op tionally, the functional derivative may include additional amino acids.

In an embodiment the CPPs used in this invention are positively charged and/or cyclized. Cy clized CPPs have the advantage of being less reactive and more stable than linear CPPs, which is advantageous within the concept of this invention. Cyclic peptides are more stable towards enzymatic cleavage than linear CPPs. As used herein, the term "cyclized" is not to be construed as relating to a peptide having one ring system only, i.e., the present invention is not limited to monocyclic peptides. Accordingly, the present invention includes cyclopeptides wherein two or more ring systems are covalently linked to each other. Furthermore, the cyclopeptides may also comprise amino acids that are not part of the ring system, i.e. the invention includes branched cyclopeptides. Preferably, the cyclopeptides are monocyclic peptides, and more preferably un branched monocyclic peptides. Further, the CPPs can be composed of L-amino acids, D-amino acids, or mixtures thereof, wherein for linear CPPs, D-amino acids are preferred. In an embodiment, the CPPs comprise a majority of lysine and/or arginine moieties, which have isoelectric points of around 9.5 and 11, respectively. Due to their additional amino or guanidine group, these two amino acids are positively charged under neutral and even under weakly basic conditions. Accordingly, a CPP mostly comprising moieties of said two specific amino acids is positively charged under neutral and weakly basic conditions as well. Herein, the term "majority" means that at least 30%, preferably at least 50%, more preferably at least 60%, and particularly at least 70% of the amino acids forming the CPP molecule are lysine and/or arginine moieties. Thereby, it is ensured that the CPPs have a positive charge under neutral and weakly basic conditions, i.e., have an isoelectric point of more than 7.0. Therefore, in a specific embodiment of the present invention, the CPPs have an isoelectric point of more than 7.0, preferably of more than 7.5, more preferably of more than 8.0 and particularly preferably of more than 8.5. In this context, the isoelectric point of the CPP is the arithmetic mean of the isoelectric points of the amino acids forming the CPP.

In a specific embodiment of the present invention, the CPPs comprise between 2 to 19, prefera bly between 3 to 16, more preferably between 4 to 14, and particularly preferably between 6 to 12 arginine moieties as well as one or more moieties selected from the group consisting of tyro sine, threonine, serine, lysine, aspartic acid, glutamic acid, glutamine, asparagine and cysteine. For example, the CPP may comprise nine arginine moieties and one cysteine moiety in a ring system, and are referred to as a cyclic cysteine R9 derivative (such as SEQ ID NO: 8; RRRRRRRRRC). Another preferred example is a cyclopeptide comprising nine arginine moie ties and one lysine moiety in the ring system, which is referred to as cyclic R9K derivative (SEQ ID NO: 9; RRRRRRRRRK).

The CPPs of this invention include dimerized CPPs, wherein homo- and heterodimers are within the scope of this disclosure. Dimerization of CPPs can be effected by any means known in the art. In a particular embodiment, CPPs are dimerized via the tripeptide KAK.

The amino acids forming the cyclopeptides are not limited to proteinogenic amino acids. Herein, the amino acids may be selected from any amino acids known in the art, and may include the respective D-enantiomer, L-enantiomer, or any mixture thereof.

CPPs may include peptide-mimetics of the CPPs mentioned above. Peptido-mimetics include depsipeptides and peptoids of the CPPs disclosed herein. A depsipeptide CPP is a CPP wherein at least one peptide bond was replaced by an ester bond. Active agent

According to this invention, “peptides” have at least one peptide bond, linking at least two amino acids together. “Polypeptides” have at least eight amino acids. “Proteins” have at least 30 amino acids. This invention is particularly useful for “polypeptides” and “proteins” as active agents. Suitable active agents may be cyclic peptides. Cyclic peptides have improved resistance to gas tric fluid. Preferred active agents have excellent stability in gastric and/or intestinal fluids. In an embodiment, the active agent exhibits a degradation half-life in simulated gastric and/or intesti nal fluid at 37 °C of at least 10 minutes, preferably at least 20 minutes or at least 30 minutes.

For testing the stability in simulated gastric (comprising pepsin) and intestinal fluid (comprising pancreatin), dissolved peptides can be incubated in fast state simulated gastric fluid or in fast state simulated intestinal fluid (FaSSGF and FaSSIF) prepared according to USP and incubated on a shaker at 37 °C for one hour. Samples can be withdrawn after 5, 10, 15, 30, 45 and 60 min and analyzed by HPLC or LC-MS. From the obtained profile a degradation half-life can be cal culated.

Optionally, the active agent is a peptidic active agent. Useful peptidic active agents include pep tides and proteins, such as polypeptides having at least six amino acids. Preferred active agents have molecular weights in the range of from 600 to 200.000 g/mol, preferably from 1.000 to 150.000 g/mol, or from 2.000 to 80.000 g/mol. Preferred active agents may be selected from anti-cancer agents (e.g. rituximab, trastuzumab, nivolumab); immune-modulatory agents (e.g. adalimumab, eternacept, cytokines, interferons, glatiramer acetate); hormones such as insulin, GLP-1 analogues such as exenatide and liraglutide, somatostatin and its analogues such as oc treotide and pasireotide, hGh, and; glycopeptide antibiotics e.g. vancomycin and daptomycin; peptide drugs for hepatitis treatment such as Myrcludex B; and peptides and antibodies acting on cellular receptors such as glucagon, leuprolide, octreotide, vasopressin, and cetuximab.

Suitable active agents include, without limitation, vancomycin, glatiramer acetate, bulevirtide, octreotide, insulins, and liraglutide, as well as other GLP (glucagon-like peptide)-analogues such as exenatide, lixisenatide, albiglutide, dulaglutide, taspoglutide, and semaglutide, and anti bodies (e.g. etanercept; pegfilgrastim; adalimumab, infliximab, rituximab, epoietin alfa, tra- tuzumab, ranibizumab, beta-interferon, omalizumab). Other examples include pharmaceutically active agents selected from the group consisting of hormones, such as human growth hormone, growth hormone releasing hormone, growth hormone releasing peptide, interferons, colony stimulating factors, interleukins, macrophage activating factor, macrophage peptide, B cell fac tor, T cell factor, protein A, allergy inhibitor, cell necrosis glycoproteins, immunotoxin, lympho- toxin, tumor necrosis factor, tumor suppressors, metastasis growth factor, alpha-1 antitrypsin, albumin and fragment polypeptides thereof, apolipoprotein-E, erythropoietin, factor VII, factor VIII, factor IX, plasminogen activating factor, urokinase, streptokinase, protein C, C-reactive pro tein, renin inhibitor, collagenase inhibitor, superoxide dismutase, platelet-derived growth factor, epidermal growth factor, osteogenic growth factor, bone stimulating protein, insulin, atriopeptin, cartilage inducing factor, connective tissue activating factor, follicle stimulating hormone, lutein izing hormone, luteinizing hormone releasing hormone, nerve growth factors, parathyroid hor mone, relaxin, secretin, somatomedin, insulin-like growth factor, adrenocortical hormone, gluca gon, cholecystokinin, pancreatic polypeptide, gastrin releasing peptide, corticotropin releasing factor, thyroid stimulating hormone, monoclonal or polyclonal antibodies against various viruses, bacteria, or toxins, virus-derived vaccine antigens, cyclosporine, rifampycin, lopinavir, ritonavir, telavancin, oritavancin, dalbavancin, bisphosphonates, itraconazole, danazol, paclitaxel, naproxen, capsaicin, albuterol sulfate, terbutaline sulfate, diphenhydramine hydrochloride, chlorpheniramine maleate, loratidine hydrochloride, fexofenadine hydrochloride, phenylbuta zone, nifedipine, carbamazepine, betamethasone, dexamethasone, prednisone, hydrocortisone, 17 beta-estradiol, ketoconazole, mefenamic acid, beclomethasone, alprazolam, midazolam, miconazole, ibuprofen, ketoprofen, prednisolone, methylprednisone, phenytoin, testosterone, flunisolide, diflunisal, budesonide, fluticasone, glucagon-like peptide, C-Peptide, calcitonin, lutenizing hormone, prolactin, adrenocorticotropic hormone, leuprolide, interferon alpha-2b, in terferon beta-la, sargramostim, aldesleukin, interferon alpha-2a, interferon alpha-n3alpha-pro- teinase inhibitor, etidronate, nafarelin, chorionic gonadotropin, prostaglandin E2, epoprostenol, acarbose, metformin, desmopressin, cyclodextrin, antibiotics, antifungal drugs, steroids, anti cancer drugs, analgesics, anti-inflammatory agents, anthelmintics, anti-arrhythmic agents, peni cillins, anticoagulants, antidepressants, antidiabetic agents, antiepileptics, antihistamines, anti hypertensive agents, antimuscarinic agents, antimycobacterial agents, antineoplastic agents, CNS-active agents, immunosuppressants, antithyroid agents, antiviral agents, anxiolytic seda tives, hypnotics, neuroleptics, astringents, beta-adrenoceptor blocking agents, blood products and substitutes, cardiacinotropic agents, contrast media, corticosteroids, cough suppressants, expectorants, mucolytics, diuretics, dopaminergics, antiparkinsonian agents, haemostatics, im munological agents, lipid regulating agents, muscle relaxants, parasympathomimetics, parathy roid calcitonin, prostaglandins, radiopharmaceuticals, sex hormones, anti-allergic agents, stimu lants, anoretics, sympathomimetics, thyroid agents, vasidilators, xanthines, heparins, therapeu tic oligonucleotides, somatostatins and analogues thereof, and pharmacologically acceptable organic and inorganic salts or metal complexes thereof. Optionally, the active agent may be se lected from pasireotide, bremelanotide, oxytocin, ziconotide, corticorelin, enfuvirtide, eptifiba- tide, buserelin, goserelin, leuprolide, lanreotide, glatiramer acetate, pentagastrin and combina tions thereof. In an embodiment, the pharmaceutical composition is free of nucleic acid active agents, such as DNA and RNA.

Examples of such peptidic active agents are somatostatin receptor agonists such as pasire- otide, lanreotide, octreotide or combinations thereof. Another preferred group is the glucagon like peptide-1 (GLP-1) receptor agonists such as liraglutide, exenatide, lixisenatide, albiglutide, dulaglutide, taspoglutide, semaglutide, and derivatives and combinations thereof. The peptidic active agent may include GLP-1 receptor agonists and/or derivatives thereof. The derivatives may be those disclosed in EP 3479841 A2, which is incorporated by reference as if fully set forth herein. Improving therapy adherence is particularly useful for somatostatin receptor and GLP-1 receptor agonists because the patient must administer these active agents very regularly for a satisfactory effect.

Method of treatment

In an aspect, the invention includes a method of treatment of a patient, comprising administer ing to said patient an effective amount of an active agent in a composition according to this dis closure.

The method includes enhancing absolute and/or relative oral bioavailability of an active agent. The invention includes a pharmaceutical composition as described herein for use in therapy.

Method of making

In an aspect, the invention includes a method of making a pharmaceutical composition, com prising the steps of a. reacting at least one CPP (cell penetrating peptide) with at least one lipid to obtain a lipid conjugate, b. optionally purifying the lipid conjugate to obtain a purified lipid conjugate, c. optionally lyophilizing the lipid conjugate to obtain a lipid conjugate lyophilisate, d. adding active agent, e. incorporating lipid conjugate and active agent into a pharmaceutical dosage form, wherein the amount of lipid conjugate is from 0.1 to 100 mol% relative to the total amount of li pid in the composition.

The method of this invention may include the step of lyophilizing the lipid conjugate to obtain a lyophilisate. Preferred lyophilisates have limited water content, preferably of not more than 5 wt.-%, or less than 3 wt.-%. The water content can be determined by Karl-Fischer-titration or automated systems such as Water Content Analyzer. Compared to liquid formulations lyophi- lized lipid conjugates have better long-term stability.

The step of lyophilizing the lipid conjugate may include: c1. preparing a mixture of the lipid conjugate and at least one lyoprotector.

There is an optimum amount range for lyoprotector in relation to the amount of lipid conjugate in the mixture. Preferably, the amount of lyoprotector ranges from 0.01 to 2 g lyoprotector per gram of lipid conjugate, preferably from 0.02 to 1 g per gram of lipid conjugate, or from 0.03 to 0.5 g per gram of lipid conjugate. In preferred embodiments, a minimum value is at least 0.03 g per 1 g of lipid conjugate. A maximum value may be 0.3, 0.2 or 0.1 g lyoprotector per g of lipid conjugate. In embodiments, these limitations concerning the amount of lyoprotector relative to the amount of lipid apply to the pharmaceutical composition.

Alternatively or in addition, the active agent may be lyophilized. In an embodiment, the pharma ceutical composition comprises a mixture of lyophilized lipid conjugate and lyophilized active agent.

The lyoprotector may be selected from the saccharides, preferably monosaccharides or disac charides, including sugars and sugar alcohols. The lyoprotector may be selected from sucrose, mannitol, glucose, trehalose, lactose, palatinose and combinations thereof.

The step of incorporating lipid conjugate and active agent into a pharmaceutical dosage form may include, without limitation, the preparation of tablets, pills, capsules, pellets, liquids (includ ing suspensions), powder, effervescent formulations, pastes, lozenges, chewing gums, gels, sprays or granules.

Brief description of figures

Figure 1A Chemical structure of activated lipid Tfp-PEGi3-DSPE.

Figure 1B Chemical structure of activated lipid Mal-PEGi2-DSPE. Figure 2 Diagram of particle size and PDI of lipid conjugates and octreotide formed in cit rate buffer, and changes of size and PDI over time

Figure 3 Diagram of zeta potential measured at 10 minutes, 3 hours and 24 hours after preparation of particles

Figure 4 Diagram of absolute oral bioavailability of octreotide in compositions with lipid conjugate, without lipid conjugate and in CPP-lipid liposomes

Figure 5 Plasma concentration curves and resulting diagram of absolute oral bioavailability of octreotide in compositions with lipid conjugate and without lipid conjugate

Figure 6 Plasma concentration curves and resulting diagram of absolute oral bioavailability of pasireotide in compositions with lipid conjugate and without lipid conjugate

Figure 7 Plasma concentration curves of pasireotide after administration of active agent with (black symbols) and without lipid conjugates (white symbols)

Figure 8 Synthesis of exemplary conjugates

Figure 9 Synthesis of exemplary conjugates

Figure 10 Structure of a modified phospholipid used to make a conjugate.

Figure 11 Example conjugate and modified phospholipid

Figure 12 Further example conjugate and modified phospholipid

Figure 13 Comparison of zetapotentials of both octreotide and exenatide containing SEDDS

Figure 14 Comparison of particles sizes of both octreotide and exenatide containing SEDDS

Figure 15 Comparison of PDI values of both octreotide and exenatide containing SEDDS

Figure 16 Example of a conjugated lipid

Figure 17 Diagrams of size, PDI and zetapotential of micelles containing a conjugated lipid

Figure 18 Diagram of absolute oral bioavailability of octreotide in compositions with lipid conjugate SEDDS and without lipid conjugate Figure 19 Diagram of absolute oral bioavailability of exenatide in compositions with lipid conjugate SEDDS, lipid conjugate micelles and without lipid conjugate.

Examples

Procedures

Zetasizer measurements

The particle size, PDI and zeta potential were determined at room temperature using a zetasizer Nano ZS from Malvern™ (Malvern Instruments Ltd., Worcestershire, United Kingdom). Size and PDI were measured after dilution to a lipid concentration of 0.07 mg/ml with a 10 mM phosphate buffer with a pH of 7.4 using the automatic mode. The zeta potential was determined after dilution to a lipid concentration of 0.14 mg/ml by a 50 mM phosphate buffer with a pH of 7.4. The default settings of the automatic mode of the zetasizer Nano ZS from Malvern™ (Mal vern Instruments Ltd., Worcestershire, United Kingdom) were the following: number of measure ments = 3; run duration = 10 s; number of runs = 10; equilibration time = 60 s; refractive index solvent 1.330; refractive index polystyrene cuvette 1.590; viscosity = 0.8872 mPa s; tempera ture = 25 °C; dielectric constant = 78.5 F/m; backscattering mode (173°); automatic voltage se lection; Smoluchowski equation.

1. Conjugate synthesis

The synthesis of the lipid conjugate consists of the solid-phase peptide synthesis of lysinyl- nona-arginine utilizing the Fmoc/tBu strategy. Purity is controlled after loading of lysine on a solid phase resin, after coupling of five arginines, and after completion of the solid-phase syn thesis. The peptide is cleaved from the resin with side-chain protection groups intact using HFIP/DCM and purified via HPLC. The head-to-tail cyclization of the side-chain protected pep tide is performed in solution (ACN/DCM) using HATU/DIEA, which impedes racemization, fol lowed by deprotection with TFA/water/anisole and precipitation with MTBE to obtain the cyclo- (Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Lys) as intermediate. Purification of the peptide interme diate is performed by HPLC on a Phenyl/Hexyl column.

The peptide intermediate is conjugated in solution (DMF/water) with 1.2 equivalents of the sec ond intermediate (2R)-3-((((4,46-dioxo-46-(2,3,5,6-tetrafluorophenoxy)- 7, 10, 13, 16, 19,22,25,28,31 ,34, 37,40, 43-tridecaoxa-3-azahexatetracontyl)oxy)(hydroxy)-phos- phoryl)oxy)propane-1,2-diyl distearate and purified via HPLC on a Phenyl/Hexyl column with concurrent ion exchange to acetic acid.

The resulting lipid conjugate is shown in Figure 11. 2. Synthesis of the lipid conjugates

3 equivalents of CPP (cyclic R9-peptides) and 10 equivalents of DIPEA were added to malei- mido-PEG(12)-distearoylphosphatidylethanolamine (Iris Biotech) (shown in Figure 1B) or PEG(13)-distearoylphosphatidylethanolamine-tetrafluorophenyl ester (Iris Biotech) (shown in Figure 1A) dissolved in DMF in a concentration of 5 mg/ml. The reaction mixture was stirred overnight at room temperature. The reaction mixture was diluted with a 1:2 mixture of ACN/H2O and purification was performed via HPLC using a Chromolithe® Performance RP-C18e column (100 x 3 mm). Water and acetonitrile containing 0.05 % TFA were used as eluents with a flow rate of 2 ml/min.

Figure 12 shows the resulting lipid conjugate structure.

3. Administration of octreotide to beagle dogs

The lipid conjugate produced according to example 2 was used in lyophilized form. The lyophi- lized lipid conjugate was blended with lyophilized octreotide and suspended in citrate buffer. Upon suspension, the lipid conjugate formed particles. The particles had sizes of about 10 to 20 nm with a PDI in the range of from 0.17 to 0.20 (Figure 2). Particles were remarkably stable over a period of 24 hours. Figure 3 shows that the zeta potential of the particles was stable over 24 hours. The zeta potential was in a range of about 3.5 mV to about 7.0 mV.

A bioavailability study was performed at LPT, Hamburg, Germany (Study No 36229) by analyz ing the systemic absolute bioavailability of octreotide. The pharmaceutical composition compris ing 5 mg lipid conjugate and 1.5 mg octreotide, prepared in citrate buffer (100 mM, pH 5.5) was administered to four beagle dogs by gavage. The amount of active agent in plasma was meas ured and compared to administration of active agent alone in a dose of 1.5 mg, and with active agent in CPP-lipid-conjugate liposomes prepared in citrate buffer (100 mM, pH 5.5) according to WO 2018/178395 A1.

The results are shown in Figures 4 and 5. Figure 5 illustrates the plasma concentration curves and the resulting absolute bioavailabilities. The absolute bioavailability of oral octreotide in creased more than 4-fold due to the lipid-conjugate in the composition. The absolute bioavaila bility, i.e. compared to intravenous bolus administration of 0.1 mg/dog, was essentially the same as bioavailability of the same active agent in the liposome. 4. Administration of pasireotide to beagle dogs

The experiment of example 3 was repeated with 1.5 mg pasireotide as active agent, comparing the active agent in citrate buffer (100 mM, pH 5.5) with and without 5 mg of lipid conjugate.

The result is shown in Figure 6. The bioavailability was more than doubled due to the lipid con jugate in the composition.

Figure 7 shows the pharmacokinetics of pasireotide in plasma of beagle dogs after administra tion of free active agent (white symbols) and active agent with lipid conjugate (black symbols).

5. Pharmacokinetic evaluation of octreotide lipid conjugate

The effect of lipid conjugate as absorption enhancer for octreotide was investigated in a study in beagle dogs at Charles River, Evreux, France (Study No 47656 PAC), by analyzing the systemic absolute bioavailability of octreotide. Animals were treated with a combination of octreotide/lipid conjugate or octreotide alone, prepared in citrate buffer (100 mM, pH 5.5), by single oral admin istration via gavage (table). For determination of the absolute bioavailability plasma concentra tions after intravenous bolus injection were determined. Between the cycles, a washout phase of one week was performed.

The following observations were included in the overall study design:

Body weight, food consumption, and clinical observations:

Morbidity and mortality: each animal was checked for mortality and morbidity at least once a day during the study, including weekends and public holidays. Clinical signs: each animal was ob served at least once a day, during pre-treatment and on the day of treatment, for the recording of clinical signs.

No signs of toxicity were observed throughout the study.

First, animals received an intravenous bolus injection of 0.01 mg/kg octreotide. The consecutive administrations were given orally by gavage. After a wash-out of one week animals #1 and #2 received 0.12 mg/kg free octreotide and animals #3 and #4 received 0.12 mg/kg octreotide with 0.3 mg/kg lipid-conjugate. After a wash-out of one week animals #1 and #2 received 0.12 mg/kg free octreotide with 1 mg/kg lipid-conjugate and animals #3 and #4 received 0.12 mg/kg oc treotide with 3 mg/kg lipid-conjugate. After a wash-out of one week animals #1 and #2 received 0.12 mg/kg free octreotide with 10 mg/kg lipid-conjugate and animals #3 and #4 received 0.12 mg/kg octreotide with 30 mg/kg lipid-conjugate. After a wash-out of one week animals #1 and #2 received 0.012 mg/kg free octreotide with 1 mg/kg lipid-conjugate and animals #3 and #4 re ceived 1.2 mg/kg octreotide with 1 mg/kg lipid-conjugate.

The lipid conjugate increased bioavailability of oral octreotide up to more than 3-fold. With re spect to the determined bioavailability of octreotide and to the efforts to use the minimal re quired amount of lipid conjugate for the desired formulation, a dose range of 0.3 - 1.0 mg/kg conjugate is considered to be the optimal dose. Effective octreotide plasma concentrations were reached with 0.12 mg/kg octreotide. The respective calculated absolute bioavailabilities are listed in the table below. Additionally, a 10-fold increase in octreotide dose (1.2 mg/kg) revealed approximate dose linearity when administered with 1 mg/kg lipid-conjugate.

6. Preparation of conjugates

Figure 8 illustrates the synthesis of exemplary conjugates. The linker used in this example was SM(PEG)8, a PEGylated, long-chain SMCC crosslinker, and succinimidyl([N-maleimidopropio- namido]-ethyleneglycol)ester, respectively. R9-CPP stands for either the linear or cyclic nona- arginine peptide. In case of the cyclic it is cyclized via a lysine (R9K) and coupled at the side chain amino function of this lysine.

For coupling of cyclic CPP to the bifunctional PEG-linker, as a first step the cyclized CPP was coupled by an additional lysine to the linker (1.). For this reaction an excess of the CPP was used. In the second step this intermediate product was coupled to the thiol modified phospho lipid (2.). The modified phospholipid was 1 ,2-dipalmitoyl-sn-glycero-3-phosphothioethanol, so dium salt.

Figures 9 illustrates the synthesis of exemplary conjugates.

Cysteine-modified penetratin was coupled to the headgroup-modified phospholipid which is shown in Figure 10. Its chemical name is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [maleimide(polyethylene glycol)-2000], ammonium salt.

Figure 11 shows an exemplary conjugate and a modified phospholipid used to make the conju gate. A further conjugate comprising cyclic R9C was prepared. The conjugate prepared and the modi fied lipid used to make it are shown in Figure 12.

7. Preparation of SEDDS

SEDDS containing either octreotide (c = 0.21 mg/ml) or exenatide (c = 0.14 mg/ml) with a lipid conjugate concentration of c = 1.5 mg/ml were prepared as follows:

The oily component (corn oil; 310 mg, 10.33 mg/ml) was weighted in a vial. Subsequently, the required amounts of lipid-conjugate (as shown in Figure 11) and the respective API were added. Afterwards, the SEDDS were prepared by the addition of the required amount of citrate buffer (pH = 5.5). Upon suspension, the lipid-conjugate and the oily component formed SEDDS. The SEDDS had sizes of about 150 to 200 nm with a PDI in the range of 0.3 to 0.4 (Figures 14 and 15) and a strong positive zeta potential of about +8 mV (Figure 13). The concentration of oc treotide was 0.21 mg/ml, and exenatide concentration was 0.14 mg/ml. The number of meas urements was n=3 for zeta potential and PDI, n=6 for particle size.

The encapsulation efficiency was 98.06% for octreotide and 67.55% for exenatide. The encap sulation efficiency of SEDDS containing either octreotide (c = 0.21 mg/ml) or exenatide (c =

0.14 mg/ml) with a lipid conjugate concentration of c = 1.5 mg/ml was determined as follows:

After preparation of SEDDS, a volume of 500 pi of the respective formulation was loaded on a Sephadex G-25 gel filtration column (NAP-5 size exclusion column). Elution was performed with a volume of 1 ml of water. The amount of the respective API in this eluted fraction was com pared with the API content of the unpurified fraction under consideration of potential dilution ef fects. Concentrations were determined by UPLC-MS/MS quantification.

8. Bioavailability of octreotide SEDDS formulation

The administration of octreotide in SEDDS formulation was investigated in a study in beagle dogs at Charles River, Evreux, France (Study No 47656 PAC and 48482 PAC). Individual sys temic absolute bioavailability of octreotide in SEDDS (dose: 1.5 mg/animal) was evaluated by comparison to intravenous administration of 0.1 mg/animal of free drug, and oral administration of free drug at a dose of 1.5 mg/animal. Animals were treated with octreotide in SEDDs formula tion, or octreotide alone, prepared in citrate buffer (100 mM, pH 5.5), by single oral administra tion via gavage. For determination of the absolute bioavailability plasma concentrations after in travenous bolus injection (in PBS) were determined. Between the cycles, a wash-out phase of one week was performed. Bioavailability of the SEDDS formulation was 2.8%, whereas free drug bioavailability was 2.1% (Figure 18). Thus, performance of the SEDDS formulation was about 25% improved by the SEDDS.

9. Preparation of C16-TAT micelles

The C16-TAT lipid-conjugate (see Figure 16) was used in lyophilized form. The lyophilized lipid- conjugate was blended with lyophilized octreotide and suspended in citrate buffer (pH = 5.5). Upon suspension, the lipid conjugate formed micelles. The particles had sizes of about 40 to 50 nm with a PDI in the range of from 0.3 to 0.5 (Figure 17A and B). The zeta potential was in a range of about +10 mV to about +15 mV (Figure 17C). Figure 17 shows the results of measure ment of size, PDI and zetapotential of C16-TAT-micelles (c = 1 mg/ml), containing 0.2 mg/ml oc treotide), n=3.

10. Preparation of exenatide formulations

The co-administration of lipid conjugate with the peptide therapeutic exenatide was investigated in a study in beagle dogs at Charles River, Evreux, France (Study No 48482 PAC). Individual systemic absolute bioavailability of exenatide in SEDDS (dose: 1 mg/animal) and micellar for mulation (only lipid-conjugate in lyophilized form; dose: 1 mg/animal) was evaluated by compari son to intravenous administration of 0.1 mg/animal of free drug, and oral administration of free drug at a dose of 1 mg/animal. Animals were treated with a combination of exenatide/lipid con jugate, exenatide in SEDDs formulation, or exenatide alone, prepared in citrate buffer (100 mM, pH 5.5), by single oral administration via gavage. For determination of the absolute bioavailabil ity plasma concentrations after intravenous bolus injection (in PBS) were determined. Between the cycles, a wash-out phase of one week was performed. Compared to free drug, the bioavail ability of exenatide in a lipid-conjugate micelle formulation with Aprotinin was increased by fac tor 3.9 (F = 0.093%), and in lipid-conjugate SEDDS formulation by a factor of 4.2 (F=0.10%) in beagle dogs (Figure 19).

The SEDDS were prepared as described in example 7. In the bioavailability study in beagles, each dog received 7 ml of the SEDDS formulation.

For preparation of the micellar formulation, the lipid-conjugate was used in lyophilized form. The lyophilized lipid-conjugate was blended with lyophilized exenatide (c = 0.14 mg/ml) and the pro tease inhibitor Aprotinin (c = 3.5 mg/ml) and suspended in citrate buffer (pH = 5.5). Upon sus pension, the lipid conjugate formed micelles. In the bioavailability study in beagles, each dog re ceived 7 ml of the micelle formulation. Plasma concentrations of all investigated peptide therapeutics were determined by quantifica tion using a validated UPLC-MS/MS assay (according to the pertinent recommendations for bio- analytical method development of the US FDA and EMA). All assays were performed on a Wa ters Xevo TQ-XS triple quadrupole tandem mass spectrometer coupled to a Waters Acquity classic UPLC using C18 columns, heated electrospray ionization (ESI), and selected ion moni toring in the positive ion mode.