JP2020033372A - Oral administration - Google Patents

Abstract

To provide therapeutically effective biopharmaceuticals which can be administered via the oral route.SOLUTION: A drug is a compound comprising: a moiety (I), which imparts desired therapeutic activity; and an amino acid sequence corresponding to a moiety (II), which binds to albumin and comprises albumin binding protein G, or an albumin binding domain, fragment or derivative thereof; with the proviso that the moiety (I) is not selected from an exendin sequence, an exendin analog sequence, an exendin active fragment sequence or an exendin analog active fragment.SELECTED DRAWING: None

Description

  The present invention is within the field of biopharmaceutical administration. More specifically, the invention provides for oral administration of a compound comprising a moiety that confers a desired therapeutic activity; and a polypeptide moiety that binds albumin.

Oral Delivery of Protein Therapeutics Most of the currently marketed protein and peptide therapeutics are administered by the parenteral route, i.e., without passing through the gastrointestinal tract, such as by intravenous, intramuscular or subcutaneous injection. . Intravenous administration directly into the systemic circulation results in 100% bioavailability and rapid onset of drug action. However, the risk of side effects increases as the drug concentration in the blood rises immediately. Moreover, administration by any injection method is associated with low patient compliance due to pain and discomfort. Self-administration is often not possible, and treatment must therefore be performed at the clinic. The latter is particularly problematic if the drug has a short half-life and requires frequent repeated administrations to maintain a sufficient level of therapeutic effect. In addition, the clinical treatment of patients, and sometimes the need for hospitalization, is also costly to society. Thus, while simplifying administration is a major driver in developing drugs intended for alternative delivery routes, such as oral, intranasal, pulmonary, transdermal, or rectal, any such alternative delivery route Related to certain advantages and limitations. Oral administration continues to be one of the most convenient routes of administration, especially for the treatment of pediatric patients. In addition, oral formulations do not require production under sterile conditions, thereby lowering the manufacturing cost per unit of drug (Non-Patent Document 1). For some protein therapeutics, the oral delivery route may still be more physiological, as has been proposed for insulin (Non-Patent Document 2).

  In fact, oral delivery of conventional low molecular weight drugs is well established. However, oral delivery of larger, less stable, and often polar peptide and protein therapeutics, for example, may result in the drug being 1) resistant to the acidic environment of the stomach, 2) digestion Other challenges are faced, such as being resistant to intraluminal enzymatic degradation and 3) being able to cross the intestinal epithelium and reach the circulatory system. Various approaches have been attempted to address these issues, either by modifying the protein itself, or by optimizing the formulation or drug carrier system.

Factors Affecting Oral Bioavailability The bioavailability of an orally administered protein therapeutic can be determined, for example, by molecular weight, amino acid sequence, hydrophobicity, isoelectric point (pI), solubility, and pH stability. Physiology and other physiological properties of the protein, as well as the biological barriers found in the gastrointestinal tract, namely the proteolytic environment and the generally poor absorption of large molecules from the intestinal wall. You.

The physiochemical environment of the gastrointestinal tract varies depending on the individual's feeding status. Factors that change between the fasting and feeding stages include gastric pH, gastrointestinal fluid composition, and volume. In humans, the pH of the stomach in the fed state is approximately 1-2, but rises to 3-7 in the fasted state. The pH varies throughout the small intestine, but averages about 5 and 6.5 in fed and fasted states, respectively (Non-Patent Document 3). The difference in pH affects the activity level of the proteolytic enzyme, each associated with a specific optimal pH. Pepsin, the major protease in the stomach, has optimal activity at about pH 2, whereas intestinal trypsin and chymotrypsin have optimal activity at about pH 8. Moreover, gastric emptying is a rate-limiting step. Foods, especially fatty foods, slow gastric emptying and thus slow the rate of drug absorption (Non-Patent Document 4), prolonging the time the drug is exposed to proteolytic enzymes. Therefore, if a drug is taken with or without a significant volume of liquid, or various types of liquids, during or between meals, the bioavailability of the drug may be affected There is.

  Low absorption from the intestinal wall continues to be a major factor limiting the bioavailability of orally delivered protein therapeutics. An ingested drug, like any nutrient, has two options: a transcellular pathway that utilizes a pathway through cells, or a paracellular pathway that utilizes a pathway between adjacent cells through tight junctions. There is an option to cross the intestinal wall by using any of the routes. Low molecular weight substances having a molecular weight of less than 500 Da can be passed using either route (Non-Patent Document 5). The ability of a drug having a higher molecular weight to cross the intestinal wall depends on the physiochemical properties of the drug, such as, for example, charge, lipophilicity, and hydrophilicity. In the case of lipophilic drugs, the transcellular route takes precedence, while the hydrophilic drug can pass through the paracellular route (Non-Patent Document 1). However, because the size of the intercellular space is between 10 ° and 30 ° to 50 °, it has been suggested that paracellular transport is generally limited to molecules with a radius of less than 15 ° (about 3.5 kDa) (non-patented). Reference 6). As in the transcellular route, small molecular weight substances are easily passed by passive diffusion. However, larger molecular weight substances are limited to active processes that require energy expenditure, such as, for example, phagocytosis (non-specific "cell drinking") or transcytosis (receptor-mediated transport). You.

  Finally, bioavailability can further vary among patients, such as age (in fetal, neonatal, and elderly populations, drugs are generally metabolized more slowly), gastrointestinal health, and systemic In addition, intra-patient variability, ie, variability in the same patient over time, such as pathology (eg, hepatic failure, reduced renal function).

  The bioavailability of orally administered proteins and peptides because they must allow for the delivery of therapeutically effective doses, reduce manufacturing costs, and, to a lesser extent, patient-to-patient and intra-patient variability. It is important to increase. Strategies to improve the oral bioavailability of protein therapeutics include altering physiochemical properties such as, for example, hydrophobicity, charge, pH stability, and solubility; Inclusion of enhancers; and the use of formulation vehicles such as, for example, emulsions, liposomes, microspheres, or nanoparticles (as summarized in [7]).

Prolonged half-life of proteins in vivo Considering the relatively low bioavailability of orally administered peptide and protein drugs, the ability of bioactive forms to pass through the epigut membrane in a biologically active form It is appropriate to maintain an in vivo plasma half-life of minutes. Several strategies for preventing rapid renal clearance have been described in the literature and are known to those skilled in the art. These strategies include fusion, conjugate or association with albumin, their antibodies or fragments, or conjugate to one or several polyethylene glycol (PEG) derivatives. It has also been reported that PEGylation promotes absorption from mucous membranes, stabilizes peptide drugs, and prevents degradation by proteases (Non-Patent Document 8). In vivo, associating with molecules that exhibit a long half-life after administration can be directly fused or conjugated to them prior to administration to retain small size and prevent partial proteolysis of molecules that exhibit a long half-life. May be more favorable than

Association with serum albumin to increase protein half-life in vivo Serum albumin is the most abundant protein in mammalian serum (35-50 g / l in humans, ie 0.53-0.75 mM) And some of the covalently linked peptides or proteins to carrier molecules that are expected to allow for association to serum albumin in vivo, eg, US Pat. The strategy is explained. The first document describes, inter alia, the use of an albumin binding peptide or protein derived from streptococcal protein G (SpG) to increase the half-life of other proteins. The concept is to fuse albumin binding peptides / proteins from bacteria to therapeutically important peptides / proteins that have been shown to be rapidly cleared from the blood. When the resulting fusion protein binds to serum albumin in vivo, it takes advantage of its longer half-life to increase the net half-life of the fused therapeutically important peptide / protein. U.S. Patent Nos. 5,064,026 and Dennis et al. Relate to the same concept, but the authors utilize a relatively short peptide to bind serum albumin. Peptides were selected from a peptide library by phage display. U.S. Patent Application (Dennis), also published as U.S. Patent No. 6,064,072, also discloses the use of constructs containing peptide ligands identified by phage display technology, such peptide ligands binding to serum albumin. Conjugated to a bioactive compound for targeting a tumor.

Albumin Binding Domain of Bacterial Receptor Protein Streptococcal protein G (SpG) is a bifunctional receptor present on the surface of a given Streptococcus strain and is capable of binding both IgG and serum albumin (non- Patent Document 10). The structure is highly repetitive with several structurally and functionally distinct domains (Non-Patent Document 11), these domains are more precisely three Ig-binding domains and three serum albumin binding domains. (Non-Patent Document 12). It has been determined that the structure of one of the three serum albumin binding domains in SpG exhibits a three-helix bundle fold (Non-Patent Document 13; Non-Patent Document 14). The 46 amino acid motif was defined as ABD (albumin binding domain) and was subsequently named G148-GA3 (GA for protein G-related albumin binding).

  Bacterial albumin binding domains other than those present in protein G have also been identified, some of which are structurally similar to those of protein G. Examples of proteins containing such an albumin binding domain are PAB, PPL, MAG, and ZAG proteins (Non-Patent Document 15). Structural and functional studies of such albumin binding domains have been performed and reported, for example, by Johansson and coworkers (Non-Patent Document 16).

  In addition to the three-helix bundle protein described above, there are other unrelated bacterial proteins that bind to albumin. For example, the streptococcal protein family named "M protein" includes those that bind to albumin (see, for example, Non-Patent Document 17). Non-limiting examples are proteins M1 / Emm1, M3 / Emm3, M12 / Emm12, EmmL55 / Emm55, Emm49 / EmmL49, and H.

Engineered ABD Mutants Rozak et al. Have reported the generation of artificial variants of G148-GA3, which have been selected and studied for various species specificities and stability (15). In contrast, Jonsson et al. Have developed an artificial mutant of G148-GA3 with significantly improved affinity for human serum albumin (Non-Patent Document 18; Patent Document 4).

Although a small number of T-cell and B-cell epitopes have been experimentally identified within the albumin binding region of streptococcal protein G strain 148 (G148), such epitopes have led to the albumin binding domain G148. Has become less suitable for use in pharmaceutical compositions for human administration. As described in US Pat. No. 6,059,086, a new ABD variant has been developed to reduce immunostimulatory properties, while retaining a high potential for albumin binding, while reducing the potential for the presence of B-cell and T-cell epitopes.

WO91 / 01743 WO 01/45746 US2004 / 0001828 WO2009 / 016043 WO2012 / 004384

Salama et al., Adv Drug Deliv Rev. 58: 15-28, 2006 Hoffman and Ziv, Clin Pharmacokinet. 33: 285-301, 1997 Klein, AAPS J .; 12: 397-406, 2010 Singh, Clin Pharmacokinet. 37: 213-55, 1999 Mueller, Curr Issues Mol Biol. 13: 13-24, 2011 Rubas et al., J Pharm Sci. 85: 165-9, 1996 Park et al., Reactive and Functional Polymers, 71: 280-287, 2011. Meibohm, Pharmacokinetics and Pharmacodynamics of Biotech Drugs, Wiley-VCH, 2006 J Biol Chem 277: 35035-43, 2002 Bjoerck et al., Mol Immunol 24: 1113, 1987. Guss et al., EMBO J 5: 1567, 1986. Olsson et al., Eur J Biochem 168: 319, 1987. Kraulis et al., FEBS Lett 378: 190, 1996. Johansson et al. Biol. Chem. 277: 8114-20, 2002 Rozak et al., Biochemistry 45: 3263-3271, 2006. Johansson et al., J Mol Biol 266: 859-865, 1997. Table 2 in Navarre and Schneewind, MMBR 63: 174-229, 1999. Jonsson et al., Prot Eng Des Sel 21: 515-27, 2008. Goetsch et al., Clin Diagn Lab Immunol 10: 125-32, 2003.

  As is apparent from the above background, there remains a need for therapeutically effective biopharmaceuticals that can be administered via the oral route.

  Various aspects of the present invention address this need by allowing oral administration of a molecule as further defined below. Accordingly, the invention is directed to molecules for use in such oral administration treatments; pharmaceutical compositions comprising such molecules and formulated for oral administration; and such molecules or pharmaceutical compositions. Is orally administered to a subject in need of such treatment.

I Compounds for Use In a first aspect,
The present invention provides a compound for use in treatment by oral administration, wherein the compound comprises:
-A moiety (I) that confers a desired therapeutic activity; and-binds to albumin and is M1 / Emm1, M3 / Emm3, M12 / Emm12, EmmL55 / Emm55, Emm49 / EmmL49, H, G, MAG, ZAG, PPL, And an amino acid sequence corresponding to a portion (II) comprising a naturally occurring albumin binding protein selected from PAB and any one of the albumin binding domains, fragments or derivatives thereof,
Provided that the compound (I) is not selected from an exendin sequence, a sequence of an exendin analog, a sequence of a fragment having exendin activity, or a fragment having exendin analog activity.

  The compounds as defined above comprise at least two moieties (I) and (II), which may be covalently linked using, for example, known organic chemistry methods, or Where one or both moieties are polypeptides, they may be expressed as one or more fusion polypeptides in a recombinant expression system for the polypeptides, or may be directly or with a linker comprising multiple amino acids. May be combined in any other manner. For a discussion of linking albumin binding moieties with other moieties, eg, to provide compounds as defined above, see, eg, PCT Publications WO 2010/054699 and WO 2012/004384, which are incorporated herein by reference. I want to be.

Portion (I) that confers desired therapeutic activity
In one embodiment of the invention, some of the compounds, designated Part (I), comprise human endogenous enzymes, hormones, growth factors, chemokines, cytokines, blood coagulation and complement factors, innate immune defense and regulatory peptides A component selected from the group consisting of, for example, insulin, insulin analogs, IL-2, IL-5, GLP-1, BNP, IL1-RA, KGF, Stemgen®, GH, G-CSF, A component selected from the group consisting of CTLA-4, myostatin, factor VII, factor VIII, and factor IX, and derivatives thereof.

In another embodiment, moiety (I) is an organism selected from the group consisting of: modulin, bacterial toxin, hormone (but exendin), innate immune defense and regulatory peptides, enzymes, and activating proteins. Includes chemically active non-human proteins.

  In yet other embodiments, moiety (I) comprises a binding polypeptide capable of selective interaction with a target molecule. Such binding polypeptides include, for example, antibodies and fragments and domains thereof that substantially retain antibody binding activity; microbodies, maxibodies, avimers, and other small disulfide-binding proteins; Protease inhibition such as staphylococcal protein A and their domains, other 3 helix domains, lipocalin, ankyrin repeat domain, cellulose binding domain, gamma crystallin, green fluorescent protein, human cytotoxic T cell binding antigen 4, Kunitz domain Agents, PDZ domain, SH3 domain, peptide aptamer, staphylococcal nuclease, tendamistat, fibronectin type III domain, transferrin, zinc finger, and It may be selected from the group consisting of Notokishin.

  In some examples of such embodiments, the binding polypeptide comprises a variant of protein Z, which is then derived from domain B of staphylococcal protein A, which is described in Nilsson B et al., Protein Engineering 1 : 107-133, 1987. Such variants having affinity for a number of different targets are described in a number of previous publications, including, but not limited to, WO 95/19374; Nord et al., Nat Biotech, all of which are incorporated herein by reference. (1997) 15: 772-777; and WO 2009/080811, selected from a library and further processed. In such an embodiment of the compound for use according to the invention, the variant of protein Z corresponding to part (I) is a scaffold amino acid sequence selected from SEQ ID NO: 719, SEQ ID NO: 720 and SEQ ID NO: 721 Wherein X represents any amino acid residue. As explained in the above-mentioned literature and the PCT publication, all amino acid positions including X are involved in the binding function of protein Z variant, and to which target the Z variant is designed to bind Expected to vary depending on In these embodiments, preferably, the scaffold amino acid sequence of part (I) comprises SEQ ID NO: 719 or SEQ ID NO: 720.

  In an embodiment of the invention, wherein the moiety (I) comprises a binding polypeptide capable of selective interaction with the target molecule, said target molecule is CD14, CD19, CD20, CD22, CD30, CD33, CD37, CD40, CD52, CD56, CD70, CD138, cMet, HER1, HER2, HER3, HER4, CAIX, CEA, IL-2 receptor, IGF1R, VEGFR2, MUC1, PDGFR-beta, PSMA, tag-72, FOLR1, mesothelin, CA6, Tumor-related or other cell surface-related antigens such as GPNMB, integrins, and ephA2; TNF-α, IL-1α, IL-1β, IL-1Ra, IL-5, IL-6, IL-13, IL-17A , IL-18, IL-23, IL-36, G-CSF, GM-CS And cytokines such as their receptors; IL-8, CCL-2, and CCL11, and chemokines such as their receptors; complement factors such as C3 and factor D; such as HGF and myostatin Growth factors; hormones such as GH, insulin, and somatostatin; peptides such as Aβ peptide of Alzheimer's disease; other disease-related amyloid peptides; hypersensitivity mediators such as histamine and IgE; von Willebrand factor Blood coagulation factors; and toxins such as bacterial toxins and snake venoms.

In an alternative embodiment, portion (I) comprises a non-proteinaceous component with therapeutic activity. Particularly important examples include cytotoxics and anti-inflammatory drugs, as albumin has been shown to accumulate in tumor tissue and at sites of inflammation (Kratz and Beyer, Drug Delivery 5: 281- 99, 1998; Wunder et al., J. Immunol. 170: 4793-801, 2003). This, in turn, is a rationale for oral delivery of such compounds with an albumin binding moiety for targeting and accumulation at relevant tumor tissues or sites of inflammation. Non-limiting examples of cytotoxic agents include calicheamicin, auristatin, doxorubicin, maytansinoids, taxanes, ecteinasidine, geldanamycin, methotrexate, camptothecin, cyclophosphamide, cyclosporine, and derivatives thereof, and It is a combination of Non-limiting examples of anti-inflammatory drugs include non-steroidal anti-inflammatory drugs (NSAIDs), cytokine inhibitory anti-inflammatory drugs (CSAIDs), corticosteroids, methotrexate, prednisone, cyclosporine, moroniside cinnamic acid. ), Leflunomide, and derivatives thereof, and combinations thereof.

  In such embodiments, the non-proteinaceous moiety (I) and the albumin binding moiety (II) may be non-covalently associated, but are currently covalently linked together. Is preferred.

  Conjugation of the non-proteinaceous moiety (I) to the albumin binding moiety (II) may increase the solubility and therefore the otherwise poorly soluble compounds that are not suitable for oral administration May increase the bioavailability of

Part (II) that binds to albumin
As defined herein, compounds for use in treatment by oral administration bind to albumin and have M1 / Emm1, M3 / Emm3, M12 / Emm12, EmmL55 / Emm55, Emm49 / EmmL49, H, G , MAG, ZAG, PPL, and PAB, or an amino acid sequence corresponding to portion (II) comprising an albumin binding domain, fragment or derivative of any one of them.

  As described in the background section and in the literature by Rozak et al. And Johansson et al., Cited herein, many of the above albumin binding proteins contain an albumin binding domain called the GA domain. In one embodiment of the invention, part (II) of the compound comprises a naturally occurring GA domain or a derivative thereof. Specific examples of such useful GA domains are domain GA1, domain GA2, and domain GA3 of protein G from streptococcal strain G148, and derivatives thereof. In one specific embodiment, part (II) comprises domain GA3 of protein G from streptococcal strain G148. This albumin binding domain is also often referred to in the literature as "ABD" or "ABDwt" and has the amino acid sequence set forth in SEQ ID NO: 515 in the accompanying list. In another embodiment, portion (II) comprises a derivative of domain GA3 of protein G from streptococcal strain G148. Some of such derivatives have been developed, for example, as described in WO 2009/016043 and WO 2012/004384, which are incorporated herein by reference above.

Part (II) containing ABD derivative disclosed in WO2009 / 016043
Thus, with reference to WO 2009/016043, part (II) of the compound for use in the treatment by oral administration according to the present invention may comprise an albumin binding motif, which has the amino acid sequence:
GVSDX ₅ YKX ₈ X ₉ I X ₁₁ X ₁₂ AX ₁₄ TVEGVX ₂₀ ALX ₂₃ X ₂₄ X ₂₅ I
Consisting of
Wherein, independently of each other,
X ₅ is selected from Y and F;
X ₈ is selected from N, R, and S;
X ₉ is, V, selected I, L, M, F, and from Y;
X ₁₁ is selected from N, S, E, and D;
X ₁₂ is selected from R, K, and N;
X ₁₄ is selected from K and R;
X ₂₀ is selected from D, N, Q, E, H, S, R, and K;
X ₂₃ is selected from K, I, and T;
X ₂₄ is selected from A, S, T, G, H, L, and D; and X ₂₅ is selected from H, E, and D.

  The definition of the class of sequences related to albumin binding polypeptides for use in part (II) in the present compounds described above has been identified and characterized for a number of identified and characterized as detailed in the experimental section of WO2009 / 016043. Based on statistical analysis of albumin binding polypeptide. Briefly, the mutants are selected from a large pool of random mutants of the parent polypeptide sequence of ABDwt (SEQ ID NO: 515), wherein the selection comprises, for example, albumin in a phage display or other selection experiment. It is based on the interaction. The identified albumin binding motif, or "ABM", corresponds to the albumin binding region of the parent scaffold, which makes up two alpha helices within the domain of a three helix bundle protein. Although the original amino acid residues of the two ABM helices in the parent scaffold already constitute a binding surface for interaction with albumin, the binding surface has been modified by the substitution according to the invention to provide an alternative To provide albumin binding ability.

In one embodiment, _{X 5} is Y.

In one embodiment, X ₈ is selected from N and R, may in particular be R.

In one embodiment, X ₉ is L.

In one embodiment, X ₁₁ is selected from N and S, and may in particular be N.

In one embodiment, X ₁₂ is selected from R and K, for example, X ₁₂ is R or X ₁₂ is K.

In one embodiment, X ₁₄ is K.

In one embodiment, X ₂₀ is selected from D, N, Q, E, H, S, and R, and may especially be E.

In one embodiment, X ₂₃ is selected from K and I, and in particular may be K.

In one embodiment, X ₂₄ is selected from A, S, T, G, H, and L.

In a more specific embodiment, X ₂₄ is L.

In an even more specific embodiment, X ₂₃ X ₂₄ is KL.

In other even more specific embodiments, X ₂₃ X ₂₄ is TL.

In one embodiment, X ₂₄ is selected from A, S, T, G, and H.

In a more specific embodiment, X ₂₄ is selected from A, S, T, G, and H, and X ₂₃ is I.

In one embodiment, X ₂₅ is H.

  As described in detail in the experimental section of WO 2009/016043, the selection of albumin binding mutants led to the identification of significant amounts of individual albumin binding motif (ABM) sequences. These sequences constitute individual embodiments of the ABM sequence in the definition of the albumin binding amino acid sequence as part (II) in the context of the present invention. The sequence of the individual albumin binding motif is shown as SEQ ID NOs: 1-257 in FIG. In certain embodiments, the ABM consists of an amino acid sequence selected from SEQ ID NOs: 1-257. In a more specific embodiment, the ABM sequence is SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 155, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, and SEQ ID NO: 245. In an even more specific embodiment, the ABM sequence is selected from SEQ ID NO: 3, SEQ ID NO: 53, and SEQ ID NO: 239.

  In embodiments of the albumin binding moiety (II), the ABM may form part of a three-helix bundle protein domain. For example, ABM may substantially constitute or form part of two alpha helices having loops connected to each other within the three-helix bundle protein domain.

  In certain embodiments, such a three-helix bundle protein domain is selected from the group consisting of three helix domains of a bacterial receptor protein. Non-limiting examples of such bacterial receptor proteins are selected from the group consisting of albumin binding receptor proteins from the streptococcal species Peptostreptococcus and Finegoldia, such as protein G, It is selected from the group consisting of MAG, ZAG, PPL, and PAB. In a specific embodiment of the invention, the ABM forms part of a protein G, such as, for example, protein G from streptococcal strain G148. In various variants of this embodiment, the three-helix bundle protein domain of which the ABM forms a part is selected from the group consisting of domain GA1, domain GA2, and domain GA3 of protein G from streptococcal strain G148, Domain GA3.

  In an alternative embodiment, the ABM forms part of one or more of the five three-helix domains of protein A of the bacterial receptor protein from Staphylococcus aureus; The three-helix bundle protein domain is selected from the group consisting of domains A, B, C, D, and E of protein A. In other similar embodiments, the ABM forms part of a protein Z derived from domain B of protein A from Staphylococcus aureus.

In embodiments where the ABM “forms” a three-helix bundle protein domain, “forms” is a sequence of the ABM such that the ABM is replaced with a similar structural motif in the original domain. Is "inserted" or "grafted" into the sequence of a naturally occurring (or otherwise native) three-helix bundle domain. For example, without wishing to be limited by theory, it is believed that the ABM constitutes two of the three helices of the three-helix bundle, and thus the ABM may have two such helices in any three-helix bundle. Two helix motifs. As will be appreciated by those skilled in the art, the replacement of two helices of a three-helix bundle domain with two ABM helices must be made without affecting the basic structure of the polypeptide. Must. That is, the entire fold of the Cα main chain of the polypeptide according to this embodiment is substantially the same as the fold of the three-helix bundle protein domain that forms a part thereof, for example, having the same secondary structure element in the same order. Are expected to be the same. Thus, when the polypeptide according to this embodiment of the invention folds in the same way as the original domain, the ABM according to the invention "forms" a three-helix bundle domain, which is the basic structural These characteristics are common, and include, for example, characteristics that result in a similar CD spectrum. Those skilled in the art are aware of other relevant parameters.

In one embodiment, the albumin binding polypeptide is a three helix bundle protein domain, which comprises an albumin binding motif as defined above and additional sequences that make up the remainder of the three helix structure. Thus, in this embodiment, part (II) comprises the amino acid sequence:
LAEAKX _{a Xb} AX _c X _d ELX _e KY- [ABM] -LAALP
Comprising an albumin binding domain having
Where:
[ABM] is an albumin binding motif as defined above in this section,
Independent of each other,
_Xa is selected from V and E;
X _b is selected from L, E, and D;
_Xc is selected from N, L, and I;
X _d is selected from R and K; and X _e is selected from D and K.

In one embodiment, X _a is V.

In one embodiment, X _b is L.

In one embodiment, X _C is N.

In one embodiment, _Xd is R.

In one embodiment, _Xe is D.

Again, as described in detail in the experimental section of WO 2009/016043, the selection and sequencing of a number of albumin binding variants led to the identification of the sequence of the individual albumin binding domain. These sequences constitute individual embodiments of the albumin binding domain comprised in part (II) of the compound for use in the oral administration treatment of the present invention. The sequences of these individual albumin binding domains are shown in FIG. 1 as SEQ ID NOs: 258-514. Albumin binding domains having an amino acid sequence having 85% or greater identity to a sequence selected from SEQ ID NOs: 258-514 are also encompassed by the definitions herein. In certain embodiments, the sequence of the albumin binding domain is SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 266, SEQ ID NO: 272, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 303, SEQ ID NO: 306, SEQ ID NO: 310, SEQ ID NO: 310 No. 311, SEQ ID No. 312, SEQ ID No. 412, SEQ ID No. 496, SEQ ID No. 497, SEQ ID No. 498, SEQ ID No. 499, SEQ ID No. 500, SEQ ID No. 501 and SEQ ID No. 502 and 85% or more identical thereto Are selected from sequences having the same characteristics. In a more specific embodiment of this aspect of the invention, the sequence of the albumin binding polypeptide has SEQ ID NO: 260, SEQ ID NO: 310, and SEQ ID NO: 496, and 85% or greater identity thereto. Selected from the array.

Part (II) containing ABD derivatives disclosed in WO2012 / 004384
Referring instead to WO2012 / 004384, part (II) of the compound for use in the treatment by oral administration according to the present invention may alternatively comprise an albumin binding domain, which in turn comprises:
i) LAX ₃ AKX ₆ X ₇ ANX ₁₀ ELDX ₁₄ YGVSDF YKRLIX ₂₆ KAKT VEGVEALKX ₃₉ X ₄₀ ILX ₄₃ X ₄₄ LP
Wherein, independently of each other,
X ₃ is selected from E, S, Q, and C;
X ₆ is selected from E, S, and C;
X ₇ is selected from A and S;
X ₁₀ is selected from A, S, and R;
X ₁₄ is selected from A, S, C, and K;
X ₂₆ is selected from D and E;
X ₃₉ is selected from D and E;
X ₄₀ is selected from A and E;
X ₄₃ is selected from A and K;
_X44 is selected from A, S, and E;
L at position 45 is present or absent; and P at position 46 is present or absent;
And ii) an amino acid sequence having at least 95% identity to the sequence defined in i),
Including an amino acid sequence selected from:

  Albumin binding domains according to this definition, for example, exhibit a range of features that make them suitable for use as fusion or conjugate partners for therapeutic molecules for administration to humans. The advantages of this class of albumin binding domains are described in detail in WO2012 / 004384.

In one embodiment, X ₆ is E.

In another embodiment, X ₃ is S.

In another embodiment, X ₃ is E.

In another embodiment, X ₇ is A.

In another embodiment, X ₁₄ is S.

In another embodiment, X ₁₄ is C.

In another embodiment, X ₁₀ is A.

In another embodiment, X ₁₀ is S.

In another embodiment, X ₂₆ is D.

In another embodiment, X ₂₆ is E.

In another embodiment, _X39 is D.

In another embodiment, X ₃₉ is E.

In another embodiment, X ₄₀ is A.

In another embodiment, X ₄₃ is A.

In another embodiment, X ₄₄ is A.

In another embodiment, X ₄₄ is S.

  In another embodiment, there is an L residue at position 45.

  In another embodiment, there is a P residue at position 46.

  In another embodiment, there is no P residue at position 46.

In other embodiments, the albumin binding domain according to the definition of this section, X ₇ is subject to the conditions that it is not L, E or D.

  Albumin binding domains for use in part (II) according to the definitions in this section may be made to conjugate with suitable conjugate partners as described in detail in WO2012 / 004384.

  In one embodiment, the amino acid sequence of the albumin binding domain of part (II) is selected from any one of SEQ ID NOs: 516-659 and 679-718, for example, from any one of SEQ ID NOs: 516-659. Selected. More specifically, the amino acid sequence is represented by SEQ ID NOs: 519 to 520, SEQ ID NOs: 522 to 523, SEQ ID NOs: 525 to 526, SEQ ID NOs: 528 to 529, SEQ ID NOs: 531 to 532, SEQ ID NOs: 534 to 535, SEQ ID NOs: 537 to 537 538, SEQ ID NOs: 540-541, SEQ ID NOs: 543-544, SEQ ID NOs: 546-547, SEQ ID NOs: 549-550, SEQ ID NOs: 552-553, SEQ ID NOs: 556-557, SEQ ID NOs: 564-565, SEQ ID NOs: 679-685 and Selected from SEQ ID NOs: 707-718. Therefore, the amino acid sequence is represented by SEQ ID NOs: 519-520, SEQ IDs: 522-523, SEQ IDs: 525-526, SEQ IDs: 528-529, SEQ IDs: 531-532, SEQ IDs: 534-535, SEQ IDs: 537-538, SEQ ID NOs: 540 to 541, SEQ ID NOs: 543 to 544, SEQ ID NOs: 546 to 547, SEQ ID NOs: 549 to 550, SEQ ID NOs: 552 to 553, SEQ ID NOs: 556 to 557, and SEQ ID NOs: 564 to 565.

In one embodiment, the albumin binding domain according to this definition further comprises one or more additional amino acid residues located at the N-terminal and / or C-terminal of the sequence defined in i). These additional amino acid residues may be useful in enhancing albumin binding by the domain or improving the structural stability of the folded albumin binding domain, for example, in the production of polypeptides, It may be equally useful for one or more of purification, in vivo or in vitro stabilization, ligation, labeling, or detection, as well as other purposes for any combination thereof. Such additional amino acid residues may be, for example, chemically linked to moiety (I) to confer a therapeutic effect; or may be complexed with a chromatographic resin to obtain an affinity matrix or with a radiometal. Or one or more amino acid residues added for the purpose of chemically linking to the chelating moiety for

  Thus, in one embodiment, amino acids that occur immediately before or immediately after the alpha helix at the N-terminus or C-terminus of amino acid sequence i) can affect structural stability. An example of an amino acid residue that may contribute to improved structural stability is a serine residue located at the N-terminus of the amino acid sequence i) defined above. In some cases, the serine residue at the N-terminus is replaced by a regular SXXE capping box by hydrogen bonding between the gamma oxygen of the serine side chain and the NH of the polypeptide backbone of the glutamic acid residue. There is a possibility of forming a capping box). This N-terminal capping may contribute to the stabilization of the first alpha helix of the three helix domains that make up the albumin binding domain according to this definition.

  Thus, in one embodiment, the additional amino acids comprise at least one serine residue at the N-terminus of the domain. In other words, one or more serine residues may be present before the amino acid sequence. In other embodiments, the additional amino acids include a glycine residue at the N-terminus of the domain. It is understood that one, two, three, four or any suitable number of amino acid residues may be present before the amino acid sequence i). Thus, before the amino acid sequence, there is a single serine residue, a single glycine residue, or a combination of the two, such as a glycine-serine (GS) combination or a glycine-serine-serine (GSS) combination. It may be. Examples of albumin binding domains containing an additional amino residue at the N-terminus are set forth in SEQ ID NOs: 660-678, such as SEQ ID NOs: 660-663 and SEQ ID NOs: 677-678. In yet other embodiments, the additional amino acid residue comprises glutamic acid at the N-terminus as defined in sequence i).

  Similarly, C-terminal capping may be used to improve the stability of the third alpha helix of the three helix domains that make up the albumin binding domain. If a proline residue is present at the C-terminus of the amino acid sequence defined in i), the proline residue may at least partially function as a capping residue. In such cases, if both the epsilon amino group of the lysine residue and the carbonyl group of the amino acid located two and three residues before the lysine in the polypeptide backbone, for example, both L45 and P46, i ), The lysine residue after the proline residue at the C-terminus further stabilizes the third helix of the albumin binding domain by hydrogen bonding with the carbonyl groups of the leucine and alanine residues of the amino acid sequence defined in May contribute to Thus, in one embodiment, the additional amino acids comprise a lysine residue at the C-terminus of the domain.

The additional amino acids may be for the production of the albumin binding domain. In particular, if the albumin binding domain according to the embodiment where P46 is present is produced by chemical peptide synthesis, one or more optional amino acid residues following the C-terminal proline may benefit. . Such additional amino acid residues may prevent the formation of undesired substances such as, for example, diketopiperazines during the synthetic dipeptidation step. One example of such an amino acid residue is glycine. Thus, in one embodiment, the additional amino acids include a glycine residue at the C-terminus of the domain, immediately after the proline residue or after an additional lysine and / or glycine residue as described above. Alternatively, where a proline residue is present at the C-terminus of amino acid sequence i), polypeptide production may benefit from amidation of such proline residues. In this case, the C-terminal proline contains an additional amine group at the carboxyl carbon. In one embodiment of the domains described in this section, particularly in one embodiment of the domain whose C-terminus is terminated with proline or another amino acid known to racemize during peptide synthesis, the C Addition of glycine to the terminus, or amidation of proline, if present, can also address possible problems in racemizing C-terminal amino acid residues. If the domain to be amidated in this manner is intended to be produced by recombinant means rather than chemical synthesis, amidation of the C-terminal amino acid can be accomplished by several methods known in the art, such as PAM amidating enzyme. Can be performed by a method using

  Examples of albumin binding domains containing additional amino acid residues at the C-terminus are set forth in SEQ ID NOs: 660-667, for example, SEQ ID NOs: 663-665. One skilled in the art understands how to achieve C-terminal modification, such as by matrices for the synthesis of different types of pre-made peptides.

  In other embodiments, the additional amino acid residues include cysteine residues at the N-terminal and / or C-terminal of the domain. Such a cysteine residue may be present immediately before and / or immediately after the amino acid sequence defined in i), or before and / or before any other additional amino acid residues described above. It may be present later. Examples of albumin binding domains that include cysteine residues at the N- and / or C-terminus of the polypeptide chain are set forth in SEQ ID NOs: 664-665 (C-terminus) and SEQ ID NOs: 666-667 (N-terminus). By adding a cysteine residue to the polypeptide chain, a thiol group may be added to a site intended for conjugate of the albumin binding domain. Alternatively, selenocysteine residues can be introduced at the C-terminus of the polypeptide chain in a manner similar to cysteine residue introduction to facilitate site-specific conjugates (Cheng et al., Nat Prot 1: 2). P. 2006).

  In one embodiment, the albumin binding domain contains no more than two cysteine residues. In other embodiments, the albumin binding domain contains only one cysteine residue.

In general applicable aspects some embodiments of the portion (II), the albumin binding domain in part (II) of the compound for use according to the present invention, is _the K _D value of the interaction, the maximum To 1 × 10 ⁻⁸ M, that is, 10 nM. In some embodiments, the K _D value of the interaction is at most 1 × 10 ⁻⁹ M, at most 1 × 10 ⁻¹⁰ M, at most 1 × 10 ⁻¹¹ M, or at most 1 × 10 ⁻¹² M. M.

  In one embodiment, the albumin binding domain within portion (II) binds human serum albumin. Alternatively or additionally, in one embodiment, the albumin binding domain binds albumin from a species other than the human species, such as, for example, albumin from mouse, rat, dog, and cynomolgus macaque.

As explained broadly above, the albumin binding moiety (II) may comprise an amino acid sequence selected from SEQ ID NOs: 258-718 or a subset thereof, or may comprise an albumin binding motif in a larger albumin binding domain May include a sequence selected from SEQ ID NOs: 1 to 257. As one skilled in the art will appreciate, the function of any polypeptide, such as, for example, the albumin binding capacity of these polypeptide domains, will depend on the tertiary structure of the polypeptide. However, it is also possible to change the sequence of the amino acids in the α-helix polypeptides without affecting their structure (Taverna and Goldstein, J Mol Biol 315 (3): 479-84, 2002). He et al., Proc Natl Acad Sci;
USA 105 (38): 14412-17, 2008). Thus, modified variants of the naturally occurring albumin proteins or derivatives thereof disclosed in detail above are also envisioned as candidates for the albumin binding domain comprised in portion (II). For example, an amino acid residue belonging to a predetermined functional class (for example, hydrophobic, hydrophilic, polar, etc.) of an amino acid residue can be exchanged for another amino acid residue belonging to the same functional class.

  Thus, portion (II) may include variants of the disclosed albumin binding proteins that show only minor differences in comparison to SEQ ID NOs: 1-718. One such definition is an albumin binding domain having an amino acid sequence that has at least 85% identity to a sequence selected from SEQ ID NOs: 258-718. In some embodiments, the albumin binding domain is at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, relative to a sequence selected from SEQ ID NOs: 258-718. It may have a sequence with at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity.

  The terms "% identical" or "% identity", as used herein and in the claims, are calculated as follows. Align the query sequence against the target sequence using the CLUSTAL W algorithm (Thompson, JD, Higgins, DG and Gibson, TJ., Nucleic Acids Research, 22: 4673-4680 (1994). Year)). The comparison is performed in the window corresponding to the shortest of the arranged arrays. The shortest of the aligned sequences may in some cases be a target sequence, such as, for example, the albumin binding domain disclosed herein. In another example, the query sequence may constitute the shortest of the ordered sequences. The query sequence may consist, for example, of at least 10 amino acid residues, such as at least 20 amino acid residues, such as at least 30 amino acid residues, such as at least 40 amino acid residues, such as 45 amino acid residues. The amino acid residues at each position are compared and the percentage of positions in the query sequence that have identical counterparts in the target sequence are reported as% identity.

The terms "albumin binding" and "binding affinity for albumin" as used herein refer to a property of a polypeptide that can be tested by the use of surface plasmon resonance technology, for example, a Biacore instrument. For example, as described in the examples below, albumin binding affinity was determined in an experiment in which albumin or a fragment thereof was immobilized on a sensor chip of an instrument and a sample containing the polypeptide to be tested was passed over the chip. May be tested. Alternatively, the polypeptide to be tested is immobilized on the sensor chip of the instrument, and a sample containing albumin or fragments thereof is passed over the chip. In this regard, the albumin may be a serum albumin from a mammal such as, for example, human serum albumin. One skilled in the art may then interpret the results obtained from such experiments to establish at least a qualitative measure of the binding affinity of the polypeptide to albumin. For example, where a quantitative measure is required to determine the _KD value of the interaction, surface plasmon resonance can also be used. The binding value may be defined by, for example, a Biacore 2000 device (GE Healthcare). Albumin is preferably immobilized on a sensor chip for measurement, and a sample of the polypeptide whose affinity is to be determined is prepared by serial dilution and injected. Then, for example, equipment manufacturer BIAevaluation4.1 software supplied by (GE Healthcare) 1: may calculate the _{K D} values from the result using 1 Langmuir binding model.

II Pharmaceutical composition In a second aspect,
The present invention
a) a compound,
-A moiety (I) that confers a desired therapeutic activity; and-binds to albumin and is M1 / Emm1, M3 / Emm3, M12 / Emm12, EmmL55 / Emm55, Emm49 / EmmL49, H, G, MAG, ZAG, PPL, And an amino acid sequence corresponding to a portion (II) comprising a naturally occurring albumin binding protein selected from PAB and any one of the albumin binding domains, fragments or derivatives thereof,
With the proviso that the moiety (I) is not selected from an exendin sequence, a sequence of an exendin analog, a sequence of a fragment having exendin activity or a fragment having exendin analog activity;
b) a pharmaceutical composition for oral administration comprising at least one pharmaceutically acceptable excipient.

  Thus, this second aspect of the invention provides a pharmaceutical composition comprising, as a constituent, a) a compound as defined for the first aspect of the invention. The compound, when present in a pharmaceutical composition for oral administration, exhibits any one or more of the properties, features, features, and / or embodiments described above with respect to the first aspect of the invention. Any combination may be used. For brevity, this information will not be repeated word-for-word with respect to this second aspect, but is incorporated by reference into the above disclosure.

  The pharmaceutical composition further comprises b) at least one pharmaceutically acceptable excipient. "Excipients" are inert substances used as diluents or vehicles in drug formulations. The excipient may facilitate administration or manufacture, improve delivery of the product, enhance constant release and bioavailability of the drug, enhance stability, aid in product identification, or otherwise. It is mixed with one or more compounds having therapeutic activity to enhance the characteristics of the product. Additives include binders, diluents / fillers, lubricants, lubricants, disintegrants, abrasives, colorants, suspending agents, film formers, and coatings, plasticizers, dispersants, preservatives, fragrances, Can be classified as sweeteners.

  In some embodiments of the pharmaceutical composition of the present invention, the pharmaceutical composition of the present invention comprises at least one kind of compound for increasing the oral bioavailability of the moiety (I) that confers the desired therapeutic activity. The components of the present invention are further included. In such embodiments, the component of interest may be selected from the group consisting of a protease inhibitor, an absorption enhancer, a mucoadhesive polymer, a formulation vehicle, and any combination thereof. In the following sections, the use of such components and their scientific rationale are described with respect to general strategies for improving the oral bioavailability of therapeutic agents.

  The resistance of pharmaceutical compositions to the acidic and enzymatic environment of the gastrointestinal tract can be attributed to enzymes that target related peptides and proteins active in the stomach (eg, pepsin) and enzymes that target related peptides and proteins active in the intestine (E.g., trypsin, chymotrypsin, and carboxypeptidase) can be enhanced by adding one or more inhibitors (cocktails or individually targeted). Such inhibitors include trypsin and α-chymotrypsin inhibitors such as pancreatin inhibitors, soybean trypsin inhibitors, FK-448, camostat mesylate, aprotinin, chicken and duck ovomucoid, carboxymethylcellulose, and Bowman-Bourke inhibition Or a conjugate of a mucoadhesive polymer and a protease inhibitor (Non-Patent Document 7).

To increase the absorption of the polypeptide from the intestinal wall and improve the therapeutic effect, the pharmaceutical composition may include an absorption enhancer that makes the epithelial barrier more permeable. The absorption enhancer can, for example, disrupt transmembrane lipid bilayers to improve transcellular transport, or act as a chelator to break tight junctions to facilitate paracellular transport. . Non-limiting examples of absorption enhancers for use in this aspect of the invention include detergents, surfactants, bile salts, calcium chelators, fatty acids, medium chain glycerides, salicylates, alkanoylcholines, N-acetyl Α-amino acids, N-acetylated non-α-amino acids, chitosan, phospholipids, sodium caprate, acylcarnitine, and closed zone toxin (Non-Patent Document 1).

  As an additional or alternative component in pharmaceutical compositions, mucoadhesive polymers have the potential to protect against proteolysis, but primarily to increase residence time at drug absorption sites to achieve site specific delivery to the mucosa. It is applied to lengthen and further improve membrane penetration, both of which promote increased absorption from the intestinal wall. Non-limiting examples for use in the pharmaceutical compositions of the present invention include poly (methacrylic acid-g-ethylene glycol) [P (MAA-g-EG)] hydrogel microparticles, lecithin-bound alginate microparticles, thiolated polymers ( Thiomers), gastrointestinal mucoadhesive patch systems (GI-MAPS) and conjugates of mucoadhesive polymers with protease inhibitors (Park et al., 2011, supra).

  Formulation media such as emulsions, liposomes, microspheres, nanospheres, nanocapsules, or complete encapsulation help protect against proteolysis, achieve a defined controlled release rate, and also enhance enhanced delivery from the intestinal wall there's a possibility that. Such formulation vehicles also constitute alternative or complementary components for use in the pharmaceutical compositions of the present invention. In particular, nanoparticles having altered surface properties or linked to a target molecule may be used. Modification of the nanoparticle surface can be achieved, for example, either by coating with a hydrophilic stabilizer, a bioadhesive polymer or surfactant, or by incorporating a hydrophilic copolymer into the nanoparticle formulation. Examples of such hydrophilic polymers include PEG and chitosan (des Rieux et al., J Control Release. 116: 1-27, 2006). The design of targeted nanoparticles can be achieved by linking a ligand, such as a lectin or RGD (arginine-glycine-aspartate) derivative, to the receptor, expressed in intestinal cells or M cells in the epithelial layer of the intestinal wall. (Des Rieux et al., 2006, supra). M cells also provide a route of delivery to the lymphatic system (Rubas and Grass, Advanced Drug Delivery Reviews, 7: 15-69, 1991).

  Pharmaceutical compositions of the present invention include, for example, solid forms, such as, for example, pills, tablets, capsules, powders or granules; semi-solid forms, such as, for example, pastes; or liquids, such as, for example, elixirs, solutions or suspensions. May be administered orally. It is presently preferred in solid form, such as chitosan, alginate, microcrystalline cellulose, lactose, saccharose, starch, gelatin, lactose, polyethylene glycol, polyvinylpyrrolidone (PVP), magnesium stearate, Additives such as calcium stearate and sodium starch glycolate may be included. Liquid form preparations may contain additives, for example, sweetening or flavoring agents, emulsifying or suspending agents, or diluents, for example, water, ethanol, propylene glycol, and glycerin.

The formulation may be intended for immediate release, delayed release or controlled release applications. Tablets or capsules intended for immediate release are expected to disintegrate rapidly and release all actives in the upper gastrointestinal tract, ie, in the stomach. On the other hand, tablets or capsules intended for delayed or controlled release can be designed for time-dependent release (depot formulation) or site-specific release (eg intestinal). Time-dependent release may be based, for example, on degradation or diffusion controlled release depending on the composition of the matrix or membrane. Site-specific release may be based, for example, on pH sensitivity or enzyme sensitivity. Particularly preferred for the formulation of the pharmaceutical composition according to the invention are enteric-coated capsules intended for release in the small intestine or colon. Such enteric capsules are expected to be stable at high acidic pH in the stomach but rapidly dissolve at lower acidic pH in the intestinal tract. Examples of pH-sensitive enteric film-forming agents include, for example, hydroxypropylmethylcellulose phthalate (HPMCP), cellulose acetate phthalate (CAP), cellulose acetate trimellitate (CAT), hydroxypropylmethyl acetate succinate (HPMCAS), polyvinyl Cellulose polymers such as acetate phthalate (PVAP) and other polymers such as, for example, Eudragit® derivatives, shellac (SH), chitosan, and chitin.

III Method of Treatment In a third aspect,
The present invention is a method of treating a mammalian subject in need of such treatment, comprising orally administering the compound, wherein the compound comprises:
-A moiety (I) that confers a desired therapeutic activity; and-binds to albumin and is M1 / Emm1, M3 / Emm3, M12 / Emm12, EmmL55 / Emm55, Emm49 / EmmL49, H, G, MAG, ZAG, PPL, And an amino acid sequence corresponding to a portion (II) comprising a naturally occurring albumin binding protein selected from PAB and any one of the albumin binding domains, fragments or derivatives thereof,
Provided that the method (I) is not selected from an exendin sequence, an exendin analog sequence, a sequence of a fragment having exendin activity or a fragment having exendin analog activity.

  Thus, this third aspect of the invention provides a method of treatment comprising orally administering a compound as defined for the first aspect of the invention. Optionally, the compound may be administered as present in a pharmaceutical composition as defined for the second aspect of the invention. The compounds and pharmaceutical compositions each exhibit any one or more of the characteristics, features, features, and / or embodiments described above with respect to the first and second aspects of the invention in any combination. It may be something. For the sake of brevity, this information will not be repeated word-for-word with respect to this third aspect, but is incorporated by reference into the above disclosure.

  In one embodiment, the treatment method according to the invention is performed according to the specified dosing regimen. It is expected that the optimal dosage regimen will depend on the potency of the moiety that confers a therapeutic effect, the bioavailability of the compounds described herein, and the nature of the disease to be treated. However, compounds containing an albumin binding moiety (II) that are believed to extend the half-life of the compounds described herein are not expected to require a single high dose to achieve a given level of therapeutic effect. However, due to the prolonged circulatory residence time, it is possible to administer a smaller number of doses to increase the concentration of the compound and ultimately achieve a sustained desired therapeutic effect. In other words, after oral administration, the bioavailability may be lower than the bioavailability of the short-lived therapeutic. Such repeated administrations may be made at least twice a month, once a week, twice a week, three times a week, once a day, twice a day, for example at least three times a day.

For a given disease, it may be desirable to administer a bolus dose, followed by repeated administration of lower doses. The bolus dose may be at least once a day, twice a day, three times a day, four times a day, or at least five times a day, multiple doses of a drug formulated for oral use. Alternatively, large bolus doses may be administered via another route, such as by intravenous or subcutaneous injection. For the purpose of achieving a sustained therapeutic effect, subsequent administrations may be performed at least twice a month, once a week, twice a week, three times a week, once a day, twice a day, three times a day, for example at least It may be done four times a day.

Definitions and Uses of Terms As used herein, "bioavailability" refers to the proportion of an administered dose of an active pharmaceutical agent that reaches systemic circulation. By definition, the bioavailability of an intravenously administered drug is 100%. However, if the drug is administered via other routes, for example by the oral route, bioavailability is reduced due to metabolism and incomplete absorption. Absolute bioavailability compares the bioavailability of the active drug in the systemic circulation after non-iv administration with the bioavailability of the same drug after intravenous administration . It is calculated as the ratio of drug absorbed by non-iv administration to the corresponding intravenous administration of the same drug. This comparison must be normalized (eg, to take into account different doses or different body weights of the subject). To determine the absolute bioavailability of a drug, pharmacokinetic studies must be performed to obtain a plasma drug concentration-time plot of the drug after each of the intravenous and non-iv doses . Absolute bioavailability is the area under the curve (AUC) corrected for non-intravenous dose divided by the intravenous AUC.

  The present invention will now be further illustrated by the following non-limiting examples.

2 is a table providing a short list of various amino acid sequences discussed in the text herein. Continuation of FIG. 1-1. Continuation of FIG. 1-2. Continuation of FIG. 1-3. Continuation of FIG. 1-4. Continuation of FIG. 1-5. Continuation of FIG. 1-6. Continuation of FIG. 1-7. Continuation of FIG. 1-8. Continuation of FIG. 1-9. Continuation of FIG. 1-10. Continuation of FIG. 1-11. Continuation of FIG. 1-12. Continuation of FIG. 1-13. Continuation of FIG. 1-14. Continuation of FIG. 1-15. Continuation of FIG. 1-16. Continuation of FIG. 1-17. Continuation of FIG. 1-18. Continuation of FIG. 1-19. Continuation of FIG. 1-20. Continuation of FIG. Continuation of FIG. 1-22. Continuation of FIG. 1-23. 2 shows the results of SDS-PAGE analysis of purified polypeptide variants produced as described in Example 1. Lanes 1-2: 25 and 50 μg of PEP04419, respectively; Lanes 3-4: 25 and 50 μg of PEP10986, respectively; and Lanes 5-6: 25 and 50 μg of PEP03973, respectively. Lane M: Novex® Sharp prestained protein standard (molecular weight: 3.5, 10, 15, 20, 30, 40, 50, 60, 80, 110, 160, and 260 kDa). Figure 4 shows the pharmacokinetic profiles after oral administration of PEP03973 (open squares), PEP10986 (open triangles), and PEP04419 (open circles) to mice, respectively, as described in Example 2. FIG. 3A shows serum concentrations measured over time (mean of the values of three animals per time point). FIG. 3A shows the same data as FIG. 3A but adjusted for variations in the administered dose of the three polypeptides (ie, at each time point: [measured serum concentration] / [administered dose]). 5 shows the pharmacokinetic profile of PEP10896 in serum samples obtained from rats following oral gavage (open squares) or intraduodenal administration (open circles) as described in Example 3. The concentration of PEP10896 was determined by ELISA and shows the mean nM +/− SD values. 9 shows the pharmacokinetic profile after repeated intraduodenal administration of PEP10896 as described in Example 4. Polypeptides were given at time points 0, 2, and 24 hours, marked by arrows in the graph. The concentration of PEP10896 was determined by ELISA and shows the mean nM +/− SD values (open circles). Figure 4 shows the pharmacokinetic profile after a single intraduodenal administration determined in Example 3 for comparison (open squares).

[Example 1]
Cloning, Production, and Characterization of Polypeptides Materials and Methods To illustrate the present invention, three polypeptides, designated PEP03973, PEP10986, and PEP04419, respectively, were prepared for oral administration in mice. As previously described by Feldwisch et al. (J. Mol. Biol. 398: 232-247, 2010; referred to in the literature as ABY-025), PEP03997 and PEP10986 each convert the protein A variant of Z (a derivative of domain B of staphylococcal protein A; Nilsson B et al., 1987, supra) contains a different albumin binding moiety fused at the N-terminus, whereas PEP04419 is an MMA-DOTA (maleimide-monoamide -1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) at the C-terminus. Therefore, PEP04419 did not contain any albumin binding moieties and was tested for comparison. PEP03973 contains a wild-type albumin binding domain (ABDwt, SEQ ID NO: 515; ie, the GA3 domain of protein G from streptococcal strain G148; Kraulis et al., 1996 supra; Johansson et al., 2002 supra), as opposed to PEP10986. Contains a derivative of this GA3 domain (SEQ ID NO: 528).

  Cloning and culturing of polypeptide variants: DNA encoding polypeptides PEP03973 and PEP10986, respectively, were converted into an expression vector containing a T7 promoter, multiple cloning sites, and a kanamycin resistance gene using standard molecular biology techniques. Cloned. The expression vector encodes the amino acid GSSLQ at the N-terminus of the Z variant sequence, with the Z variant and albumin binding domain sequence separated by amino acids VD and VDSS in PEP03973 and PEP10986, respectively.

E. coli BL21 (DE3) cultures transformed with plasmids to express PEP03997 and PEP10986, respectively, were supplemented with 50 μg / ml kanamycin and 0.3 ml / l antifoam (Breox FMT30). The cells were inoculated into 800 ml of the prepared TSB + YE medium and grown at 37 ° C. until the OD ₆₀₀ reached approximately 2. Protein expression was then induced by adding 1 M IPTG to a final concentration of 0.2 mM. The culture was performed using Hedvig (Belach Bioteknik, Stockholm, Sweden) of a multifermentor system. Five hours after induction, cultures were harvested by centrifugation at 15900 × g for 20 minutes. The supernatant was discarded and the cell pellet was collected and stored at -20C. The level of protein expression was determined using SDS-PAGE and visual inspection of the stained gel.

Purification of polypeptide variants: Pelleted bacterial cells containing soluble PEP03973 and PEP10986 were each purified from TST buffer (25 mM Tris-HCl, 1 mM EDTA, 200 mM, supplemented with 20 U / ml Benzonase®). NaCl, 0.05% Tween 20, pH 8) and disrupted by sonication. The lysate was clarified by centrifugation and loaded onto 100 ml of affinity agarose packed on an XK50 column (GE Healthcare) previously equilibrated with TST-buffer. The column was washed with TST buffer 6 column volumes (CV), washed with subsequently to CV of 6 volumes of 5 mM _NH 4 of Ac (pH5.5), 3 bound proteins CV Elution was carried out with twice the volume of 0.1 M HAc. The flow rate was 15 ml / min and the signal at 280 nm was monitored. Fractions containing PEP03973 and PEP10986 were each identified by SDS-PAGE analysis. Relevant fractions were pooled, acetonitrile (ACN) was added to a final concentration of 10%, Source 15RPC pre-equilibrated with RPC eluate A (0.1% TFA, 10% ACN, 90% water) (GE Healthcare) was loaded on a FineLine column packed with 125 ml. After washing the column with 5 volumes of RPC eluate A of CV, RPC eluate B (0.1% TFA, 80%) with a linear concentration gradient of 0 to 60% within the range of 10 volumes of CV. % ACN, 20% water) to elute the bound proteins. The flow rate was 30 ml / min and the signal at 280 nm was monitored. Fractions containing pure PEP03973 and PEP10986 were each identified by SDS-PAGE analysis and pooled separately.

  Purified PEP03973 and PEP10986 were buffer-exchanged using 500 ml of Sephadex G25m (GE Healthcare) packed in an XK50 column (GE Healthcare) and 50 mM NaAc (pH 4.5) and 50 mM sodium phosphate (pH 7.5), respectively. 0). Finally, concentration was performed using a 15 ml Amicon Ultra centrifugal filter unit (Millipore) with 3 kDa MWCO.

PEP04419 was produced substantially as described by Feldwisch et al. (J. Mol. Biol. 2010, supra). And as described for PEP03973 and PEP10986 above, transferred and purified PEP04419, buffer _NH 4 exchanged by the 25mM of Ac, HCl of 6.25 mM, the NaCl (pH 4.9) of 112.5 mm, concentrated did.

  Analysis of purified polypeptide variants: Protein concentration was determined by measuring absorbance at 280 nm using a NanoDrop® ND-1000 spectrophotometer.

For SDS-PAGE analysis, concentrated PEP03973, PEP10986, and PEP04419 were diluted to 5 mg / ml, mixed with 4 × LDS sample buffer, incubated at 70 ° C. for 15 minutes, and 10-well NuPAGE®. Loaded on 4-12% bistris gel. The gel was run in a MES SDS running buffer on a Novex Mini-Cell using Novex® Sharp prestained protein standards as molecular weight markers and Coomassie Blue for staining.

  To verify the identity of purified PEP03973, PEP10986, and PEP04419, LC / MS-analysis was performed using an Agilent 1100 LC / MSD system equipped with API-ESI and a single quadrupole mass analyzer. 25 μg was loaded onto a Zorbax 300SB-C8 narrow aperture column (2.1 × 150 mm, 3.5 μm) at a flow rate of 0.5 ml / min. The protein was eluted at 0.5 ml / min over 15 minutes using a linear gradient of eluent B from 10 to 70%. Separation was performed at 30 ° C. The ion signal and absorbance at 280 and 220 nm were monitored. The molecular weight of the purified protein was determined by analysis of the ion signal.

Results As assessed by SDS-PAGE analysis, the produced polypeptides PEP03973, PEP10986, and PEP04419 were estimated to have a purity greater than 98% (FIG. 1). Table 1 summarizes the concentrations prepared, as determined by absorbance measurements at 280 nm, and the correct molecular weights verified by LC / MS-analysis.

[Example 2]
Pharmacokinetic Analysis of Polypeptide Variants Orally Administered to Mice Materials and Methods Oral Administration: 65 μmol each of polypeptide variants PEP03973, PEP10986, and PEP04419 produced as described in Example 1. Fasted male NMRI mice (Charles River, Germany) were orally administered at time point 0 by gavage at doses of / kg body weight, 92 μmol / kg body weight, and 298 μmol / kg body weight. Approximately 30 minutes after dosing, food was again provided. Serum samples were obtained by cardiac puncture of anesthetized mice at 1, 3, 8, 24, 48, and 72 hours after administration of PEP03997 and PEP10986, and at 15, 30, 60, 180, and 480 minutes after administration of PEP04419. did. At each time point, three mice were discarded. Serum concentrations of each protein were determined by a sandwich ELISA assay specific for each polypeptide variant.

PEP03973 ELISA Assay: ELISA plates (Greiner 96-well half-area plate, cat # 675074) were prepared in-house goats in 1 μg / ml (50 μl / well) carbonate buffer (Sigma, cat # C3041). Coated with anti-protein Z immunoglobulin (Ig) at 4 ° C. overnight. The next day, plates were washed with PBS (2.68 mM KCl, 0.47 mM KH ₂ PO ₄ , 137 mM Na
Cl, 8.1 mM Na ₂ HPO ₄ , pH 7.4) + 0.5% casein (Sigma, Cat # C8654), blocked for 1.5 hours with PBSC. Serum samples and purified PEP03973, used as a standard, were serially diluted 3-fold with PBSC, titrated in duplicate at a total volume of 50 μl / well, and incubated for an additional 1.5 hours. In-house produced rabbit anti-protein Z / ABDwt Ig diluted to 1 μg / ml in PBSC was added at 50 μl / well, followed by DELFIA Eu-N1-anti-rabbit IgG diluted to 20 ng / ml in PBSC (Perkin Elmer catalog). No. AD0105) was added at 50 μl / well to detect bound PEP03973. Antibody incubation periods were 1.5 hours and 1 hour, respectively, and washes were performed after each step. Subsequently, after gently stirring for 5 minutes, 50 μl / well of an enrichment solution (Perkin Elmer catalog 4001-0010) was added and the time-resolved fluorescence (TRF) was read on an ELISA reader (Victor ³ , Perkin Elmer). Non-linear regression (GraphPad Prism5) was used to calculate the concentration of PEP03973 in serum samples from a standard curve of PEP03973.

  PEP10986 ELISA assay: The concentration of PEP10986 in serum samples was determined by an ELISA assay similar to that described above for PEP039973, but coating was performed with 8 μg / ml of in-house produced goat anti-protein ZIg. The first detection step was performed using an in-house produced rabbit Ig specific for the albumin binding domain identified above as SEQ ID NO: 2 at a concentration of 2 μg / ml.

PEP04419 ELISA Assay: ELISA plates (Costar 96-well half area plate, catalog no. 3690) are coated overnight at 4 ° C. with 2 μg / ml (50 μl / well) goat Ig against protein Z in carbonate buffer at 4 ° C. did. The following day, plates were blocked with PBSC for 1.5 hours. Serum samples and purified PEP04419, used as a standard, were serially diluted 2-fold in PBSC, titrated in duplicate at a total volume of 50 μl / well, and incubated for an additional 1.5 hours. Plates were washed four times with PBS + 0.05% Tween (PBST). Binding with 50 μl / well rabbit anti-protein Z Ig (0.125 μg / ml in PBSC) followed by 50 μl / well anti-rabbit IgG-HRP (Dako, Cat # P0448) diluted 1: 10000 in PBSC. PEP04419 was detected. Each antibody was incubated for 1 hour and washing was performed after each step. After the last wash, the reaction was developed with 50 μl / well of TMB Immunopure (Thermo Scientific, Cat. No.34021) and stopped by the addition of 50 μl / well of H ₂ SO ₄ . The absorbance at 450 nm was read with an ELISA reader (Victor ³ , Perkin Elmer). The concentration of PEP04419 in the serum samples was calculated using non-linear regression (GraphPad Prism 5) from the standard curve of PEP04419.

Results As can be seen from FIG. 2, all three polypeptides administered were absorbed after administration via the oral route. Surprisingly, the polypeptides containing the albumin binding moiety, ie, PEP03973 and PEP10986, were at least as well absorbed, albeit nearly twice as large as the PEP04419 of the polypeptide without the albumin binding moiety. Moreover, the polypeptides containing the albumin binding moiety exhibited a significantly longer residence time as compared to the polypeptides without the albumin binding moiety, whereas the polypeptides without the albumin binding moiety showed a sample taken at 8 hours post-dose. Was not detectable in the cells (see FIGS. 3A and 3B). The area under the curve (AUC) was 106, 96, and 7 nMh for PEP03973, PEP10986, and PEP041944, respectively. The predicted pharmacokinetic profile indicated that PEP03973 and PEP10986 retained their albumin binding capacity upon oral administration.

[Example 3]
Pharmacokinetic Analysis of Oral and Intraduodenal Administration of PEP10896 in Rats Materials and Methods Oral Administration: Fasted polypeptide PEP10986, prepared as described in Example 1, at a dose of 67 μmol / kg body weight. Male Sprague Dawley rats (Charles River, Germany) were orally administered by gavage at time point 0. Approximately 30 minutes after dosing, food was again provided. Blood samples were taken under isoflurane anesthesia at 1, 3, 8, 24, 72, 120, and 168 hours post-dose and serum prepared by standard procedures. The serum concentration of PEP10896 was measured by the sandwich ELISA assay described in Example 2.

  Intraduodenal administration: An indwelling catheter (IITC Life Science, catalog number S2C6) was surgically inserted 10-15 mm from its start into the duodenum of anesthetized male Sprague Dawley rats (Charles River) and penetrated subcutaneously into the back. After a 3-5 day recovery period, the polypeptide PEP10986, prepared as described in Example 1, was administered directly to the duodenum of fasted rats at a time point of 0 at a dose of 67 μmol / kg body weight. Approximately 30 minutes after dosing, food was again provided. Blood samples were collected under isoflurane anesthesia at 1, 3, 8, 24, 72, 120, and 168 hours after administration of PEP10986, and serum was prepared by standard procedures. The serum concentration of PEP10896 was measured by the sandwich ELISA assay described in Example 2.

Results The pharmacokinetic profile of PEP10896 obtained after oral and intraduodenal administration is shown in FIG. The results showed that PEP10896 was absorbed via the oral route in rats both after oral gavage and after intraduodenal administration. Intraduodenal administration contributed to increased intestinal absorption as indicated by a higher Cmax (3 h) concentration compared to oral gavage (11.3 nM vs 0.71 nM). An improved absorption resulted in an AUC that was at least 15 times higher. A long-term pharmacokinetic profile with a half-life similar to albumin indicated that PEP10986 retained albumin binding capacity after oral ingestion, either by gavage or intraduodenal administration.

[Example 4]
Pharmacokinetic analysis of repeated intraduodenal administration of PEP10896 in rats Materials and Methods Repeated intraduodenal administration:
The polypeptide variant PEP10986 prepared as described in Example 1 was administered directly to the duodenum of rats fasted as described in Example 3. PEP10986 was administered three times at time points of 0, 2, and 24 hours at a dose of 67 μmol / kg body weight. Food was provided again approximately 30 minutes after each dose. Blood samples were collected under isoflurane anesthesia at 1, 3, 5, 24, 27, 96, 144, and 192 hours after administration of PEP10986 and serum prepared according to standard procedures. The serum concentration of PEP10896 was measured by the sandwich ELISA assay described in Example 2.

Results The pharmacokinetic profile of PEP10896 obtained after repeated intraduodenal administration is shown in FIG. To highlight the difference between single dose and repeated dose, the results of intraduodenal administration described in Example 3 were included in FIG. Repeated administration increased the concentration of polypeptide PEP10896 in rat serum and prolonged the pharmacokinetic profile, indicating that PEP10986 retained albumin binding ability after oral ingestion. Two higher increases in concentration after the third dose indicated that higher serum levels could be achieved with repeated doses.

List of embodiments by item A compound for use in treatment by oral administration, said compound comprising:
-A moiety (I) that confers a desired therapeutic activity; and-binds to albumin and is M1 / Emm1, M3 / Emm3, M12 / Emm12, EmmL55 / Emm55, Emm49 / EmmL49, H, G, MAG, ZAG, PPL, And an amino acid sequence corresponding to a portion (II) comprising a naturally occurring albumin binding protein selected from PAB and any one of the albumin binding domains, fragments or derivatives thereof,
Provided that the compound (I) is not selected from an exendin sequence, an exendin analog sequence, a sequence of a fragment having exendin activity, or a fragment having exendin analog activity.

  2. Said moiety (I) is a component selected from the group consisting of human endogenous enzymes, hormones, growth factors, chemokines, cytokines, blood clotting and complement factors, innate immune defense and regulatory peptides, eg insulin, insulin-like Body, IL-2, IL-5, GLP-1, BNP, IL1-RA, KGF, Stemgen®, GH, G-CSF, CTLA-4, myostatin, factor VII, factor VIII, and factor A compound for use according to item 1, comprising a component selected from the group consisting of factor IX, and derivatives thereof.

  3. The item 1 wherein said moiety (I) comprises a biologically active non-human protein selected from the group consisting of modulin, bacterial toxins, hormones, innate immune defense and regulatory peptides, enzymes, and activating proteins. Compounds for the described use.

  4. The compound for use according to item 1, wherein said moiety (I) comprises a binding polypeptide capable of selective interaction with a target molecule.

  5. Binding polypeptides include antibodies that substantially retain antibody binding activity and fragments and domains thereof; microbodies, maxibodies, avimers, and other small disulfide-binding proteins; and staphylococcal protein A and their domains, other 3 helix domain, lipocalin, ankyrin repeat domain, cellulose binding domain, gamma crystallin, green fluorescent protein, human cytotoxic T cell binding antigen 4, protease inhibitor such as Kunitz domain, PDZ domain, SH3 domain, peptide aptamer, Derived from a scaffold selected from the group consisting of staphylococcal nuclease, tendamistat, fibronectin type III domain, transferrin, zinc finger, and conotoxin It is selected from the group consisting of coupling proteins, the compounds for use according to item 4.

  6. The binding polypeptide comprises a variant of protein Z derived from domain B of staphylococcal protein A, wherein the variant comprises a scaffold amino acid selected from SEQ ID NO: 719, SEQ ID NO: 720, and SEQ ID NO: 721 A compound for use according to item 5, comprising the sequence, wherein X represents any amino acid residue.

7. The target molecule is CD14, CD19, CD20, CD22, CD30, CD33.
CD37, CD40, CD52, CD56, CD70, CD138, cMet, HER1, HER2, HER3, HER4, CAIX, CEA, IL-2 receptor, IGF1R, VEGFR2, MUC1, PDGFR-beta, PSMA, tag-72, FOLR1 Tumor-related or other cell surface-related antigens, such as, mesothelin, CA6, GPNMB, integrin, and ephA2; TNF-α, IL-1α, IL-1β, IL-1Ra, IL-5, IL-6, IL- 13, cytokines such as IL-17A, IL-18, IL-23, IL-36, G-CSF, GM-CSF, and their receptors; IL-8, CCL-2, and CCL11, and their Chemokines such as receptors; complement factors such as C3 and factor D; HGF Growth factors such as and myostatin; hormones such as GH, insulin and somatostatin; peptides such as the Aβ peptide of Alzheimer's disease; other disease-related amyloid peptides; hypersensitivity mediators such as histamine and IgE; A compound for use according to any one of items 4 to 6, selected from the group consisting of a blood clotting factor such as a factor; and a toxin such as a bacterial toxin and a snake venom.

  8. Said moiety (I) comprises: a) cytotoxic substances such as calicheamicin, auristatin, doxorubicin, maytansinoids, taxanes, ecteinascidin, geldanamycin, methotrexate, camptothecin, cyclophosphamide, cyclosporin and derivatives thereof And b) anti-inflammatory drugs, such as non-steroidal anti-inflammatory drugs, cytokine inhibitory anti-inflammatory drugs, corticosteroids, methotrexate, prednisone, cyclosporine, moroniside cinnamate, leflunomide, and derivatives thereof. And a non-proteinaceous component selected from the group consisting of combinations thereof for use according to item 1.

  9. 9. The compound for use according to any one of items 1 to 8, wherein said moiety (II) comprises a naturally occurring GA domain or a derivative thereof.

  10. The use according to item 9, wherein the portion (II) comprises a GA domain selected from the group consisting of domain GA1, domain GA2, and domain GA3 of protein G derived from streptococcal strain G148, and derivatives thereof. Compound.

  11. 11. A compound for use according to item 10, wherein said portion (II) comprises domain GA3 of protein G from streptococcal strain G148 or a derivative thereof.

  12. 12. The compound for use according to item 11, wherein said portion (II) comprises domain GA3 of protein G from streptococcal strain G148 having the amino acid sequence set forth in SEQ ID NO: 515.

13. Said part (II) comprises an albumin binding motif, said motif comprising the amino acid sequence:
GVSDX ₅ YKX ₈ X ₉ I X ₁₁ X ₁₂ AX ₁₄ TVEGVX ₂₀ ALX ₂₃ X ₂₄ X ₂₅ I
Consisting of
Wherein, independently of each other,
X ₅ is selected from Y and F;
X ₈ is selected from N, R, and S;
X ₉ is, V, selected I, L, M, F, and from Y;
X ₁₁ is selected from N, S, E, and D;
X ₁₂ is selected from R, K, and N;
X ₁₄ is selected from K and R;
X ₂₀ is selected from D, N, Q, E, H, S, R, and K;
X ₂₃ is selected from K, I, and T;
X ₂₄ is, A, S, T, G , H, L, and it is selected from D; and X ₂₅ is, H, E, and are selected from D, according to any one of claims 1 to 11 Compound for use.

14． 14. The compound for use according to item 13, wherein X ₅ is Y.

15. X ₈ is selected from N and R, especially R, the compounds for use according to any one of claims 13 to 14.

16. X ₉ is L, the compound for use according to any one of claims 13 to 15.

17． 17. A compound for use according to any one of items 13 to 16, wherein X ₁₁ is selected from N and S, especially N.

18. X ₁₂ is selected from R and _{K, X 12} is either R, or K, the compounds for use according to any one of claims 13 to 17.

19. 19. A compound for use according to any of items 13 to 18, wherein X ₁₄ is K.

20. _20. A compound for use according to any one of items 13 to 19, wherein X20 is selected from D, N, Q, E, H, R and S, and in particular is E.

21. X ₂₃ is selected from K and I, particularly K, the compounds for use according to any one of claims 13 to 20.

22. X ₂₄ is, A, S, T, G , is selected from H, and the L, especially L, the compounds for use according to any one of claims 13 to 21.

23. X ₂₄ is L, the compound for use according to item 22.

24. A compound for use according to item 23, wherein X ₂₃ X ₂₄ is KL.

25. X ₂₃ X ₂₄ is TL, the compounds for use according to item 23.

26. X ₂₄ is, A, S, T, is selected G, and the H, the compounds for use according to item 22.

27. X ₂₃ is I, the compounds for use according to item 26.

28. 28. The compound for use according to any one of items 13 to 27, wherein X ₂₅ is H.

29. The albumin binding motif consists of an amino acid sequence selected from SEQ ID NOs: 1 to 257,
SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 1
55, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244 and SEQ ID NO: 245
29. A compound for use according to any one of items 13 to 28, consisting of an amino acid sequence selected from SEQ ID NO: 3, SEQ ID NO: 53, and an amino acid sequence selected from SEQ ID NO: 239, among others. .

30. Part (II) has the amino acid sequence:
LAEAKX _{a Xb} AX _c X _d ELX _e KY- [ABM] -LAALP
Including
Where:
[ABM] is the albumin binding motif according to any one of items 13 to 29,
Independent of each other,
_Xa is selected from V and E;
X _b is selected from L, E, and D;
_Xc is selected from N, L, and I;
30. The compound for use according to any one of items 13-29, wherein X _d is selected from R and K; and X _e is selected from D and K.

31. _Xa is V. The compound for use according to item 30, wherein Xa is V.

32. 32. The compound for use according to any one of items 30-31, wherein _Xb is L.

33. 33. The compound for use according to any one of items 30-32, wherein _Xc is N.

34. 34. The compound for use according to any one of items 30-33, wherein X _d is R.

35. X _e is D, the compounds for use according to any one of claims 30 to 34.

36. Said part (II) comprises a derivative of domain GA3 of protein G from streptococcal strain G148, said derivative having one definition selected from:
i) the amino acid sequence is selected from SEQ ID NOs: 258-514;
SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 266, SEQ ID NO: 272, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 303, SEQ ID NO: 306, SEQ ID NO: 310, SEQ ID NO: 311, SEQ ID NO: 312, SEQ ID NO: 412, SEQ ID NO: 496, SEQ ID NO: 497, SEQ ID NO: 498, SEQ ID NO: 499, SEQ ID NO: 500, SEQ ID NO: 501 and SEQ ID NO: 502,
Selected from among SEQ ID NO: 260, SEQ ID NO: 310, and SEQ ID NO: 496;
ii) The amino acid sequence according to item 11, wherein the amino acid sequence comprises an amino acid sequence satisfying one definition selected from being an amino acid sequence having 85% or more identity to the sequence described in (i). For the use of the compound.

37. Said part (II) comprises a derivative of domain GA3 of protein G from streptococcal strain G148, said derivative comprising:
i) LAX ₃ AKX ₆ X ₇ ANX ₁₀ ELDX ₁₄ YGVSDF YKRRLIX ₂₆ KAKT VEGVEALKX ₃₉ X ₄₀ ILX ₄₃ X ₄₄ LP
Wherein, independently of each other,
X ₃ is selected from E, S, Q, and C;
X ₆ is selected from E, S, and C;
X ₇ is selected from A and S;
X ₁₀ is selected from A, S, and R;
X ₁₄ is selected from A, S, C, and K;
X ₂₆ is selected from D and E;
X ₃₉ is selected from D and E;
X ₄₀ is selected from A and E;
X ₄₃ is selected from A and K;
_X44 is selected from A, S, and E;
L at position 45 is present or absent; and P at position 46 is present or absent;
And ii) an amino acid sequence having at least 95% identity to the sequence defined in i),
A compound for use according to item 11, comprising an amino acid sequence selected from:

38. X ₆ is E, the compounds for use according to item 37

39. X ₃ is either S, or E, the compounds for use according to any one of claims 37-38.

40. X ₇ is A, the compounds for use according to any one of claims 37 to 39.

41. X ₁₄ is either S, or is C, compound for use according to any one of claims 37 to 40.

42. 42. The compound for use according to any one of items 37-41, wherein X ₁₀ is A or S.

43. 43. The compound for use according to any one of items 37-42, wherein X ₂₆ is D or E.

44. 45. The compound for use according to any one of items 37-43, wherein X ₃₉ is D or E.

45. 45. The compound for use according to any one of items 37-44, wherein X ₄₀ is A.

46. _45. The compound for use according to any one of items 37-45, wherein X ₄₃ is A.

47. X ₄₄ is either A, or S, the compounds for use according to any one of claims 37-46.

  48. The compound for use according to any one of items 37-47, wherein L at position 45 is present.

  49. The compound for use according to any one of items 37-48, wherein P at position 49.46 is present.

50. The derivative is an amino acid sequence selected from the group consisting of SEQ ID NOs: 516-659 and SEQ ID NOs: 679-718, in particular,
SEQ ID NOs: 519-520, SEQ IDs: 522-523, SEQ IDs: 525-526, SEQ IDs: 528-529, SEQ IDs: 531-532, SEQ IDs: 534-535, SEQ IDs: 537-538, SEQ IDs: 540-541, SEQ IDs: 543-544, SEQ ID NOs: 546-547, SEQ ID NOs: 549-550, SEQ ID NOs: 552-553, SEQ ID NOs: 556-557, SEQ ID NOs: 564-565, SEQ ID NOs: 679-685, and SEQ ID NOs: 707-718.
Or an amino acid sequence selected from the group consisting of SEQ ID NOs: 516-659, in particular,
SEQ ID NOs: 519-520, SEQ IDs: 522-523, SEQ IDs: 525-526, SEQ IDs: 528-529, SEQ IDs: 531-532, SEQ IDs: 534-535, SEQ IDs: 537-538, SEQ IDs: 540-541, SEQ IDs: 543-544, SEQ ID NOs: 546-547, SEQ ID NOs: 549-550, SEQ ID NOs: 552-553, SEQ ID NOs: 556-557 and SEQ ID NOs: 564-565
38. A compound for use according to item 37, comprising an amino acid sequence selected from the group consisting of:

  51. 51. A compound for use according to any one of items 37 to 50, further comprising one or more additional amino acid residues located at the N-terminus and / or C-terminus of the sequence defined in i). .

52. 52. The compound for use according to item 51, wherein the derivative comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 660-665 and SEQ ID NOs: 677-678.

53. Portion (II) is, _{K D} of the interaction, maximum 1 × ^{10 -8} M, such as at the most ^{1 × 10 -9 M, 1 ×} 10 -11 such as at the most 1 × ^{10 -10} M, for example, the maximum 53. A compound for use according to any one of the preceding items, wherein the compound binds to albumin so as to be at most M, for example 1 × 10 ⁻¹² M.

54. a) a compound,
-A moiety (I) that confers a desired therapeutic activity; and-binds to albumin and is M1 / Emm1, M3 / Emm3, M12 / Emm12, EmmL55 / Emm55, Emm49 / EmmL49, H, G, MAG, ZAG, PPL, And an amino acid sequence corresponding to a portion (II) comprising a naturally occurring albumin binding protein selected from PAB and any one of the albumin binding domains, fragments or derivatives thereof,
With the proviso that the moiety (I) is not selected from an exendin sequence, a sequence of an exendin analog, a sequence of a fragment having exendin activity or a fragment having exendin analog activity;
b) a pharmaceutical composition for oral administration comprising at least one pharmaceutically acceptable excipient.

  55. 55. The pharmaceutical composition according to item 54, wherein the compound is the compound according to any one of items 2 to 53.

56. 56. The pharmaceutical composition according to any of the items 54-55, further comprising at least one component for increasing the oral bioavailability of the therapeutic activity.

  57. 57. The pharmaceutical composition according to item 56, wherein said component is selected from the group consisting of a protease inhibitor, an absorption enhancer, a mucoadhesive polymer, a formulation vehicle, and any combination thereof.

  58. Item present in a form selected from solid forms such as pills, tablets, capsules, powders or granules; semi-solid forms such as pastes; and liquid forms such as elixirs, solutions or suspensions. 58. The pharmaceutical composition according to any one of 54 to 57.

  59. 59. The pharmaceutical composition according to any one of items 54 to 58, which is in the form of a formulation designed for immediate release, delayed release or controlled release.

  60. 60. The pharmaceutical composition according to any one of the items 54-59, which is formulated as an enteric capsule.

61. A method of treating a mammalian subject in need of such treatment, comprising administering the compound orally, wherein the compound comprises:
-A moiety (I) that confers a desired therapeutic activity; and-binds to albumin and is M1 / Emm1, M3 / Emm3, M12 / Emm12, EmmL55 / Emm55, Emm49 / EmmL49, H, G, MAG, ZAG, PPL, And an amino acid sequence corresponding to a portion (II) comprising a naturally occurring albumin binding protein selected from PAB and any one of the albumin binding domains, fragments or derivatives thereof,
Provided that the portion (I) is not selected from an exendin sequence, an exendin analog sequence, a sequence of a fragment having exendin activity, or a fragment having exendin analog activity.

  62. 61. A method of treating a mammalian subject in need of such treatment, comprising orally administering the pharmaceutical composition of any one of items 54-60.

  63. 63. The method according to any one of items 61 to 62, wherein the compound is the compound according to any one of items 2 to 53.

Claims (17)

A compound for use in treatment by oral administration, said compound comprising:
-A moiety (I) that confers a desired therapeutic activity; and-binds to albumin and is M1 / Emm1, M3 / Emm3, M12 / Emm12, EmmL55 / Emm55, Emm49 / EmmL49, H, G, MAG, ZAG, PPL, And an amino acid sequence corresponding to a portion (II) comprising a naturally occurring albumin binding protein selected from PAB and any one of the albumin binding domains, fragments or derivatives thereof,
Provided that the compound (I) is not selected from an exendin sequence, an exendin analog sequence, a sequence of a fragment having exendin activity, or a fragment having exendin analog activity.   The compound for use according to claim 1, wherein said moiety (I) comprises a binding polypeptide capable of selective interaction with a target molecule.   The binding polypeptide is a variant of protein Z derived from domain B of staphylococcal protein A, wherein the variant is a scaffold amino acid sequence selected from SEQ ID NO: 719, SEQ ID NO: 720, and SEQ ID NO: 721 3. The compound for use according to claim 2, wherein X represents any amino acid residue.   The compound for use according to claim 3, wherein said portion (II) comprises the domain GA3 of protein G from streptococcal strain G148 having the amino acid sequence as set forth in SEQ ID NO: 515. Said part (II) comprises an albumin binding motif, said motif comprising the amino acid sequence:
GVSDX ₅ YKX ₈ X ₉ I X ₁₁ X ₁₂ AX ₁₄ TVEGVX ₂₀ ALX ₂₃ X ₂₄ X ₂₅ I
Consisting of
Wherein, independently of each other,
X ₅ is selected from Y and F;
X ₈ is selected from N, R, and S;
X ₉ is, V, selected I, L, M, F, and from Y;
X ₁₁ is selected from N, S, E, and D;
X ₁₂ is selected from R, K, and N;
X ₁₄ is selected from K and R;
X ₂₀ is selected from D, N, Q, E, H, S, R, and K;
X ₂₃ is selected from K, I, and T;
X ₂₄ is selected from A, S, T, G, H, L, and D; and X ₂₅ is selected from H, E, and D. For the use of the compound. The albumin binding motif consists of an amino acid sequence selected from SEQ ID NOs: 1 to 257, and is preferably SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 155, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244 and SEQ ID NO: 245
The compound for use according to claim 5, consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 53, and SEQ ID NO: 239. Part (II) has the amino acid sequence:
LAEAKX _{a Xb} AX _c X _d ELX _e KY- [ABM] -LAALP
Including
Where:
[ABM] is the albumin binding motif according to claim 5 or 6,
Independent of each other,
_Xa is selected from V and E;
X _b is selected from L, E, and D;
_Xc is selected from N, L, and I;
7. The compound for use according to claim 5 or 6, wherein X _d is selected from R and K; and X _e is selected from D and K. Said part (II) comprises a derivative of domain GA3 of protein G from streptococcal strain G148, said derivative having one definition selected from:
i) the amino acid sequence is selected from SEQ ID NOs: 258-514;
SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 266, SEQ ID NO: 272, SEQ ID NO: 282, SEQ ID NO: 284, SEQ ID NO: 303, SEQ ID NO: 306, SEQ ID NO: 310, SEQ ID NO: 311, SEQ ID NO: 312, SEQ ID NO: 412, SEQ ID NO: 496, SEQ ID NO: 497, SEQ ID NO: 498, SEQ ID NO: 499, SEQ ID NO: 500, SEQ ID NO: 501 and SEQ ID NO: 502,
Selected from among SEQ ID NO: 260, SEQ ID NO: 310, and SEQ ID NO: 496;
ii) The amino acid sequence according to claim 3, wherein the amino acid sequence satisfies one definition selected from being an amino acid sequence having 85% or more identity to the sequence described in (i). Compounds for the described use. Said part (II) comprises a derivative of domain GA3 of protein G from streptococcal strain G148, said derivative comprising:
i) LAX ₃ AKX ₆ X ₇ ANX ₁₀ ELDX ₁₄ YGVSDF YKRRLIX ₂₆ KAKT VEGVEALKX ₃₉ X ₄₀ ILX ₄₃ X ₄₄ LP
Wherein, independently of each other,
X ₃ is selected from E, S, Q, and C;
X ₆ is selected from E, S, and C;
X ₇ is selected from A and S;
X ₁₀ is selected from A, S, and R;
X ₁₄ is selected from A, S, C, and K;
X ₂₆ is selected from D and E;
X ₃₉ is selected from D and E;
X ₄₀ is selected from A and E;
X ₄₃ is selected from A and K;
_X44 is selected from A, S, and E;
L at position 45 is present or absent; and P at position 46 is present or absent;
And ii) an amino acid sequence having at least 95% identity to the sequence defined in i),
A compound for use according to claim 3, comprising an amino acid sequence selected from: The derivative is an amino acid sequence selected from the group consisting of SEQ ID NOs: 516-659 and SEQ ID NOs: 679-718, especially
SEQ ID NOs: 519-520, SEQ IDs: 522-523, SEQ IDs: 525-526, SEQ IDs: 528-529, SEQ IDs: 531-532, SEQ IDs: 534-535, SEQ IDs: 537-538, SEQ IDs: 540-541, SEQ IDs: 543-544, SEQ ID NOs: 546-547, SEQ ID NOs: 549-550, SEQ ID NOs: 552-553, SEQ ID NOs: 556-557, SEQ ID NOs: 564-565, SEQ ID NOs: 679-685, and SEQ ID NOs: 707-718.
Or an amino acid sequence selected from the group consisting of SEQ ID NOs: 516-659, especially
SEQ ID NOs: 519-520, SEQ IDs: 522-523, SEQ IDs: 525-526, SEQ IDs: 528-529, SEQ IDs: 531-532, SEQ IDs: 534-535, SEQ IDs: 537-538, SEQ IDs: 540-541, SEQ IDs: 543-544, SEQ ID NOs: 546-547, SEQ ID NOs: 549-550, SEQ ID NOs: 552-553, SEQ ID NOs: 556-557 and SEQ ID NOs: 564-565.
10. A compound for use according to claim 9, comprising an amino acid sequence selected from the group consisting of: a) a compound,
-A moiety (I) that confers a desired therapeutic activity; and-binds to albumin and is M1 / Emm1, M3 / Emm3, M12 / Emm12, EmmL55 / Emm55, Emm49 / EmmL49, H, G, MAG, ZAG, PPL, And an amino acid sequence corresponding to a portion (II) comprising a naturally occurring albumin binding protein selected from PAB and any one of the albumin binding domains, fragments or derivatives thereof,
With the proviso that the moiety (I) is not selected from an exendin sequence, a sequence of an exendin analog, a sequence of a fragment having exendin activity or a fragment having exendin analog activity;
b) a pharmaceutical composition for oral administration comprising at least one pharmaceutically acceptable excipient.   The pharmaceutical composition according to claim 11, wherein the compound is a compound according to any one of claims 2 to 10.   13. The pharmaceutical composition according to claim 11 or 12, further comprising at least one component for increasing the oral bioavailability of said therapeutic activity.   The pharmaceutical composition according to any one of claims 11 to 13, which is formulated as an enteric capsule. A method of treating a mammalian subject in need of such treatment, comprising administering the compound orally, wherein the compound comprises:
-A moiety (I) that confers a desired therapeutic activity; and-binds to albumin and is M1 / Emm1, M3 / Emm3, M12 / Emm12, EmmL55 / Emm55, Emm49 / EmmL49, H, G, MAG, ZAG, PPL, And an amino acid sequence corresponding to a portion (II) comprising a naturally occurring albumin binding protein selected from PAB and any one of the albumin binding domains, fragments or derivatives thereof,
Provided that the portion (I) is not selected from an exendin sequence, an exendin analog sequence, a sequence of a fragment having exendin activity, or a fragment having exendin analog activity.   A method for treating a mammalian subject in need of such treatment, comprising orally administering the pharmaceutical composition of any one of claims 11 to 14.   The method according to claim 15 or 16, wherein the compound is a compound according to any one of claims 2 to 10.