WO2015032981A1

WO2015032981A1 - Erythropoietin conjugates having oral bioavailability

Info

Publication number: WO2015032981A1
Application number: PCT/EP2014/069198
Authority: WO
Inventors: Mateja Cegnar; Barbara Podobnik; Vladka Gaberc Porekar; Mateja Novak Stagoj; Spela JALEN; Radovan Komel; Simon CASERMAN
Original assignee: Lek Pharmaceuticals dd
Current assignee: Lek Pharmaceuticals dd
Priority date: 2013-09-09
Filing date: 2014-09-09
Publication date: 2015-03-12
Anticipated expiration: 2016-03-09

Abstract

The present invention relates to erythropoietin (EPO) conjugates having a high bioavailability and efficacy, especially when administered perorally. Also provided are methods for preparing the EPO conjugates, their use for treating various diseases, and pharmaceutical compositions comprising said EPO conjugates, alone or in combination with absorption enhancers and/or coating polymers.

Description

Erythropoietin conjugates having oral bioavailability Abstract

Background

Erythropoietin (EPO) is a glycoprotein hormone, which is mainly produced in the kidney, but also to a certain extent in the liver. It controls erythropoiesis (red blood cell production) and acts as a cytokine for erythrocyte (red blood cell) precursors in the bone marrow. EPO exerts its biological effect of stimulating the proliferation and differentiation of erythroid progenitors by binding to specific cell-surface receptors, i.e., erythropoietin receptors (EPO-Rs) that are most abundant on erythroid progenitors located primarily in the bone marrow. EPO-Rs are also located in non- hematopoietic tissues where they mediate other biological functions.

EPO increases hemoglobin serum levels thus reducing the need for blood transfusion. It is used in the treatment of anemia, for example resulting from chronic kidney disease and

myelodysplasia, from cancer therapy (for example chemotherapy and/or radiation), as well as from other critical illnesses (for example heart failure).

Due to its low bioavailability after oral administration, the use of EPO is limited to invasive, parenteral administration. Therefore, there are currently no oral formulations of EPO available. Major constraints in this regard are the poor absorption as well as the rapid and extensive degradation of EPO in the gastrointestinal tract. The poor absorption is mostly due to EPO's large molecular size and low lipophilicity, which limits its permeability across the intestinal epithelium.

EPO can be produced biosynthetically using recombinant DNA technology. The recombinant human EPO (rhEPO) dosage form currently available on the market is a ready for injection liquid vial (syringe), which is usually administered 2-3 times weekly. To achieve a therapeutic effect of parenterally administered EPO, cumulative doses are required that significantly exceed levels of endogenous EPO. These high serum levels result in prolonged circulation times of EPO and unspecific binding to non-targeted tissue, which may lead to undesired side effects. In fact, severe side effects have been recently reported in chemotherapy and/or radiation receiving cancer patients taking anti-anemia drugs. These side effects, i.e., increased risk of death and tumor growth, were attributed to the high serum levels of parenterally administered EPO, which enable EPO to act on other non-target (undesired) tissues. The parenteral administration, as practiced with the current EPO dosage forms on the market, is also inconvenient for patients, especially in case of chronic therapies. Apart from the inconvenience for the patients, parenteral delivery systems are also more expensive in terms of production and drug administration through health care professionals.

Therefore, there is a need to develop new and improved EPO delivery systems, which enable a low dose long-term therapy and the delivery of EPO into the systemic circulation in an adequate amount, not exceeding the amount necessary for therapeutic effects, so that the adverse effects related to high EPO levels following parenteral administration are reduced.

From the standpoint of patient compliance, especially in case of chronic therapies, the peroral route would be the most convenient. It is also contemplated that the peroral delivery of EPO would benefit from entering the systemic circulation via the portal vein, which would more closely resemble the physiological pathway of endogenous EPO that is produced in the liver.

US 2006/01 1 1279 provides conjugates of EPO with polyethylene glycol (PEG) moieties. The conjugates are linked via an intact glycosyl linking group interposed between, and covalently attached to the peptide and the modifying group. The conjugates are formed from glycosylated peptides by the action of a glycosyltransferase. In example 7, an EPO-PEG derivative was intravenously administered into rats and compared with non-pegylated EPO. In this example, the pegylated EPO showed a longer distribution time in the circulation than the non pegylated EPO. However, a longer circulation time may be indicative for a lower biological activity, because circulating EPO is not present at the place of action.

Consequently, there is an ongoing need for an effective peroral delivery system for EPO, which avoids the known disadvantages associated with the parenteral EPO formulations as described above, and which enhances patient compliance.

The present invention overcomes the aforesaid problems by providing EPO conjugates having a high bioavailability and efficacy, which are suitable for oral administration.

Detailed description

The present invention provides novel modified EPO derivatives comprising one or more lipophilic side chains referred to herein as EPO conjugates. It has been surprisingly found that a better oral bioavailability of EPO can be achieved by this covalent modification of EPO. In addition to the high bioavailability after oral administration, the novel EPO conjugates also display high efficacy. Pharmaceutical compositions of the novel EPO conjugates are further provided herein, which involve the use of an absorption enhancer and/or a coating with a coating polymer. Both, conjugation and formulation with an absorption enhancer and/or a coating with a coating polymer are able to promote the permeability of the protein across the intestinal epithelia in a synergistic manner.

The types of conjugations provided by the present invention may also be applied to other therapeutic proteins, similarly resulting in increased bioavailability of these therapeutic proteins after peroral administration. These other therapeutic proteins include, but are not limited to, Granulocyte colony- stimulating factor (G-CSF), Granulocyte macrophage colony-stimulating factor (GM-CSF), Insulin like Growth Factor (IGF), Interleukine 17, Interleukine 2, Interferon alpha, Interferon beta, Parathyroid hormone (PTH), Human Growth Hormone (HGH), Fibroblast growth factor (FGF), Relaxin, Salmon Calcitonin and Fab fragments. The resulting conjugates may further be formulated into pharmaceutical compositions corresponding to the compositions provided herein.

EPO Conjugates

The term "EPO" as used herein refers to erythropoietin and may be a natural or recombinant EPO, recombinant human EPO (rhEPO), or any protein or peptide having in vivo biological activity of the EPO glycoprotein, e.g., a wild type or mutant EPO, an EPO peptidomimetic, or an EPO fragment. The species from which EPO is derived can be animal, mammal or human species. Human EPO is, however, preferred. Human EPO comprises the known human erythropoietin having 165 amino acids after posttranslational cleavage of the N-terminal signal peptide of 27 amino acids and the C-terminal arginine. It further comprises human EPO in it's naturally glycosylated state, i.e., including all carbohydrate side chains. The abbreviation "P" as used herein stands for EPO as defined above.

The invention provides novel EPO conjugates comprising EPO and one or more lipophilic side chain(s). The one or more lipophilic side chain(s) may be attached to the oamino group of the N-terminal amino acid and/or to the ε-amino group of lysine residues present on the EPO amino- acid sequence, e.g., via an amide bond.

Alternatively, the lipophilic side chain may be attached to the sugar components present in glycoproteins such as EPO.

Each of the one or more lipophilic side chain(s) attached to the EPO comprises one or more moieties "B" and/or a branched or straight chain alkylene moiety -(CH₂)_m-■ While each of the one or more lipophilic side chain(s) attached to the EPO may comprise up to 6 moieties "B", it is equally preferred that each of the one or more lipophilic side chain(s) attached to the EPO comprises only one moiety "B".

The moiety B is a bivalent aromatic moiety selected from arylene or heteroarylene. The term "arylene" refers to a bivalent aromatic moiety comprising 4, 5, 6 or 7 ring carbon atoms. The term "heteroarylene" refers to an arylene group comprising 1 , 2, 3 or 4 hetero-ring atoms each independently selected from oxygen, nitrogen, and sulfur. The arylene or heteroarylene moiety may, each and independently, be unsubstituted, or may be substituted with one or two substituents each independently selected from straight chain or branched Ci_₄- alkyl, -OH, -SH, -NH₂, -CHO, and -COOH.

The alkylene moiety -(CH₂)_m- is branched or straight chain alkylene moiety having 4 to 20 carbon atoms, or from 5 to 1 1 carbon atoms, or having 5, 7, 9 or 1 1 carbon atoms (i.e., m is an integer from 4 to 20, or from 5 to 1 1 , or m is 5, 7, 9 or 1 1 ). The lipophilic side chain(s) may further comprise a polar end group Y and/or one or more linker(s) each independently selected from the group consisting of L and X.

If present, the linker L is selected from -NHCO-, -CONH-, -CH=N-, or -NHCH₂-; the linker X is selected from -CH₂NH-, -NHCO-, -CONH-, -COO-, -SS-, -S-CH₂- and -NN-; and the polar end group Y is a polar group selected from -COOH, -CHO and -COOCH₃. While it is preferred that a polar group Y is present in the EPO conjugates of the invention, it is equally preferred that no polar group Y is present therein.

The number of lipophilic side chains attached to EPO ranges from 1 to 6, for example, 1 , 2, 3, 4, 5 or 6 side chains may be present in the EPO conjugates according to the invention. If more than one lipophilic side chains are present in the EPO conjugates of the invention, e.g., if 2 or more side chains are present, these lipophilic side chains may either be the same or different. In one embodiment, the one or more lipophilic side chain(s) attached to the EPO comprise(s) one or more of a bivalent aromatic moiety B, wherein B is arylene. Said arylene moiety may have 4, 5, 6 or 7 ring carbon atoms. Said arylene moiety is either unsubstituted, or is substituted with one or two substituents each independently selected from -CH₃, -OH, -SH, -NH₂, -CHO, and -COOH. The substituent -CH₃ is preferred. Preferably, B is a phenylene, toluylene, or xylene moiety. More preferably, B is an o-, m- or p-phenylene moiety, most preferably a p-phenylene moiety.

In another embodiment, the one or more lipophilic side chain(s) attached to the EPO comprise(s) one or more of a bivalent aromatic moiety B and/or a branched or straight chain alkylene moiety -(CH₂)m-, and further one or more linker(s) each independently selected from the group consisting of L and X, and/or a polar end group Y, wherein

L represents -NHCO-;

B represents an arylene group having 6 carbon atoms, and is preferably a p-phenylene group; X represents -CH₂NH-;

m is an integer from 4 to 20, or from 5 to 1 1 , or is 5, 7, 9 or 1 1 ;

Y is selected from -COOH, -CHO, and -COOCH₃; preferably Y is -COOH.

In one embodiment, the EPO conjugates of the present invention are represented by the following formula

P-[(L)-B- (X)-(CH₂)_m-Y]_n,

wherein

P represents EPO;

L represents a covalent linkage between P and B, and is selected from -NHCO-, -NHCH₂- more preferably L represents -NHCO-;

B represents a hetero- or homo-bifunctional linker, enabling attachment of the lipophilic moiety to the protein. B represents a bivalent aromatic moiety, preferably an arylene or heteroarylene moiety with 4, 5, 6 or 7 ring carbon atoms, more preferably 6 carbon atoms, and optionally, one oxygen, nitrogen, or sulfur ring atom, which arylene or heteroarylene group may be unsubstituted or substituted with one or two groups selected from straight chain or branched d-4-alkyl, -OH, -SH, -NH₂, -CHO, -COOH. A preferred substituent for the arylene or heteroarylene group is methyl. Preferably, B represents a phenylene, toluylene, xylene or pyridylene group. More preferably, B is an o-, m- or p-phenylene group, most preferably a p-phenylene group;

X represents a covalent linkage between B and the lipophilic moiety, wherein X is

either -CH₂NH-, -NHCO-, -CONH-, -COO-, -SS-, -S-CH₂- or -NN-, preferably X

represents -CH₂NH-, -NHCO-, or -CONH-, more preferably X represents -CH₂NH-;

(CH₂)m represents a branched or straight chain alkylene moiety having 4 to 20 carbon atoms, or from 5 to 1 1 carbon atoms, or having 5, 7, 9 or 1 1 carbon atoms (i.e., m is an integer from 4 to 20, or from 5 to 1 1 , or m is 5, 7, 9 or 1 1 );

Y represents a polar group selected from -COOH, -CHO, and -COOCH₃. Preferably, Y is - COOH; and

n represents the number of lipophilic side chains attached to EPO, preferably attached via an amide bond to the oamino group of the N-terminal amino acid and/or to the ε-amino group of lysine residues present on the EPO amino-acid sequence, n may be selected from 1 to 6.

Preferably n is 2 or 3. The values for n equal to 2 or 3 are preferred in order to carry out the fine tuning of lipophilicity. In one embodiment, the EPO conjugate has the general formula

P[(L)-B-(X)-(CH₂)_m-Y]_n,

wherein

P represents EPO;

L, B, X, m and Y are defined as in any one of the above embodiments, and

n represents the number of lipophilic side chains attached to EPO and ranges from 1 to 6.The lipophilic side chain [(L)-B-(X)-(CH₂)_m-Y] may be attached via an amide bond to the oamino group of the N-terminal amino acid and/or to the ε-amino group of lysine residues present on the EPO amino-acid sequence.

In a specific embodiment, the EPO conjugates are represented by the following formulas:

(a) rhEPO-[NHCO-p-phenylene-CH₂-NH-(CH₂)₅-COOH]_n,

(b) rhEPO-[NHCO-p-phenylene-CH₂-NH-(CH₂)₇-COOH]_n,

(c) rhEPO-[NHCO-p-phenylene-CH₂-NH-(CH₂)₉-COOH]_n, or

(d) rhEPO-[NHCO-p-phenylene-CH₂-NH-(CH₂)₁₁-COOH]_n wherein n independently represents 1 , 2 or 3.

While not strictly mandatory, the sequence of the structural elements according to any of the above formulas is preferred as allows an attachment of the one or more lipophilic side chain(s) to the oamino group of the N-terminal amino acid and/or to the ε-amino group of lysine residue present on the EPO amino-acid sequence, e.g., via an amide bond , and via an aromatic moiety B to the linker X. The partial group -(CH₂)_m-Y can be introduced, e.g., by a ω-amino carboxylic acid.

However, while it is preferred that the lipophilic side chains comprise the structural elements in the order given in the above formulas, it is equally preferred that the parameters as defined above (i.e., L, B, X, -(CH₂)_m-, n and Y) are to be understood in that they are independent of each other and can be selected and combined freely. If they occur repeatedly within one molecule, for example in the case that more than one side chains are present per molecule erythropoietin, e.g., if n is 2 or more, the parameters may be different in each side chain. Additionally, the bivalent linker parameters L, B and X may be present in the orientation as depicted in the above formulas, but also in reversed orientation.

Further, the lipophilic side chain(s) of the EPO conjugates as defined above typically comprises one bivalent aromatic moiety B. However, more than one bivalent aromatic moieties B, may be present as well, e.g., 2, 3, 4, 5 or 6 bivalent aromatic moieties B may be present. Moreover, the covalent linkage(s) L and/or X in the lipophilic side chain of the EPO conjugate as defined above may be present or omitted.

P as used in any of the above embodiments represents erythropoietin (EPO). The term erythropoietin, or abbreviated EPO, comprises any natural or recombinant EPO, recombinant human EPO (rhEPO), or any protein or peptide having in vivo biological activity of EPO glycoprotein, i.e. wild type or mutant EPO, EPO peptidomimetic, or EPO fragment. The species from which the EPO is derived comprise animal, mammal or human species, especially human EPO.

Human EPO comprises the known human erythropoietin having 165 amino acids after posttranslational cleavage of the N-terminal signal peptide of 27 amino acids and the C-terminal arginine. It may further comprise the human erythropoietin in the naturally glycosylated state, i.e. including all carbohydrate side chains. In the EPO conjugates as defined above, P is preferably a human recombinant EPO (rhEPO).

In the EPO conjugates of the present invention, increasing values for m (e.g., m = 5, 7, 9 or 1 1 ) correlate with an increase in lipophilicity of the EPO conjugates. Fine tuning of the lipophilicity of the EPO conjugates according to the present invention may further be accomplished by varying the number of the lipophilic side chains (n) per molecule of EPO (preferably, n = 1 , 2 or 3). The EPO conjugates according to the present invention possess many advantages. For example, the novel EPO conjugates are more lipophilic, less susceptible to enzymatic degradation and retain the in vivo biological activity of native or endogenous EPO of causing bone marrow cells to increase the production of reticulocytes (red blood cell progenitors) and red blood cells. In comparison with unmodified EPO, the present EPO conjugates have an increased permeability across the intestinal membrane. Therefore, the present EPO conjugates are particularly suited for peroral administration and/or for the development of advanced oral drug delivery systems. The oral administration of the EPO conjugates provided herein results in plasma concentrations of EPO, which are lower than after parenteral administration, but higher than in untreated patients. The plasma concentrations of EPO achieved after oral administration of the EPO conjugates provided herein thus cause a therapeutic effect, but reduce the occurrence of side effects.

A further advantage of the EPO conjugates according to the present invention lays in its closer resemblance to endogenous EPO entering the systemic circulation via the portal vein.

Endogenous EPO is produced by the peritubular capillary endothelial cells in the kidney and liver. The liver accounts for 10-20% of EPO production and the liver can naturally regulate EPO plasma levels. Enteral absorption of EPO results in better regulation of hormone levels by feed back regulation mechanisms in the liver, thereby preventing peak concentrations of the drug that are responsible for the undesired side effects. This regulation is not efficient upon peripheral administration of EPO. Hence, one of the benefits of the EPO conjugates of the invention is the reduction of severe side effects in comparison to parenterally administered EPO of the current commercial preparation.

As the EPO conjugates of the present invention can be administered orally, they achieve high patient compliance. Further, the production costs of peroral EPO formulations are reduced compared to the costs for producing parenteral EPO formulations. Absorption Enhancers / Nanocomplex formation

The invention further provides pharmaceutical compositions (formulations) comprising EPO, particularly the EPO conjugates of the invention, together with at least one absorption enhancer. Preferred absorption enhancers are cycloalkylglycosides (cycloalkylmaltosides, "CYMALs"). These chemically synthesized molecules are composed of a sugar, typically a disaccharide. The carbohydrate component is linked by an O-glycosidic bond at the "free end" of maltose (carbon atom number 1 ) to an alkyl chain, wherein the alkyl chain comprises at the most distal position of the alkyl chain, the so called ω-position, a cycloalkyl group. Independently of each other, the preferred disaccharide is maltose; the preferred alkyl chain comprises 1 to 7 methylene groups, i.e. (CH₂)i-7, and the cycloalkyl rest at the ω-position is a cyclohexyl group. Thus, the general formula for these compounds reads: ω-cycloalkyl-1-(C1-7-alkyl)-β-D-maltosides, or more specifically ω-cyclohexyl-1-(CH2)1-7-β-D-maltosides (= CYMAL®). Specific examples of these cyclohexyl alkyl maltosides are 1-cyclohexyl-1-methyl- -D-maltoside (= CYMAL®-1 ), 2- cyclohexylethyl- -D-maltoside (= CYMAL®-2), 3-cyclohexyl-1-propyl- -D-maltoside (= CYMAL®- 3), 4-cyclohexyl-1-butyl- -D-maltoside (= CYMAL®-4), 5-cyclohexyl-1-pentyl- -D-maltoside (= CYMAL®-5), 6-cyclohexyl-1-hexyl- -D-maltoside (= CYMAL®-6), 7-cyclohexyl-1-heptyl- -D- maltoside (= CYMAL®-7). Preferred "CYMALs" are CYMAL®-5, CYMAL®-6 and CYMAL®-7, and particularly preferred is CYMAL®-7.

A further group of preferred absorption enhancers are amphiphilic polymers referred to herein as "PMALs" having a highly charged cationic and anionic backbone to which an aliphatic tail is attached. The expression "PMAL" describes alternant polymers prepared by the monomers maleic acid anhydride and an oolefine having 6, 8, 10, 12, 14, 16 or 18 carbon atoms, respectively (i.e., "poly(maleic acid anhydride-alt-oolefine"), which are grafted with

dimethylamino propylamine side chains. They may be prepared by reaction of poly(maleic acid anhydride-alt-oolefine with 3-dimethylamino propylamine, whereby the anhydride group reacts with the primary amino function of the 3-dimethylamino propylamine to form an amide bond. By this way, the "non-reacting" or leaving carboxylic acid function of the original anhydride function is present as a free carboxylic acid, and simultaneously, the basic dimethylamino function is present in the side chain. Thus, a zwitterionic or ampholytic polymer ("amphipol") is obtained. Specific examples of PMALs are alternant polymers of maleic acid anhydride and oolefine having 6 to 18 carbon atoms, substituted with 3-dimethylamino propylamine (=PMAL®) such as the poly(maleic acid anhydride-alt-1-octene) 3-dimethylamino propylamine derivative (=PMAL- C6®); poly(maleic acid anhydride-alt-1-decene) 3-dimethylamino propylamine derivative (=PMAL-C8®); poly(maleic acid anhydride-alt-1-dodecene) 3-dimethylamino propylamine derivative (=PMAL-C10®); poly(maleic acid anhydride-alt-1-tetradecene) 3-dimethylamino propylamine derivative (=PMAL-C12®); poly(maleic acid anhydride-alt-1-hexadecene) 3- dimethylamino propylamine derivative (=PMAL-C14®); and poly(maleic acid anhydride-alt- 1-octadecene) 3-dimethylamino propylamine derivative (=PMAL-C16 ®). Preferably, poly(maleic acid anhydride-alt-1-dodecene) 3-dimethylamino propylamine derivative (=PMAL-C10®) is used. We have observed that PMALs can form organized assemblies ("nanocomplexes") with EPO or its conjugates. The advantage is that upon administration of these nanocomplexes, only the absorption of the EPO molecule (protein) complexed to PMAL is promoted. Amphipols have been described in protein technology processes to solubilize membrane proteins and to improve their solubility in water (Gorzelle BM et al.,. J Am Chem Soc 124, 2002). However, they have never been used as absorption promoting agents as provided herein.

The size of the nanocomplexes formed by the EPO conjugates of the invention and the at least one absorption enhancer(s) as defined above (e.g., PMALs as defined above) ranges between 6 and 30 nm. Coating/ Nanoparticle formation

The present invention further provides coated pharmaceutical compositions comprising comprising EPO, particularly the EPO conjugates of the invention, alone or in combination with at least one absorption enhancer as provided herein, which are coated with one or more coating polymer(s). The term "coated pharmaceutical compositions" is interchangeably used herein with the term "nanoparticle compositions" or "nanoparticles".

The one or more, e.g., one or two, coating polymer(s) may be coating polymer(s) selected from the group of natural or synthetic hydrophilic polymers such as polymers based on polyacrylic acid (Carbopol®, Carbomer®); polyanhydrides such as polymethyl vinyl ether/maleic anhydrides; enteric polymers such as Eudragit®, basic and acidic polysaccharides such as chitosan and its derivatives, alginic acid or its salts, pectin, hyaluronic acid, tragacanth; and polyesters such as copolymers of lactic/glycolic acids with amino acids.

The preferred polymers of the present invention include cationic natural polymers such as chitosan and its derivatives such as trimethylchitosan, and anionic polysaccharides such as alginic acid or its salts. These polymers have the advantage that they are generally non-toxic and not irritating to the mucosa, and do not require organic solvents, which would not be desirable from the point of protein stability and additionally adversely affect the patient.

The chitosan derivate trimethylchitosan may function as both, an absorption promoting agent and as part of the polymer coating. Hence, if CS, or TMC, or a combination thereof, is present in the pharmaceutical compositions according to the invention, one or more additional absorption enhancing agent (e.g., as defined above) may be present; it is, however, equally preferred that no other absorption enhancing agents are comprised in the pharmaceutical compositions of the present invention in this caseThe polymers are added to form nanoparticles providing partial protection against protein degradation, and mucoadhesivness to localize the delivery system closer to the intestinal wall.

A method for nanoparticle formation is based on self association of the ingredients in aqueous media under mild stirring conditions. Further formation of nanoparticles can be obtained by ionotropic gelation or polyelectrolyte complexation using counter ions, for example

polyphosphates, or polyionic polymers. Tripolyphosphate is a particularly suitable counterion in this regard. This process does not require the use of organic solvents, toxic ingredients or crosslinking agent and also does not employ harsh processing conditions necessary for the nanoparticle formation, therefore, the biological activity of the sensitive protein drug is preserved. The method also does not face technical and economical hurdles, therefore it is suitable for industrial scale production.

The size of the nanoparticles in the coated pharmaceutical compositions, i.e., the size after polymer coating of the EPO conjugates of the invention, alone or in combination with at least one absorption enhancer as further provided herein (i.e., nanocomplexes), ranges between 200- 400 nm. For formulations with improved mucoadhesiveness, the size of the nanoparticles should not exceed a certain threshold. For a suitable formulation with improved muco-adhesiveness, the nanoparticle size should be less than 4000 nanometers, preferably less than 1000 nanometers, more preferably less than 500 nanometers, and most preferably less than 400 nanometers. Nanoparticles from 300 to 400 nanometers are particularly preferred.

Preparation of the EPO conjugates

The present invention further provides a process for the production of the EPO conjugates according to the present invention. General preparation method

The sequence of structural elements of the lipophilic side chain as defined above might be selected freely. A skilled artisan can produce any lipophilic side chain as defined herein by means of generally known methods, and further convert a suitable starting material into an EPO conjugate according to the invention. Particular embodiments will be described in the following. The process for the preparation of the EPO conjugates, wherein the one or more lipophilic side chain(s) are attached to 1 to 6 oamino group(s) of the N-terminal amino acid and/or to the ε-amino group(s) of lysine residue present on the EPO provided herein, generally comprises four steps: (1 ) modification (conversion) of a primary amine of the EPO into an amid linker and simultaneous introduction of an aldehyde group, (2) conjugation to an amino fatty acid and reductive amination, (3) gel filtration, (4) concentration and sterile filtration.

The first reaction step involves protein modification, wherein one or more bifunctional linker moieties B are attached to 1 to 6 oamino group(s) of the N-terminal amino acid and/or to the ε-amino group(s) of lysine residues present on the EPO amino-acid sequence via reaction of EPO with a reagent comprising the aromatic moiety B substituted with an N-hydroxysuccinimidyl ester functional group (also referred to as NHS activated ester of B), and an aldehyde functional group. B has the same meaning as defined in any of the above embodiments. The N- hydroxysuccinimidyl ester reacts in an acylation reaction with the amino group of the EPO in that the N-hydroxysuccinimide serves as leaving group and the carbonyl group of the former NHS ester forms an amide bond with the amino group of the protein. The intermediate product of this step can be formulated as:

P-[NHCO-B-CHO]_n,

wherein the parameters P and B have the same meaning as defined in any of the above embodiments. Preferably, the aromatic moiety B is a phenylene moiety. In this case, the specific reagent employed in this reaction step is N-hydroxysuccinimidyl 4-formylbenzoate (SFB).

While the respective reagent may be used in equimolar amounts, it is preferred that the reagent is used in excess amounts over EPO. For example, the molar excess ratio of SFB/EPO is from 6 to 36. Preferably 6 molecules SFB per molecule EPO are used.

In a next step, the aldehyde function is condensed with the amino function of a ω-amino carboxylic acid to the corresponding Schiff's base. The ω-amino carboxylic acids may comprise 4 to 20 methylene groups: HOOC-(CH₂)4-2o-NH₂, preferably from 4 to 12 methylene groups. Preferred are ω-amino carboxylic acids having 5, 7, 9 or 1 1 methylene groups between the carboxylic acid group and the amino group, so that the total number of carbon atoms is 6, 8, 10 or 12, respectively, i.e. HOOC-(CH₂)₅-NH₂, HOOC-(CH₂)₇-NH₂, HOOC-(CH₂)₉-NH₂, and HOOC-

While the ω-amino carboxylic acid may be used in equimolar amounts, it is preferred that the ω-amino carboxylic acid is used in excess amounts over EPO. For example, the molar excess ratio of SFB/EPO may be from 30 to 120, or from 30 to 90. Preferably, 60 molecules of ω-amino carboxylic acid per molecule EPO are used. The intermediate product of this step can be formulated as:

P-[NHCO-B-CH=N-(CH₂)_m-COOH]_n,

wherein the parameters P and B have the same meaning as defined in any of the above embodiments.

The subsequent synthesis step comprises the reduction of the Schiff base to the corresponding amino function, i.e. -CH=N- into -CH₂-NH-, by a reducing agent known to the skilled artisan. For example, complex alkali borohydrides such as sodium cyanoborohydride (Na[CN-BH₃]) may be used. The final product can be formulated as:

P-[NHCO-B-CH₂-NH-(CH₂)_m-COOH]_n,

The Schiff base intermediate may be isolated before performing the reduction step. It is, however, not necessary to isolate the Schiff base intermediate. Rather, the Schiff base intermediate can be processed further directly into the final product (i.e., reduction to the final EPO conjugate as defined above). Hence, the condensation and reducing reactions can be effected in a "one pot" manner.

The final reaction mixture can be purified by separating by-products, such as non-reacted EPO, e.g., rhEPO, or excess reagents such as SFB and ω-aminocarboxylic acid, from the desired conjugated EPO product. Suitable purification techniques are known to one skilled in the art, such as column chromatography, or size exclusion chromatography with a gel ("gel filtration"). For example, a Superdex75 PrepGrade (XK 26/60) column from Sigma/Aldrich may be used for purification by gel filtration. The fractions containing the desired conjugated EPO can be collected, concentrated, for example by ultrafiltration, and filtrated with a sterile filter into a sterile state.

Alternatively, the one or more lipophilic side chains may be attached to the sugar components present on the glycoprotein EPO. In this case, the sugar components can be modified so as to obtain aldehyde reactive groups. Oxidation with sodium periodate can be used for converting a relatively non-reactive sugar hydroxyl groups into the aldehyde groups (oxidation). Modification of the specific sugar components takes place in a periodate concentration-dependent manner. Sodium periodate in low concentrations (1 mM) specifically converts sialic acids. At a higher concentration of periodate (10 mM, and more), CC bond cleavage between the two cis-hydroxyl groups on sugar units occurs in addition to oxidation of terminal sialic groups. Nevertheless, terminal sialic acids are the most prone groups for modification. Alternatively, the oxidation of sugar moieties yielding free aldehyde groups available for modification could be achieved through enzymatic reactions (e.g. glucose oxidase as described in Biotechnol Appl Biochem. 1994, (Pt1 ): 45-53). The resulting aldehyde group can be reacted with primary or secondary amines in the next step, thus leading to the formation of unstable Schiff bases. These intermediates are after reduction (e.g., with NaCNBH₃) converted to a stable amine.

Such modifications usually occur in certain locations that are remote from the 'critical' binding sites and therefore result in highly preserved biological activity of conjugates obtained after modification, which is beneficial.

Medical Use

The invention further provides the EPO conjugates and EPO conjugate compositions as defined herein for use in the treatment of anemia, for example resulting from chronic kidney disease and myelodysplasia, from the treatment of cancer, for example by chemotherapy and/or radiation, and from other critical illnesses, for example heart failure. The EPO conjugates and the pharmaceutical compositions according to the present invention are further intended for use in the treatment of a disorder related to a reduction of red blood cells, particularly for use in the treatment of a disorder related to a reduction of red blood cells, wherein the risk of side effects caused by abnormally high EPO levels is increased.

Test Methods Caco-2 cell cultures, TEER and permeability measurements

Human colon carcinoma cell line Caco-2, cultured on permeable supports, were used to determine the permeability coefficient of EPO or its conjugate in different formulations

(nanocomplexes and nanoparticles). Caco-2 cells were seeded on the tissue-culture-treated polyesters filters (growth area 1.12 cm², membrane pore size 0.4 μητι) in Transwell 12 wells per plates (Costar 3460) at a seeding density of 100,000 cells/well. DMEM (SIGMA, D5921 ;

Dulbecco's modified Eagle's medium) supplemented with 10% HIFBS (heat inactivated fetal bovine serum), 1 % glutamine and 1 % antibiotic-antimycotic solution was used as the culture medium, and added to both the donor and acceptor compartments. The medium was replaced every 48 hours. The cultures were kept in an atmosphere of 95% air and 5% C0₂ at 37 °C. Transport experiments were performed 21 days after the seeding.

TEER values (transepithelial electrical resistance) of the Caco-2 cell monolayers were monitored with a Millicell RTM-Electrical Resistance System (Millipore Corp.) connected to a pair of chopstick electrodes. To initiate the transport experiments, the culture media in the donor and acceptor compartments were aspirated and the cells were rinsed twice with pre-warmed transport media (DMEM). The cells were incubated for 3 hours with 0.5 ml transport media containing testing formulation. At predetermined time intervals (30, 60, 120, and 180 min) samples were taken from acceptor chambers for protein analysis (ELISA) and replaced with fresh DMEM buffer. Subsequently, testing formulations were carefully removed and cells were washed and replaced with fresh culture media for another 24 hours. The TEER value, which is a good indicator of Caco-2 cell monolayer integrity, was measured before and after the transport experiment (3h), and also after 24h in the culture media to study the reversibility of the effect of testing formulations on Caco-2 cell monolayer. The apparent permeability coefficient, P_app (cm/s) was calculated according to the equation (Eq.1 ):

a^{pp ~} dt ^X A x C₀ · Equation 1 where (dQ/dt) is the amount of compound transported within a given time period, A is the surface area of the insert; C₀ is the initial concentration compound on the donor side.

Permeability coefficient was calculated only for those testing molecules where TEER value of cells was not affected or showed a gradual recovery over time to the initial TEER value.

In vitro excised rat intestine (IVERI) model with muscle layer removed

The experiments were performed according to the modified procedure described by Legen et al (Legen et al., Int J Pharm 2003; 256: 161-6). Rat jejunum was obtained from female Wistar rats. All animals were starved for 18 h before the experiments. After decapitation and laparotomy, the small intestine was immediately excised and placed into the ice-cold bubbled (carbogen, 95:5 0₂/C0₂) 10 mM solution of D-glucose in standard Ringer buffer containing (in mM): 140.6 Na⁺, 5 K⁺, 1.2 Ca²⁺, 1 .2 Mg²⁺, 121 .8 CI^", 25 HC0₃ ^", 0.4 H₂P0₄ ^", 1.6 HP0₄ ^2". Jejunum was used in the experiments. The tissue was cut into 3 cm long segments, excluding visible Peyer's patches. The intestinal segments were opened along the mesenteric border, stretched onto a special insert followed by the gentle removal of serosa and layers of muscularis externa to facilitate erythropoietin detection in the acceptor chamber. The intestinal segments were then placed between the two EasyMount side-by-side diffusion chambers with the exposed tissue area of 1 cm² (Physiologic Instruments, San Diego, CA, USA). During the experiments the intestinal segments were bathed with standard Ringer buffer (pH 7.0 donor chamber and pH 7.4 acceptor chamber) supplemented with 10 mM mannitol on the mucosal side and with 10 mM D-glucose on the serosal side. 0.08% v/v of protease inhibitors cocktail (Sigma) was added to the acceptor and the donor chamber and 1 % m/v of bovine serum albumin was added to acceptor chamber only. The bathing solution was continuously gassed with carbogen gas (95:5 0₂/C0₂) and kept at 37°C.

After the 30 min incubation period, the testing formulations were added to the mucosal side to study the mucosal-to-serosal (MS) transport of erythropoietin. The final volume of the solution in each compartment was 2.5 ml. Samples of 250 μΙ were withdrawn from the acceptor and from the donor compartment at 30 min and 90 min intervals, respectively, up to 180 min and replaced with fresh Ringer buffer containing 10 mM D-glucose (acceptor) or 10 mM mannitol (donor) to maintain a constant volume. Samples were put on -80°C immediately after sampling and were then analyzed (ELISA). Obtained data were analyzed according to the Equation 1. The diffusion chambers were equipped with two pairs of Ag/AgCI electrodes for measuring trans- epithelial potential difference (PD). The experiments were performed under the open circuited conditions. The tissue viability and integrity was continuously checked by monitoring PD and (TEER) and additionally by recording the PD after the addition of D-glucose (25 mM) to the mucosal compartment at the end of the experiments.

The permeability coefficient was calculated only for those formulations where experimental tissue was viable throughout the experiment as observed by the tissue response in PD parameter after the addition of D-glucose.

Both, the permeation through the Caco-2 cell monolayer and the permeation through the intestinal membrane in vitro serve as indication for the absorption of the EPO conjugates via the intestinal epithelium in vivo, and thus, for the suitability of the EPO conjugates according to the invention for peroral administration. In Vivo pharmacological activity

Young adult female rats, Wist from Harlan, Italy (200-225 g) were treated according to the principles of the Convention ETS 123 (The convention for the protection of animals used for experimental and other scientific purposes) and the Directive 86/609/EEC (Council directive on the protection of animals used for experimental and other scientific purposes). They were housed under good hygienic condition with food and drink ad libitum. Animal conditions were certified by a veterinarian. Only those animals in acceptable health conditions were used for the test. Animals with hematological abnormalities were excluded from the study.

The test items were administered subcutaneously only once (at day 0 of the study) and blood samples were taken every second day throughout the 24 day lasting study. Blood samples were collected from the terminal vein (40 μΙ) into the pipette and immediately diluted with Cellpack reagent, Sysmex (160 μΙ, 1/5, v/v) in 0.5 ml Eppendorf tubes. Hematological parameters were evaluated with the hematological analyzer Sysmex XT-2000iV according to the Sysmex manufacturer's instructions. The most important parameter, reticulocytes, was compared between formulations administered. Body weight and clinical signs were also observed before and after the application and daily during experiment. At the end of the experiments, all animals were humanly sacrificed under C0₂ and anesthesia. Tissues and organs were macroscopically examined for potential abnormalities.

EXAMPLE 1 : Synthesis of mC6-EPO

Recombinant human EPO - rhEPO at concentration 2.5 mg/ml (15.6 ml, 1.28 μιηοΙ) was used in conjugation reactions. SFB was dissolved in 100% dimethylsulfoxide (DMSO) to a concentration of 2.0 mg/ml. All reactions were performed at pH 7.0 in phosphate buffer (150 mM NaCI, 50 mM Na phosphate) using thirty fold molar excess of the SFB over rhEPO. The concentration of DMSO in protein reaction mixture was 3.5 %. The reaction mixture was stirred slowly at 60 rpm for 16 hours at 20 °C.

In the second step conjugation with amino-fatty 6-aminocaproic acid was performed in the presence of sodium cyanborohydride (20mN NaCNBH₃). A sixty fold molar excess of 6- aminocaproic acid over rhEPO was used. The reaction mixture was stirred at 60 rpm for overnight incubation at 20°C.

The conjugate was separated from unreacted rhEPO, SFB and 6-aminocaproic acid by gel filtration on Superdex75 PrepGrade (XK 26/60) column. Elution (4.5 ml/min) was done with phosphate buffer (150 mM NaCI, 50 mM Na phosphate) containing 20 % glycerol. Fractions containing product were collected, concentrated and sterile filtrated. Purified EPO derivative, mC6EPO, was additionally analyzed by SDS-PAGE and RP-HPLC. Permeability of mC6EPO on Caco-2 cell monolayer

For permeability assay using Caco-2 cell monolayer all samples, presented in Table 1 , were diluted with cell culture medium (initial protein concentration was 2.0 - 2.5 mg/ml), yielding final testing concentration as presented in Tables 1 and 2. Table 1 : Testing concentration of conjugate mC6EPO, TEER value after 3h of treatment and 24h after the experiment (to monitor Caco-2 cells recovery), and calculated permeability for EPO and mC6EPO. Enhancement ratio (ER) of P_app for the conjugate is also presented. Sample Cone, (mg/ml) TEER 3h (%) TEER 24h (%) Papp EPO ER

(cm/s)

EPO 0.2 100 100 3.4E-10

mC6EPO 0.1 95 100 4.82E-09 14 mC6EPO 0.2 70 100 1.33E-08 39 mC6EPO 0.4 10 40* 1.76E-08 52

*poor cell recovery

Table 2: Compositions of testing formulations of mC6EPO with PMAL (PMAL-C10, [Poly (maleic anhydride-alt-1-dodecene) substituted with 3-(Dimethylamino) propylamine]) and CYMAL (CYMAL-7, 7-cyclohexyl-1-heptyl-maltoside), respectively, TEER value after 3h of treatment and 24h after the experiment, and calculated permeability for mC6EPO. Enhancement ratio (ER) of P_app is also presented. NP= Nanoparticles; CS: chitosan; TMC: trimethylchitosan; tpp:

tripolyphosphate.

*poor cell recovery

Results showed that chemical modification promoted the protein transport across Caco-2 monolayer (Table 1 and 2). As seen from Table 2, the addition of absorption enhancer PMAL- C10 had little effect on conjugate transport through Caco-2 monolayer. However, the addition of 0.03% CYMAL-7 in the formulation exerted almost 500-fold increase of protein permeability. A further increase in permeability can be observed, if the pre-mixture mC6EPO and CYMAL-7 is coated with chitosan and tripolyphosphate. All cases with increased P_app were characterized by the drop in transepithelial electrical resistance (TEER) of Caco-2 cells and were recovered gradually.

Permeability on IVERI model

For permeability assay using IVERI model, protein solutions were diluted with Ringer buffer, yielding final testing composition as presented in Table 3.

Table 3: Composition of testing formulations: EPO (0.3 mg/ml) and mC6EPO (0.3 mg/ml) in formulation with PMAL-C10 (1.0 mg/ml), tissue viability after 4h of experiment, and calculated permeability for EPO and mC6EPO with PMAL (ditto). Factor of relative P_app increase for formulation is also presented.

EXAMPLE 2: Synthesis of mC8-EPO

Recombinant human EPO - rhEPO at concentration 2.5 mg/ml (15.6 ml, 1.28 μιηοΙ) was used in conjugation reaction. SFB was dissolved in 100% dimethylsulfoxide (DMSO) to concentration 2.0 mg/ml. All reactions were performed at pH 7.0 in phosphate buffer (150 mM NaCI, 50 mM Na phosphate) using thirty fold molar excess of the SFB over rhEPO. The concentration of DMSO in protein reaction mixture was 3.5 %. The reaction mixture was stirred slowly at 60 rpm for 16 hours at 20 °C.

In the second step conjugation with 8-aminocaprylic acid was performed in the presence of sodium cyanborohydride (20mN NaCNBH₃). A sixty fold molar excess of 8-aminocaprylic acid over rhEPO was used. The reaction mixture was stirred at 60 rpm for overnight incubation at 20°C.

The conjugate was separated from unreacted rhEPO, SFB and 8-aminocaprylic acid by gel filtration on Superdex75 PrepGrade (XK 26/60) column. Elution (4.5 ml/min) was done with phosphate buffer (150 mM NaCI, 50 mM Na phosphate) containing 20 % glycerol. Fractions containing product were collected, concentrated and sterile filtrated. Purified EPO derivative, mC8EPO, was additionally analyzed by SDS-PAGE and RP-HPLC.

Permeability on Caco-2 cell monolayer

For permeability assay using Caco-2 cell monolayer, all samples, presented in Table 4, were diluted with cell culture medium (initial protein concentration was 2.0 - 2.5 mg/ml), yielding final testing concentration as presented in Table 4.

Table 4. Permeability of EPO and its hydrophobic conjugate mC8EPO, TEER value after 3h of treatment and 24h after the experiment (to monitor Caco-2 cells recovery). Enhancement ratio

(ER) of P_app for conjugate is also presented.

EXAMPLE 3: Hematological parameters in rats after single subcutaneous administration of EPO and its derivative mC6-EPO {in vivo experiment)

Samples presented in Table 5 were administered subcutaneously (s.c) to rats at EPO or mC6EPO doses of 4 μg/kg . The volume of administration per rat was approximately 0.1 ml. Table 5. Composition of testing sample for subcutaneous administration to rats.

The most important hematologic parameter - reticulocytes - was monitored every second day throughout the 24 day lasting study. In both s.c. groups, receiving EPO or mC6EPO respectively, the increase of reticulocytes is prominent on day 4 after application. The statistical tests confirmed the statistically relevant increase on this day in comparison to day 0. The peak is higher in EPO group; however, the group receiving derivative mC6EPO showed similar profile retaining 80 % of EPO biological activity. At the end of the study, after rats were humanly sacrificed, all organs and tissue (including gastrointestinal tract) were macroscopically observed and showed no abnormalities. Results from in vivo study showed that EPO derivative mC6EPO has in vivo biological activity of causing bone marrow cells to increase production of reticulocytes and red blood cells. In spite of surface modification of the EPO it was not predictable that the increase in reticulocytes formation was not more impaired, or that a relative activity of 80 % of reticulocytes formation, in respect of unmodified EPO, was maintained.

EXAMPLE 4: Synthesis of glyco-hexyl-EPO and glyco-caproic-EPO

GLYCO-HEXYL-EPO

GLYCO-CAPROIC-EPO

Recombinant human EPO - rhEPO at concentration 2.0 mg/ml was incubated for 30 min in the presence of 10 mM sodium periodate at room temperature. Oxidation reaction was stopped by addition of glycerol. Next 60 fold molar excess of hexylamine (or 6-amino caproic acid in the case of preparation of glycol-caproic EPO conjugate) was added to the solution and incubated for several hours at room temperature in the presence of 20 mM NaCNBH3.

The conjugate was separated from reactants by gel filtration on Superdex200 PrepGrade (XK 26/60) column. Elution at flow rate 4.5 ml/min was done in phosphate buffer (150 mM NaCI, 50 mM Na phosphate) containing 20 % glycerol. Product containing fractions were collected, concentrated and sterile filtrated. Purified conjugates were analyzed by SDS-PAGE, RP-HPLC and DEAE.

Permeability on Caco-2 cell monolayer

For permeability assay on Caco-2 cells all samples were diluted approximately 10 times with the cell culture medium, yielding final testing concentration as presented in Table 6.

Table 6. Permeability of glyco-hexyl-EPO conjugate in comparison to EPO (TEER- Trans Epithelial Electrical Resistance, values after 3h and 24h, Papp - apparent permeability, ER- permeability enhancement ratio). Cone, TEER 3h TEER 24h Papp EPO

Sample ER

(mg/ml) (%) (%) (cm/s)

EPO 0.2 100 100 3.4E-10

Glyco-hexyl-EPO

0.2 80 100 3.9E-10 1 (10 mM Nal04)

Glyco-hexyl-EPO

0.4 12 100 4.7E-09 44 (10 mM Nal04)

Glyco -caproic Exp 1 : 8.35E-10

0.2 67 100 3 EPO Exp 2: 1.06E-09

Glyco -caproic Exp 1 : 1.73E-08 50 to

0.4 12 96

EPO Exp.2: 2.04E-08 60

Permeability tests on Caco-2 model showed that Glyco-hexyl EPO and Glyco-caproic EPO conjugates exhibit enhanced permeability across the cell monolayer. Especially glycol-caproic EPO conjugate showed significant increase in Papp as enhancement ratio was up to 60 in comparison to unmodified protein.

EXAMPLE 5: In vivo PD study

In vivo pharmacodynamics study was performed with series of nanoparticle formulations prepared with EPO conjugates.

The study was performed in accordance with the study protocol and Lek SOPs.

Animals were treated according to the principles of Convention ETS 123 (convention for the protection of animals used for experimental and other scientific purposes) and Directive 86/609/EEC (Council directive on the protection of animals used for experimental and other scientific purposes).

Yung adult Winstar rats (Harlan) were used in experiments, each test group containing 6 animals.

Placebo and test items were administered by a single gavage administration. UV sterilized Latex

Free syringe with gavage needle was used for p.o. application.

Placebo and test items were administered once, i.e. on day 0.

All animals were starved for 18 hours before the application and 15-30 min after the application.

For that time the cage floor was changed with the metal grid to prevent coprophagia. After that period the animals were transposed into the normal cages with sawdust bedding and the food was offered as usual (ad libitum). The water was available ad libitum all the time.

The physiological response of rats (differences in hematological parameters) was evaluated. Blood samples were collected from the terminal tail vein, 40 μΐ of blood were taken directly into the pipette and immediately diluted with Cellpack reagent, Sysmex (160 μΐ; 1/5, v/v) in

Eppendorf 0.5 mL tubes, which were suitably labeled. Some blood samples were measured twice to check the uniformity of measurements. The following parameters were evaluated according to the Sysmex manufacturer's instructions and to the SOP (ND_Gen200609) in all animals:

Dosing of animals and blood sampling schedule are presented in Table 7. Table 7. Dosing and blood sampling schedule

Test items were prepared as freeze-dried products which were rehydrated in water before the application and prepared as a final dispersion for oral application. Test item 1 labeled Lio- mC6EPO-B"' contained nanoparticles composed of mC6EPO, CYMAL7, and

chitosan/tripolyphosphate with initial loading of mC6EPO 5% and 13x mass excess of CYMAL7 relative to mC6EPO. Test item 2 labeled Lio-mC6EPO-D contained nanoparticles composed of mC6EPO, CYMAL7, and chitosan/tripolyphosphate. Initial loading of mC6EPO was 15% and 1.5x mass excess of CYMAL7 relative to mC6EPO was used. In group 3 (Placebo group) animals received demi water given by gavage.

Data evaluation

The most responsive hematological parameter was the number of reticulocytes determined at individual time points in the blood samples. Response curves were generated by plotting Reticulocyte count (in number) against time for individual animal in the test group. Individual response was evaluated by calculating AUC (area under the curve) taking zero time point and day six time point as a baseline values.

Bioavailability (BA) of per-orally administered mC6EPO formulations was calculated relative to the average AUC values obtained in the same way as described before for subcutaneously administered mC6EPO conjugates (study P44) using the equation:

A UC Dose, A UC Dose,

BA_p.0 % ^■x lOO

avgA UC_; Dose avgA UC_{S i} 100 - Dose, Results are presented in Table 8.

Table 8. AUC values in the number of reticulocytes after subcutaneous and peroral application of different formulations of C6EPO.

In the groups receiving Test item 1 and Test item 2 individual animals showed an increase in the number of reticulocytes on the 4^th day after application, expected physiological response of erythropoietin drug. Three out of six animals responded in the group receiving Test item 1 , two out of six animals responded in the group receiving Test item 2 while no animals responded in the placebo group.

Bioavailability (BA) of mC6EPO formulations calculated was up to 1 % in individual animal after peroral application.

Claims

An erytropoietin (EPO) conjugate comprising one or more lipophilic side chains.

The EPO conjugate according to claim 1 , wherein the lipophilic side chain comprises one or more moieties B and/or a branched or straight chain alkylene moiety (CH₂)_m, wherein B is selected from arylene and heteroarylene, each and independently unsubstituted or substituted with one or two substituents independently selected from straight chain or branched C^-alkyl, -OH, -SH, -NH₂, -CHO, and -COOH; and

m is an integer from 4 to 20.

The EPO conjugate according to claim 2, wherein the lipophilic side chain further comprises a polar end group Y and/or one or more linker(s) selected from the group consisting of L and X, wherein

L is -NHCO- or -NHCH₂-;

X is selected from -CH₂NH-, -NHCO-, -CONH-, -COO-, -SS-, and -NN-; and

Y is selected from -COOH, -CHO, and -COOCH₃.

The EPO conjugate according to claims 2 or 3, wherein

B is arylene, which is unsubstituted, or substituted with one or two substituents independently selected from -CH₃, -OH, -SH, -NH₂, -CHO, and -COOH.

The EPO conjugate according to claims 3 or 4, wherein

L is -NHCO-;

B is a p-phenylene group;

X is -CH₂NH-;

m is an integer from 4 to 20, or from 5 to 11 , or m is 5, 7, 9 or 11 ; and

Y is selected from -COOH, -CHO, and -COOCH₃.

The EPO conjugate according to any one of claims 3 to 5, wherein

L is -NHCO-;

B is a p-phenylene group;

X is -CH₂NH-;

m is an integer from 5, 7, 9 or 11 ; and

Y is -COOH.

The EPO conjugate according to any one of the preceding claims, wherein the number of lipophilic side chains is 1 to 6.

8. The EPO conjugate according to any one of the preceding claims, wherein the number of lipophilic side chains is 2 to 6, preferably the number is 2 or 3.

9. The EPO conjugate according to claim 8, wherein the lipophilic side chains are the same.

10. The EPO conjugate according to claim 8, wherein the lipophilic side chains are different.

11. The EPO conjugate according to any one of the preceding claims having the general formula

P[(L)-B-(X)-(CH₂)_m-Y]_n

wherein

P represents EPO,

L, B, X, m and Y are as defined in any one of claims 2 to 6, and

n is an integer from 1 to 6.

12. The EPO conjugate according to claim 11 , wherein n is 1 , 2 or 3.

13. The EPO conjugate according any one of the preceding claims having the formula

(a) rhEPO-[NHCO-p-phenylene-CH₂-NH-(CH₂)₅-COOH]_n;

(b) rhEPO-[NHCO-p-phenylene-CH₂-NH-(CH₂)₇-COOH]_n;

(c) rhEPO-[NHCO-p-phenylene-CH₂-NH-(CH₂)₉-COOH]_n; or

(d) rhEPO-[NHCO-p-phenylene-CH₂-NH-(CH₂)₁₁-COOH]_n;

wherein n independently represents 1 , 2 or 3. 14. The EPO conjugate according to any one of the preceding claims, wherein the lipophilic side chain is attached via an amide bond to the oamino group of the N-terminal amino acid and/or to the ε-amino group of lysine residues present in the EPO amino-acid sequence. 15. A pharmaceutical composition comprising an EPO conjugate according to any one of the preceding claims, and one or more absorption enhancers.

16. The pharmaceutical composition according to claim 15, wherein the absorption enhancer is selected from cycloalkyl-alkyl-maltosides and ampholytic polymers.

17. The pharmaceutical composition according to claim 16, wherein the ampholytic polymer is selected from alternant polymers of maleic acid anhydride and an a-olefine having 8 to 18 carbon atoms, substituted with 3-dimethylamino propylamine (poly(maleic acid anydride- alt-C₈-i8-oolefine) 3-dimethylamino propylamine derivatives).

18. The pharmaceutical composition according to claims 16 or 17, wherein the

cycloalkyl-alkyl-maltosides are selected from 1-cyclohexyl-1-methyl- -D-maltoside

(= CYMAL®-1 ), 2-cyclohexyl-1-ethyl- -D-maltoside (= CYMAL®-2), 3-cyclohexyl-1-propyl- β-D-maltoside (= CYMAL®-3), 4-cyclohexyl-1-butyl- -D-maltoside (= CYMAL®-4), 5- cyclohexyl-1-pentyl- -D-maltoside (= CYMAL®-5), 6-cyclohexyl-1-hexyl- -D-maltoside (= CYMAL®-6) and 7-cyclohexyl-1-heptyl- -D-maltoside (= CYMAL®-7).

19. The pharmaceutical composition according to claims 17 or 18, wherein the alternant polymers of maleic acid anhydride and a-olefine having 8 to 18 carbon atoms substituted with 3-dimethylamino propylamine are selected from poly(maleic acid anhydride-alt-1- octene) 3-dimethylamino propylamine derivative (=PMAL-C6®), poly(maleic acid anhydride-alt-1-decene) 3-dimethylamino propylamine derivative (=PMAL-C8®), poly(maleic acid anhydride-alt-1-dodecene) 3-dimethylamino propylamine derivative (=PMAL-C10®), poly(maleic acid anhydride-alt-1-tetradecene) 3-dimethylamino propylamine derivative (=PMAL-C12®), poly(maleic acid anhydride-alt-1-hexadecene) 3- dimethylamino propylamine derivative (=PMAL-C14®) and poly(maleic acid anhydride-alt- 1-octadecene) 3-dimethylamino propylamine derivative (=PMAL-C16 ®). 20. A coated pharmaceutical composition comprising the EPO conjugate according to any one of claims 1 to 14, or the pharmaceutical composition according to any one of claims 15 to 19, and one or more coating polymers selected from polyacrylic acid, polymethyl vinyl ether/maleic acid anhydride, enteric polymers, basic and acidic polysaccharides and polyesters.

21. The coated pharmaceutical composition according to claim 20 wherein the one or more coating polymer is selected from chitosan, trimethylchitosan and alginic acid.

22. Use of the EPO conjugate according to any one of claims 1 to 14 for the production of a medicament for oral administration. 23. The EPO conjugate according to one of the claims 1 to 14, or the pharmaceutical

composition according to any one of claims 15 to 19, or of the coated pharmaceutical composition according to any one of claims 20 or 21 for use in therapy.

24. The EPO conjugate according to one of the claims 1 to 14, or the pharmaceutical

composition according to any one of claims 15 to 19, or of the coated pharmaceutical composition according to any one of claims 20 or 21 for use in the treatment of a disorder related to a reduction of red blood cells.

25. A method for preparing the EPO conjugate according to any one of claims 1 to 14,

comprising

(a) reacting 1 to 6 oamino group(s) of the N-terminal amino acid and/or of the ε-amino group(s) of the lysine residues present in the EPO amino-acid sequence with a reagent comprising a bifunctional linker moiety B containing an

N-hydroxysuccinimidyl ester functional group and an aldehyde functional group, wherein B is as defined in any one of claims 2 to 6;

(b) condensation of the aldehyde group of the product obtained in step (a) with an ω- amino carboxylic acid to form a the Schiff base; and

(c) reduction of Schiff base. 26. The method according to claim 25, wherein steps (b) and (c) are carried out in a one pot manner.

27. The method according to any one of claims 25 or 26, wherein the reagent employed in step (a) is succinimidyl 4-formylbenzoate.