Long-acting growth hormone receptor antagonists and uses thereof

Abstract

The present invention relates to long-acting growth hormone receptor antagonists comprising a growth hormone variant and an albumin-binding moiety. Methods for preparing and using the compounds in pharmaceuticals, for example, for the treatment of acromegaly, are further described.

Description

[Technical Field]

[0001] The present invention relates to compounds that are long-acting growth hormone receptor antagonists that contain a growth hormone variant and an albumin binding moiety, methods for preparing said compounds, and the use of said compounds in medicine. [Background technology]

[0002] Growth hormone (GH) is a polypeptide secreted by the anterior pituitary gland in mammals. Depending on the species, growth hormone is a protein composed of approximately 190 amino acid residues, corresponding to a molecular weight of approximately 22 kDa. Human growth hormone (hGH) is composed of 191 amino acids according to SEQ ID NO: 1. Growth hormone binds to and signals through cell surface receptors, known as growth hormone receptors (GHRs). Growth hormone plays an important role in promoting growth and maintaining normal composition, anabolism, and lipid metabolism. When the body excretes excess GH, plasma levels of insulin-like growth factor 1 (IGF-1) increase, causing body tissues and bones to grow more rapidly, leading to diseases such as acromegaly.

[0003] Acromegaly is usually diagnosed in adults between the ages of 30 and 50, and the underlying cause is most often a slow-growing pituitary adenoma. The first-line treatment is surgery. However, if surgery is not possible or adequate control of hGH and IGF-1 levels cannot be achieved, medical treatment with somatostatin analogs or recombinant hGH receptor antagonists is the next level of treatment.

[0004] Pegvisomant, a PEGylated version of the potent GH receptor antagonist B2036 (SEQ ID NO: 5), first described in WO 1997 / 011178, has been commercially available for over 20 years as Somavert® for the treatment of acromegaly. Pegvisomant is a competitive hGH antagonist consisting of the peptide backbone B2036, with variations in four to six PEG chains and lysine-mediated PEGylation positions at positions F1, K145, K140, K38, K158, K120, and K70 (Pradhananga et al., J Mol Endo (2002), 29, 11-14; US Pat. No. 5,849,535). However, it is associated with several drawbacks, including injection site reactions, immunogenicity, painful injections, and complex isolation due to heterogeneous product. Additionally, pegvisomant is obtained as a solid that is reconstituted in a buffer solution prior to injection and is known to partially aggregate when reconstituted, and the high viscosity of the reconstituted product makes it difficult to administer subcutaneously using large needles (27 1 Pegvisomant should be administered once daily to achieve sustainable IGF-1 reduction. However, when used in combination with a somatostatin analog, the dosing frequency can be reduced to 2-3 times per week.

[0005] In order to overcome these shortcomings, some attempts have been made.For example, alternative PEGylated growth hormone receptor antagonists are described in, for example, CN103087182B and US10,874,717, and alternative strategies for improving the half-life of growth hormone receptor antagonists and / or providing backbone substitution are described in, for example, US2020 / 03999340, using alpha-1 antitrypsin fusion, WO2019 / 211842, using carboxy-terminal peptide conjugates and J.Biol.Chem.2021, 296, 100588, and suggest specific backbone substitution.

[0006] Thus, there remains a need to provide growth hormone receptor antagonists with improved properties, such as, for example, improved binding affinity and / or increased half-life. The present invention provides novel long-acting growth hormone receptor antagonists with improved pharmacological and pharmacokinetic properties, as well as improved patient convenience. Summary of the Invention

[0007] The present invention relates to a long-acting growth hormone receptor antagonist (LA-GHRA) comprising a growth hormone variant and an albumin-binding moiety. In some aspects, the growth hormone variant is a polypeptide derived from human growth hormone (hGH) and comprises the following amino acid substitutions compared to hGH (SEQ ID NO: 1): H18D, H21N, L101C, G120R / G120K, R167N, D171S, E174S, I179T. In one embodiment, the albumin-binding moiety is linked to the growth hormone variant via the sulfur on the side chain of cysteine ​​at position 101 of the polypeptide.

[0008] In one aspect, a long-acting growth hormone receptor antagonist is provided, the antagonist comprising: a. a growth hormone variant comprising at least the following amino acid substitutions compared to human growth hormone (hGH) (SEQ ID NO: 1): L101C, G120R / G120K, said growth hormone variant having at least 80% sequence identity with the polypeptide of SEQ ID NO: 1; b. an albumin binding moiety configured for binding to albumin, e.g., binding to albumin with a dissociation constant (Kd) of less than 1 μM, having a molecular weight of 3 kDa or less, and covalently attached to the growth hormone variant via the sulfur residue of the cysteine ​​side chain at position 101 of the growth hormone variant; or a pharmaceutically acceptable salt thereof.

[0009] In one aspect, a long-acting growth hormone receptor antagonist is provided, the antagonist comprising: a. Growth hormone variants containing the following amino acid substitutions compared to human growth hormone (hGH) (SEQ ID NO: 1): H18D, H21N, L101C, G120R / G120K, R167N, D171S, E174S, I179T, and b.Chemical formula 2: [ka] wherein * indicates the point of attachment of the albumin binding moiety to the growth hormone variant via the sulfur residue of the cysteine ​​side chain at position 101 of the growth hormone variant, or a pharmaceutically acceptable salt thereof.

[0010] In one embodiment, the long-acting growth hormone receptor antagonist is compound 1a: [ka] or a pharmaceutically acceptable salt thereof, optionally having one or two disulfide bridges between Cys53-Cys165 and / or Cys182-Cys189.

[0011] In one embodiment, the long-acting growth hormone receptor antagonist is compound 1a: [ka] or a pharmaceutically acceptable salt thereof.

[0012] The present invention further relates to pharmaceutical compositions comprising said LA-GHRA and a pharmaceutically acceptable excipient, medical uses of said antagonists, as well as nucleic acids encoding polypeptide backbones, vectors comprising said nucleic acids, and host cells comprising said vectors.

[0013] In one aspect, a long-acting growth hormone receptor antagonist is provided as defined herein for use in the treatment or prevention of diseases caused by excessive human growth hormone and / or IGF-1 levels, such as, for example, acromegaly or gigantism.

[0014] In one aspect, a nucleic acid encoding a human growth hormone variant is provided, which comprises the following amino acid modifications compared to hGH: i)H18D, H21N, L101C, G120R, R167N, K168A, D171S, K172R, E174S, I179T (GHv-1), ii) H18D, H21N, L101C, G120R, N149D, N152D, R167N, K168A, D171S, K172R, E174S, I179T (GHv-2), or iii) H18D, H21N, L101C, G120R, N149D, N152D, R167N, D171S, E174S, I179T (GHv-3).

[0015] In one aspect, nucleic acids are provided that encode human growth hormone variants that contain the following amino acid modifications compared to hGH: H18D, H21N, L101C, G120R, R167N, K168A, D171S, K172R, E174S, I179T.

[0016] In one aspect, a vector is provided comprising a nucleic acid encoding a human growth hormone variant that comprises the following amino acid modifications compared to hGH: i)H18D, H21N, L101C, G120R, R167N, K168A, D171S, K172R, E174S, I179T (GHv-1), ii) H18D, H21N, L101C, G120R, N149D, N152D, R167N, K168A, D171S, K172R, E174S, I179T (GHv-2), or iii) H18D, H21N, L101C, G120R, N149D, N152D, R167N, D171S, E174S, I179T (GHv-3).

[0017] In one aspect, the present invention provides LA-GHRAs that bind to hGHR with improved binding affinity compared to pegvisomant.

[0018] Also, or alternatively, in a second aspect, the present invention provides LA-GHRA, which is an antagonist at the hGHR, and thus inhibits or reduces the natural response that hGH exerts on the hGHR, and exhibits improved antagonistic potency compared to pegvisomant.

[0019] Also, or alternatively, in a third aspect, the present disclosure provides LA-GHRAs that exhibit improved pharmacokinetic properties. In one aspect, the present disclosure provides LA-GHRAs that have all of the above-mentioned effects.

[0020] In one aspect, the present disclosure provides a process for preparing a human growth hormone variant disclosed herein, comprising culturing a host cell comprising a vector according to the present disclosure. [Brief explanation of the drawings]

[0021] [Figure 1] FIG. 1 shows the plasma concentration-time profiles of compound 1a after intravenous and subcutaneous administration. DETAILED DESCRIPTION OF THE INVENTION

[0022] definition In the following, Greek letters may be represented by their symbols or corresponding names, e.g., α for alpha, β for beta, ε for epsilon, γ for gamma, ω for omega, etc. The Greek letter μ may also be represented by "u", e.g., μl for ul, μM for uM, etc.

[0023] When used in the context of describing the present invention, the terms "a," "an," and "the," and similar referents, unless otherwise indicated herein or clearly contradicted by context, should be construed to cover both the singular and the plural (i.e., one or more).

[0024] Also described herein are growth hormone receptor antagonists, pharmaceutical compositions, and uses thereof, where open-ended terms such as "comprises" and "comprised" are replaced with exclusive terms such as "consisting of" and "consisted of."

[0025] The present invention relates to long-acting growth hormone receptor antagonists (LA-GHRAs) that contain a growth hormone variant and an albumin-binding moiety.

[0026] The term "hGH" refers to human growth hormone, a 191 amino acid polypeptide having the following sequence: FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF (SEQ ID NO: 1). This is the natural ligand of the human growth hormone receptor or "hGHR". SEQ ID NO: 1 is the sequence of recombinant human growth hormone, also known as somatropin.

[0027] A receptor antagonist can be defined as a compound that can bind to the receptor and inhibit or reduce the response typical of the natural ligand. As described herein, "growth hormone receptor antagonist" or "GHRA" refers to a compound that can bind to the human growth hormone receptor without activating the growth hormone receptor or reducing activation compared to the natural response of hGH.

[0028] In one embodiment, the growth hormone receptor antagonist is a growth hormone variant. The term "growth hormone variant" (GHv) refers to an hGH polypeptide that contains one or more substitutions compared to hGH.

[0029] As used herein, the term "long-acting growth hormone receptor antagonist" or "LA-GHRA" refers to a chemically modified growth hormone receptor antagonist in which an albumin binding moiety is covalently attached to the polypeptide backbone. In one embodiment, LA-GHRA refers to a chemically modified growth hormone variant in which an albumin binding moiety is covalently attached to the polypeptide backbone. As used herein, "albumin binding moiety" refers to a chemical moiety or group that replaces a hydrogen atom.

[0030] As used herein, the term "sequence identity" refers to the degree of similarity between two polypeptide sequences, expressed as a percentage (%). Sequence identity can be determined, for example, by alignment, based on the Needleman-Wunsch algorithm. This algorithm is described in Needleman, S. B. and Wunsch, C. D. (1970), Journal of Molecular Biology, 48:443-453, and in the align program by Myers and W. Miller, "Optimal Alignments in Linear Space," CABIOS (computer applications in the biosciences) (1988) 4:11-17. The term "sequence identity" is used to describe growth hormone variants, for example as part of LA-GHRA, in some embodiments having at least 81% sequence identity with the polypeptide of SEQ ID NO: 1, such as at least 82%, for example at least 83%, such as at least 84%, for example at least 85%, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, for example at least 98%, such as at least 99% sequence identity with the polypeptide of SEQ ID NO: 1.

[0031] Growth hormone variants In the following, embodiments of growth hormone variants are disclosed, which in certain embodiments should be understood to be specifically disclosed as part of long-acting growth hormone receptor antagonists.

[0032] In one embodiment, the growth hormone variant comprises the substitutions G120R / G120K and L101C, preferably G120R and L101C. In a further embodiment, the growth hormone variant further comprises one or more of the following substitutions compared to hGH: H18D, H21N, R167N, D171S, E174S, and I179T. In one embodiment, the growth hormone variant comprises the following substitutions compared to hGH: H18D, H21N, L101C, G120R, R167N, D171S, E174S, and I179T. In a particular embodiment, the growth hormone variant comprises the following substitutions compared to hGH: H18D, H21N, L101C, G120R, R167N, K168A, D171S, K172R, E174S, and I179T. In one embodiment, the growth hormone variant comprises the substitutions H18D, H21N, L101C, G120R, R167N, K168A, D171S, K172R, E174S, and I179T, as well as 1 to 10 additional substitutions, such as 1 to 5 additional substitutions or 2 to 4 additional substitutions, compared to hGH. In a further embodiment, the growth hormone variant is FPTIPLSRLFDNAMLRADRLNQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSCVYGASDSNVYDLLKDLEERIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFNADMSRVSTFLRTVQCRSVEGSCGF (SEQ ID NO: 2), which may also be referred to as [H18D, H21N, L101C, G120R, R167N, K168A, D171S, K172R, E174S, I179T]-hGH or Compound 2 or GHv-1. In one embodiment, SEQ ID NO: 2 contains one or more disulfide bridges between Cys53-Cys165 and / or Cys182-Cys189.

[0033] In a further embodiment, the growth hormone variant includes substitutions H18D, H21N, L101C, G120R, N149D, N152D, R167N, K168A, D171S, K172R, E174S, and I179T, and 1 to 10 additional substitutions, such as 1 to 5 additional substitutions or 2 to 4 additional substitutions compared to hGH. In a further embodiment, the growth hormone variant is FPTIPLSRLFDNAMLRADRLNQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSCVYGASDSNVYDLLKDLEERIQTLMGRLEDGSPRTGQIFKQTYSKFDTDSHDDDALLKNYGLLYCFNADMSRVSTFLRTVQCRSVEGSCGF (SEQ ID NO: 3). (GHv-2)

[0034] In a further embodiment, the growth hormone variant includes substitutions H18D, H21N, L101C, G120R, N149D, N152D, R167N, D171S, E174S, and I179T, and 1 to 10 additional substitutions, e.g., 1 to 5 additional substitutions or 2 to 4 additional substitutions, compared to hGH. In a further embodiment, the growth hormone variant is FPTIPLSRLFDNAMLRADRLNQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSCVYGASDSNVYDLLKDLEERIQTLMGRLEDGSPRTGQIFKQTYSKFDTDSHDDDALLKNYGLLYCFNKDMSKVSTFLRTVQCRSVEGSCGF (SEQ ID NO: 4). (GHv-3)

[0035] In one embodiment the growth hormone variant has at least 81% sequence identity with the polypeptide of SEQ ID NO: 1, such as at least 82%, such as at least 83%, for example at least 84%, such as at least 85%, for example at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99% sequence identity with the polypeptide of SEQ ID NO: 1.

[0036] In one embodiment, the growth hormone variant further comprises one or more mutations selected from the group consisting of H18D, H21N, N149D, N152D, R167N, K168A, D171S, K172R, E174S, and I179T.

[0037] In one embodiment, the growth hormone variant comprises the following amino acid modifications compared to hGH: H18D, H21N, L101C, G120R, R167N, K168A, D171S, K172R, E174S, and I179T (GHv-1); H18D, H21N, L101C, G120R, N149D, N152D, R167N, K168A, D171S, K172R, E174S, and I179T (GHv-2), or H18D, H21N, L101C, G120R, N149D, N152D, R167N, D171S, E174S, and I179T (GHv-3).

[0038] In one embodiment, the growth hormone variant in addition to L101C, G120R / G120K comprises 1 to 10 further amino acid substitutions, for example 2 to 8 or 3 to 5 further amino acid substitutions.

[0039] In one embodiment, the substitution at position 120 of the growth hormone variant is G120R.

[0040] In one embodiment, the growth hormone variant is 191 amino acids in length.

[0041] In one embodiment, the growth hormone variant is a growth hormone receptor antagonist. In one embodiment, the growth hormone variant is capable of binding to the growth hormone receptor.

[0042] Albumin binders In one embodiment, the LA-GHRA comprises an albumin-binding moiety covalently attached to the growth hormone variant via a cysteine, e.g., via a cysteine ​​at position 101. The terms "albumin binder" and "albumin-binding moiety" are used interchangeably herein. In a further embodiment, the albumin-binding moiety is capable of forming a non-covalent conjugate with a protein (e.g., albumin), thereby facilitating the circulation of the LA-GHRA in the bloodstream and having the effect of extending the duration of action of the LA-GHRA due to the fact that the conjugate of LA-GHRA and albumin degrades only slowly to release the active ingredient.

[0043] In one embodiment the albumin binding moiety has a molecular weight of 2.8kDa or less, such as 2.6kDa or less, for example 2.4kDa or less, such as 2.2kDa or less, for example 2.0kDa or less, such as 1.8kDa or less, for example 1.6kDa or less, such as 1.4kDa or less, for example 1.2kDa or less, for example 1.0kDa or less.

[0044] In one embodiment the albumin binding moiety has a molecular weight of 0.3 kDa or more, for example 1.8 kDa or less but more than 0.3 kDa.

[0045] In one aspect, the albumin binding moiety is covalently attached to the growth hormone variant via a sulfhydryl group on the side chain of a backbone cysteine ​​of the growth hormone variant. The albumin binding moiety can be attached to the growth hormone variant by reaction of a reduced cysteine ​​side chain with an activated derivative of the albumin binding moiety containing a leaving group, such as a haloacetamide, as described in WO 2011 / 089255, page 77, lines 5-19, Chemical III, or as described in Example 1 herein, Formula 4a or Formula 5a. In one embodiment, the haloacetamide is selected from chloroacetamide, bromoacetamide, and iodoacetamide, as described in Formula 3, Formula 4, and Formula 5, respectively. [ka] [ka]

[0046] The structure of an "albumin binder" or "albumin binding moiety," when designated as part of a compound of the invention, is the substitution product resulting from displacement of a leaving group, e.g., chloro-, bromo-, or iodo-, of a haloacetamide depicted herein by sulfur at position 101 of a growth hormone variant. The resulting albumin binder structure can be readily deduced from the final structure of a compound disclosed herein in terms of how the compound is formed by displacement of a halogen leaving group from the depicted haloacetamide, as shown, for example, in Examples 1 and 11.

[0047] In a further embodiment, the albumin binder is selected from: Br-Formula 6a: [ka] Br-Formula 7: [ka] Br-Formula 8: [ka] Br-Formula 9: [ka] and Br-Formula 10a: [ka]

[0048] In the above formula for the albumin binder, an amino acid that is part of the formula is an L-amino acid if the formula ends with "a".

[0049] In one aspect the albumin binding moiety is The file is JPEG0007823234000011.jpg22170.

[0050] In one embodiment there is provided a long-acting growth hormone receptor antagonist of the present disclosure, wherein the albumin binding moiety is: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] wherein the * indicates the point of attachment of the albumin binding moiety to the growth hormone variant via the sulfur residue of the cysteine ​​side chain at position 101 of the growth hormone variant.

[0051] compound In one embodiment, the present disclosure provides: a. a growth hormone variant comprising at least the following amino acid substitutions compared to human growth hormone (hGH) (SEQ ID NO: 1): L101C, G120R / G120K, said growth hormone variant having at least 80% sequence identity with the polypeptide of SEQ ID NO: 1; b. an albumin binding moiety configured for binding to albumin, e.g., binding to albumin with a dissociation constant (Kd) of less than 1 μM, having a molecular weight of 3 kDa or less, and covalently attached to the growth hormone variant via the sulfur residue of the cysteine ​​side chain at position 101 of the growth hormone variant; or a pharmaceutically acceptable salt thereof.

[0052] In one embodiment the growth hormone variant has at least 81% sequence identity with the polypeptide of SEQ ID NO: 1, such as at least 82%, such as at least 83%, for example at least 84%, such as at least 85%, for example at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99% sequence identity with the polypeptide of SEQ ID NO: 1.

[0053] In one embodiment, the LA-GHRA of the present invention is selected from the compounds disclosed herein.

[0054] Below, the formulas specifically describe compounds 1-19, and the mutations shown in parentheses refer to specific mutations compared to human growth hormone, i.e., the polypeptide of SEQ ID NO: 1. To illustrate, the notation "-S101[H18D, H21N, L101C, G120R, R167N, K168A, D171S, K172R, E174S, I179T]" specifically refers to a polypeptide having the sequence of SEQ ID NO: 1 (human growth hormone), with each of the positions in brackets "[]" mutated as specified by the single-letter amino acid code. The notation further specifies that the albumin binding moiety is attached to a cysteine ​​at position 101, when the albumin binding moiety is covalently attached to S101.

[0055] In one embodiment, the LA-GHRA is selected from the group consisting of: Compound 1: [ka] [ka] Compound 3: [ka] Compound 3a: [ka] Compound 4: [ka] Compound 4a: [ka] Compound 5: [ka] Compound 5a:

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[0056] In one embodiment, an LA-GHRA of the invention is [ka] (chemical formula 1) is.

[0057] In one embodiment, a long-acting growth hormone receptor antagonist is provided, the antagonist comprising: a. Growth hormone variants containing the following amino acid substitutions compared to human growth hormone (hGH) (SEQ ID NO: 1): H18D, H21N, L101C, G120R / G120K, R167N, D171S, E174S, I179T, and b.Chemical formula 2: [ka] wherein * indicates the point of attachment of the albumin binding moiety to the growth hormone variant via the sulfur residue of the cysteine ​​side chain at position 101 of the growth hormone variant, or a pharmaceutically acceptable salt thereof.

[0058] In one embodiment, LA-GHRA is provided, wherein the growth hormone variant comprises the following amino acid substitutions compared to human growth hormone (hGH) (SEQ ID NO: 1): H18D, H21N, L101C, G120R, R167N, K168A, D171S, K172R, E174S, and I179T.

[0059] In one embodiment, the LA-GHRA is Compound 1 (Formula 1a), also referred to as Compound 1a, described in Example 1 herein. In one embodiment, the LA-GHRA contains one or more disulfide bridges between Cys53-Cys165 and / or Cys182-Cys189. In one embodiment, Compound 1 contains one or more disulfide bridges between Cys53-Cys165 and / or Cys182-Cys189.

[0060] In one embodiment, the growth hormone variant consists of [H18D, H21N, L101C, G120R, R167N, K168A, D171S, K172R, E174S, I179T]-hGH (SEQ ID NO: 2) and the albumin binding moiety is of formula 2.

[0061] In one embodiment, the long-acting growth hormone receptor antagonist is of formula 1 [ka]

[0062] In one embodiment, the long-acting growth hormone receptor antagonist is Compound 1 (Formula 1a): [ka]

[0063] In one embodiment, there is provided a long-acting growth hormone receptor antagonist of compound 1a: [ka] or a pharmaceutically acceptable salt thereof, optionally having one or two disulfide bridges between Cys53-Cys165 and / or Cys182-Cys189.

[0064] In one embodiment, the growth hormone variant comprises one or two disulfide bridges between Cys53-Cys165 and / or Cys182-Cys189. In one embodiment, the growth hormone variant comprises two disulfide bridges between Cys53-Cys165 and Cys182-Cys189. In one embodiment, the growth hormone variant comprises a disulfide bridge between Cys53-Cys165. In one embodiment, the growth hormone variant comprises a disulfide bridge between Cys182-Cys189.

[0065] Functional properties In a first functional aspect, the LA-GHRA of the present invention, e.g., compound 1a, or its polypeptide backbone, exhibits strong binding affinity to hGHR, which can be determined by various methods known in the art, for example, as described in Examples 2 or 3 herein. Binding affinity is measured by the K D may be expressed by a low K value D It is desirable to have strong binding to hGHR corresponding to the value.

[0066] In a second functional aspect, the LA-GHRA of the present invention, e.g., compound 1a, or its polypeptide backbone, does not activate the growth hormone receptor or exhibits reduced activation compared to the native response of hGH, i.e., is a GHR antagonist. In particular, this can be determined by in vitro activity assays (antagonist mode) such as those described in Examples 4 and 5 herein. Generally, activity or potency (antagonist mode) should be as good as possible, i.e., a low IC 50 It should correspond to the value.

[0067] In a third functional aspect, the LA-GHRA of the present invention, e.g., compound 1a, or its polypeptide backbone, possesses desirable pharmacokinetic properties, such as an increased terminal half-life compared to hGH. An increased terminal half-life means that the compound in question is cleared from the body more slowly, thus extending the duration of the compound's pharmacological effect. A desirable half-life means, for example, a rate suitable for daily or weekly administration, preferably weekly administration. In certain embodiments, the pharmacokinetic properties are measured, for example, by the in vivo terminal half-life (t) in Sprague-Dawley rats after intravenous or subcutaneous administration, as described in Example 5 herein. 1 / 2 In one embodiment, the half-life in Sprague-Dawley rats is at least 5 hours, such as at least 8 hours or at least 12 hours.

[0068] In one embodiment, the growth hormone receptor antagonist binds to the hGH receptor in an SPR assay, for example, as described in Example 2 herein.

[0069] In one embodiment, the growth hormone receptor antagonist binds to the hGH receptor in a binding affinity ITC assay such as that described in Example 3 herein.

[0070] In one embodiment, the growth hormone receptor antagonist is an antagonist at the growth hormone receptor.

[0071] In one embodiment, the growth hormone receptor antagonist is capable of inhibiting hGH activity.

[0072] In one embodiment, the growth hormone receptor antagonist is capable of inhibiting hGH activity in a STAT3 activity assay such as that described in Example 4 herein.

[0073] In one embodiment, the growth hormone receptor antagonist is capable of inhibiting hGH activity in a BAF-3 GHR activity assay as described in Example 5 herein.

[0074] In one embodiment, the growth hormone receptor antagonist has improved pharmacokinetic properties.

[0075] In one embodiment, the growth hormone receptor antagonist has an increased terminal half-life as determined in rats as described in Example 6 herein.

[0076] Generation Process The LA-GHRA of the present invention can be produced by a combination of recombinant technology and subsequent chemical modification, for example, as described in Example 1 herein. Growth hormone variants such as SEQ ID NO: 2 can be prepared by recombinant methods, i.e., by culturing host cells containing a DNA sequence encoding the growth hormone variant and capable of expressing the polypeptide in a suitable nutrient medium under conditions allowing expression of the polypeptide. Non-limiting examples of host cells suitable for expressing these polypeptides include Escherichia coli, Saccharomyces cerevisiae, and mammalian BHK or CHO cell lines. Specific examples of methods for preparing SEQ ID NO: 2 are included in the experimental section. Specific examples of methods for preparing SEQ ID NOs: 3 and 4 are also included in the experimental section.

[0077] In one embodiment, there is provided a process for preparing a human growth hormone variant selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, comprising culturing a host cell containing a vector as defined herein.

[0078] In one embodiment there is provided a process for preparing a human growth hormone variant of SEQ ID NO: 2, comprising culturing a host cell containing a vector as specifically defined herein.

[0079] Pharmaceutical Composition Injectable pharmaceutical compositions containing the antagonists of the present invention can be prepared using conventional techniques in the pharmaceutical industry, involving dissolving and mixing the ingredients as needed to give the desired final product.To this end, according to one procedure, the antagonists of the present invention are dissolved in a suitable buffer at a suitable pH, so that precipitation is minimized or avoided.The injectable compositions are sterilized, for example, by sterile filtration.

[0080] Pharmaceutical compositions comprising an antagonist of the present invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient may be prepared as is well known in the art.

[0081] The term "excipient" refers broadly to any ingredient other than the active therapeutic ingredient. An excipient may be an inert, non-active, and / or non-pharmaceutically active substance.

[0082] Formulations of pharmaceutically active ingredients with various excipients are known in the art; see, e.g., Remington: The Science and Practice of Pharmacy (e.g., 19th ed. (1995) and any subsequent editions).

[0083] The composition may be a stabilized formulation. The term "stabilized formulation" refers to a formulation that has increased physical and / or chemical stability, preferably both. Generally, the formulation should be stable during use and storage (in compliance with recommended use and storage conditions) until the expiration date is reached.

[0084] The pharmaceutical composition can be administered to an individual by parenteral administration, including subcutaneous, intramuscular, intraperitoneal, or intravenous injection, using a syringe, optionally a pen-like syringe. Alternatively, parenteral administration can be performed using an infusion pump.

[0085] In one aspect of the invention, the antagonist of the invention is combined with a somatostatin analogue, e.g., in the form of a kit of parts comprising a preparation of the antagonist of the invention as a first unit dosage form and a preparation of the somatostatin analogue as a second unit dosage form.

[0086] Non-limiting examples of somatostatin analogs to be combined with the antagonists of the present invention are octreotide, lanreotide, pasireotide or derivatives thereof.

[0087] In one embodiment, a pharmaceutical composition is provided comprising a long-acting growth hormone receptor antagonist as defined herein and a pharmaceutically acceptable excipient. In certain embodiments, the long-acting growth hormone receptor antagonist is compound 1a.

[0088] Pharmaceutical indications The present invention also relates to long-acting growth hormone receptor antagonists for use as pharmaceuticals. In one embodiment, the present disclosure provides a long-acting growth hormone receptor antagonist as specifically defined herein, such as compound 1a, for use as a pharmaceutical.

[0089] Alternatively, the present invention also relates to a long-acting growth hormone variant that is an antagonist at the growth hormone receptor for use as a medicine. In a particular embodiment, the present invention relates to compound 1a for use as a medicine. In particular, the antagonist of the present invention can be used to treat diseases caused by excessive levels of growth hormone and / or IGF-1.

[0090] In certain embodiments, the long-acting growth hormone receptor antagonists of the present invention may be used to treat the following medical indications, including, but not limited to, acromegaly, gigantism, various cancer types in which IGF-1 is a growth factor, diabetes mellitus, diabetic retinopathy, and diabetic nephropathy.

[0091] In certain embodiments, the indication is acromegaly.

[0092] Nucleic acids, vectors, and host cells In one embodiment, a nucleic acid encoding a human growth hormone variant is provided that contains the following amino acid modifications compared to hGH: H18D, H21N, L101C, G120R, R167N, K168A, D171S, K172R, E174S, and I179T (GHv-1); H18D, H21N, L101C, G120R, N149D, N152D, R167N, K168A, D171S, K172R, E174S, and I179T (GHv-2), or H18D, H21N, L101C, G120R, N149D, N152D, R167N, D171S, E174S, and I179T (GHv-3).

[0093] In one embodiment, a nucleic acid encoding a human growth hormone variant is provided that contains the following amino acid modifications compared to hGH: H18D, H21N, L101C, G120R, R167N, K168A, D171S, K172R, E174S, and I179T.

[0094] In one embodiment, a vector is provided comprising a nucleic acid encoding a human growth hormone variant that comprises the following amino acid modifications compared to hGH: H18D, H21N, L101C, G120R, R167N, K168A, D171S, K172R, E174S, and I179T (GHv-1); H18D, H21N, L101C, G120R, N149D, N152D, R167N, K168A, D171S, K172R, E174S, and I179T (GHv-2), or H18D, H21N, L101C, G120R, N149D, N152D, R167N, D171S, E174S, and I179T (GHv-3).

[0095] In one embodiment, a vector is provided comprising a nucleic acid encoding a human growth hormone variant that comprises the following amino acid modifications compared to hGH: H18D, H21N, L101C, G120R, R167N, K168A, D171S, K172R, E174S, and I179T.

[0096] In one embodiment, a vector is provided, wherein the human growth hormone variant is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. In one embodiment, a vector is provided, wherein the human growth hormone variant is SEQ ID NO: 2.

[0097] In one embodiment, a host cell is provided comprising a vector as defined herein. Embodiment

[0098] The present invention is further illustrated by the following non-limiting embodiments. 1. A long-acting growth hormone receptor antagonist, a. A growth hormone variant containing the following amino acid substitutions compared to human growth hormone (hGH) (SEQ ID NO: 1): L101C, G120R / G120K, and a.Chemical formula 2 [ka] wherein * indicates the point of attachment of the albumin binding moiety to the growth hormone variant via the sulfur residue of the cysteine ​​side chain at position 101 of the growth hormone variant; or a pharmaceutically acceptable salt thereof. 2. The antagonist of embodiment 1, a. Antagonists, including growth hormone variants containing the following amino acid substitutions compared to human growth hormone (hGH) (SEQ ID NO: 1): H18D, H21N, L101C, G120R / G120K, R167N, D171S, E174S, I179T. 3. The antagonist of embodiments 1-2, wherein the growth hormone variant comprises the following amino acid substitutions compared to hGH: H18D, H21N, L101C, G120R / G120K, R167N, K168A, D171S, K172R, E174S, I179T. 4. The antagonist of any one of embodiments 1 to 3, wherein the substitution at position 120 is G120R. 5. The antagonist according to any one of embodiments 1 to 4, wherein the growth hormone variant comprises 1 to 10, for example 2 to 8 or 3 to 5, additional amino acid substitutions. 6. The antagonist of any one of embodiments 1-5, wherein the growth hormone variant consists of [H18D, H21N, L101C, G120R, R167N, K168A, D171S, K172R, E174S, I179T]-hGH (SEQ ID NO: 2) and the albumin binding moiety is of formula 2. 7. The antagonist of any one of embodiments 1 to 6, wherein the antagonist is of formula 1 [ka] 8. The antagonist of any one of embodiments 1-7, which is compound 1 (formula 1a). 9. The antagonist according to any one of embodiments 1 to 8, wherein the growth hormone variant comprises one or two disulfide bridges between Cys53-Cys165 and / or Cys182-Cys189. 10. The antagonist according to any one of embodiments 1 to 9, wherein the growth hormone variant comprises two disulfide bridges between Cys53-Cys165 and Cys182-Cys189. 11. The antagonist according to any one of embodiments 1 to 9, wherein the growth hormone variant comprises a disulfide bridge between Cys53-Cys165. 12. The antagonist according to any one of embodiments 1 to 9, wherein the growth hormone variant comprises a disulfide bridge between Cys182-Cys189. 13. The antagonist of any one of embodiments 1-12, wherein the growth hormone variant is 191 amino acids in length. 14. The antagonist of any one of embodiments 1-13, wherein the growth hormone variant is a growth hormone receptor antagonist. 15. An antagonist according to any one of embodiments 1 to 14, which is capable of binding to a growth hormone receptor. 16. The antagonist of any one of embodiments 1-15, which binds to hGH in the SPR assay described in Example 2 herein. 17. The antagonist of any one of embodiments 1-16, which binds to hGH in a binding affinity ITC assay such as that described in Example 3 herein. 18. The antagonist according to any one of embodiments 1 to 17, which is an antagonist at the growth hormone receptor. 19. An antagonist according to any one of embodiments 1 to 18, capable of inhibiting hGH activity. 20. The antagonist of any one of embodiments 1-19, which is capable of inhibiting hGH activity in a STAT3 activity assay as described in Example 4 herein. 21. The antagonist of any one of embodiments 1-20, which is capable of inhibiting hGH activity in a BAF-3 GHR activity assay as described in Example 5 herein. 22. The antagonist according to any one of embodiments 1 to 21, having improved pharmacokinetic properties. 23. The antagonist of any one of embodiments 1-22, which has an increased terminal half-life as determined in rats as described in Example 6 herein. 24. A pharmaceutical composition comprising a long-acting growth hormone receptor antagonist according to any one of embodiments 1 to 23, and one or more pharmaceutically acceptable excipients. 25. A compound according to any one of embodiments 1 to 23 for use as a medicament. 26. A compound according to any one of embodiments 1-23 for use in the treatment or prevention of diseases caused by excess human growth hormone and / or IGF-1 levels, such as, for example, acromegaly or gigantism. 27. The compound for use according to embodiment 26, in combination with a somatostatin analogue, such as octreotide, lanreotide, pasireotide or a derivative thereof. 28. The compound for use according to embodiment 27, wherein the somatostatin analogue is octreotide. 29. Use of a compound according to any one of embodiments 1 to 23 for the preparation of a medicament. 30. Use of a compound according to any one of embodiments 1-23 for the preparation of a medicament for the treatment or prevention of diseases caused by an excess of human growth hormone and / or IGF-1, such as acromegaly or gigantism. 31. A method for treating a disease caused by excessive human growth hormone and / or IGF-1 levels, such as acromegaly or gigantism, comprising administering a pharmaceutically active amount of a compound according to any one of embodiments 1-23. 32. A method for treating diseases caused by excessive human growth hormone and / or IGF-1 levels, such as acromegaly or gigantism, comprising a pharmaceutically active amount of Compound 1 (Formula 1a). 33. A nucleic acid encoding a human growth hormone variant, wherein, compared to hGH, A nucleic acid comprising the amino acid modifications H18D, H21N, L101C, G120R, R167N, K168A, D171S, K172R, E174S, I179T. 34. The nucleic acid of embodiment 33, wherein the growth hormone variant is SEQ ID NO: 2. 35. A vector comprising a nucleic acid encoding a human growth hormone variant, which, compared to hGH, A vector containing the amino acid modifications H18D, H21N, L101C, G120R, R167N, K168A, D171S, K172R, E174S, and I179T. 36. The vector of embodiment 35, wherein the human growth hormone variant is SEQ ID NO: 2. 37. A host cell comprising a vector according to any one of embodiments 35-36. 38. A process for preparing a human growth hormone variant of SEQ ID NO: 2, comprising culturing a host cell containing a vector according to any one of embodiments 35-36. 39. The process of embodiment 38, further comprising alkylation of the variant according to SEQ ID NO: 2 at Cys101 with an albumin binding moiety according to formula 2a, e.g., formula 2a, e.g., with a haloacetamide according to formula 3, formula 3a, formula 4, formula 4a, formula 5, or formula 5a. [Example]

[0099] List of Abbreviations Ado: (2[2-(amino)ethoxy]ethoxy)acetyl CAD: Charged Aerosol Detector DCM: dichloromethane, CH2Cl2, methylene chloride DIC: diisopropylcarbodiimide DIPEA: N,N-diisopropylethylamine DMF: N,N-dimethylformamide DTT: 1,4-dithiothreitol ECD: extracellular domain EDTA: Ethylenediaminetetraacetic acid eq.: equivalent Et2O: Diethyl ether FBS: fetal bovine serum Fmoc: 9H-fluoren-9-ylmethoxycarbonyl gGlu: gamma-glutamic acid, γGlu GH: growth hormone h: time hGH: human growth hormone hGHR: human growth hormone receptor hGHRA: human growth hormone receptor antagonist HPLC: High-pressure liquid chromatography IGF-1: insulin-like growth factor 1 IPTG: Isopropyl-β-D-1-thiogalactopyranoside iv: intravenous LBAmp: Lysogeny broth medium containing ampicillin LCMS: Liquid chromatography-mass spectrometry m / z: mass-to-charge ratio MALS: Multi-angle light scattering MeCN: acetonitrile Min: minutes MTT: 4-methyltrityl NHS: N-hydroxysuccinimide NMP: N-methylpyrrolidin-2-one OD: optical density OtBu: tertbutyl ester PBS: phosphate buffered saline Rcf: Relative centrifugal force rpm: rounds per minute RT: room temperature SEC: Size Exclusion Chromatography sc: subcutaneous Su: N-hydroxysuccinimide TBAmp: Terrific Broth medium containing ampicillin tBu: tert-butyl TFA: Trifluoroacetic acid TIS: Triisopropylsilane TNBS: Trinitrobenzenesulfonic acid UPLC: Ultra-high performance liquid chromatography

[0100] Common methods: LCMS method: Characterization was performed by LCMS analysis using a Waters Acquity UPLC H Class Protein BEH column (C4, 300 Å, 1.7 μm, 2.1 mm × 50 mm) equipped with a Waters Xevo G2-XS QTof detector. A water / MeCN system (linear gradient of 5 to 95% MeCN over 6 min containing 0.1% TFA) was used as the eluent. The column was operated at a flow rate of 0.4 ml / min and a temperature of 60 °C. Mass spectra were recorded in the 200–3000 Da range and deconvoluted using MassLynx version 4.2.

[0101] UPLC method: Purity was assessed by UPLC analysis using a Waters UPLC system equipped with an Acquity UPLC BEH300 C4 1.7 μm, 2.1 x 50 mm column. The system was further equipped with a Waters Acquity PDA detector setup for detection at 214 nm / 280 nm. The eluent consisted of 0.05% TFA in Milli-Q water (A) and 0.05% TFA in acetonitrile (B). A linear gradient system from 25% to 75% B was used, with a gradient run time of 6 min. The column was operated at a flow rate of 0.4 ml / min and a temperature of 40 °C. Purity was defined as the peak AUC relative to the total AUC (percent exclusive solvent peak) reported by the system software for each UV wavelength.

[0102] CAD method: Concentrations were determined using a CAD detection system. Samples were run on a Vanquish Thermo-Fisher system using a Waters CSH C4, 50 x 2.1 mm, 1.7 μm column equipped with a CAD / Corona detector. A water / MeCN system (linear gradient of 5 to 95% MeCN over 6 minutes containing 0.1% TFA) was used as the eluent. The column was operated at a flow rate of 0.45 ml / min and a temperature of 40°C. Quantification was performed by comparison with insulin aspart standards.

[0103] Example 1: Preparation of GH receptor antagonist compound 1 (formula 1a): [ka]

[0104] Compound 1a was made using the following general steps: i) GHRA peptide backbone expression in E. coli to obtain Cys101 cystamine protected mixed disulfide. ii) Synthesis of the iodoacetamide albumin binding moiety (Formula 5a). iii) Deprotection of the Cys101 mixed disulfide and nucleophilic substitution with iodoacetamide (Formula 5a).

[0105] Details of each step are given below, or alternatively, the compounds can be obtained using the procedures described in WO2011 / 089255 (see page 132, lines 17 to 137, Example page 44, page 161, line 5 to page 164, line 11).

[0106] i) Methods for preparing the GH receptor antagonist scaffold (compound 2, SEQ ID NO:2) A plasmid vector encoding Hisx6-SUMO-[H18D, H21N, L101C, G120R, R167N, K168A, D171S, K172R, E174S, I179T]-hGH was prepared and transfected into Escherichia coli (E. coli). Cells were then cultured overnight in LBAmp medium at 30°C, then transferred to TBAmp medium and cultured at 37°C until the optical density (OD) reached 1.8. Target protein expression was induced overnight at 16°C with a final concentration of 0.1 mM IPTG. Cell pellets were then collected by centrifugation and disrupted using a high-pressure cell homogenizer. The material was resuspended in a buffer solution of 20 mM Tris, 300 mM NaCl, 1 mM cysteamine-2.HCl, pH 8.0, containing 200 mM Arg at a concentration of 1:10 (w / v). The supernatant was collected by centrifugation and then chromatographically purified on a Ni Excel His-tag capture column. The column was washed with five bed volumes of 20 mM Tris, pH 8.0, 300 mM NaCl, 1 mM cystamine-2.HCl, and 200 mM Arg. The His-SUMO tag was removed using SUMO protease, a 1:1000 (w / w) dilution of the protease, at 18°C ​​overnight. NaCl was added to the digestion solution to a concentration of 1 M and loaded onto a Phenyl FF column. The column was washed with two bed volumes of 20 mM Tris, pH 7.5, 1 M NaCl, 200 mM Arg, and 1 mM cystamine-2.HCl, followed by 2.5 bed volumes of 20 mM Tris, pH 7.5, and 1 mM cystamine-2.HCl to elute the protein. The crude protein was further purified by passing it through a SEC Hiload 26 / 60 Superdex 75 column eluted with PBS buffer containing 1 mM cysteamine-2.HCl. The target protein [H18D, H21N, L101C, G120R, R167N, K168A, D171S, K172R, E174S, I179T]-hGH (compound 2) was obtained at a concentration of 5.3-6.0 mg / ml in the Cys101 cystamine-protected (mixed disulfide) form, as determined by CAD-C4 concentration analysis. The intact purified protein was analyzed using LCMS. The observed mass was consistent with the theoretical mass predicted from the amino acid sequence.The expected connectivity of the disulfide bonds was demonstrated by peptide mapping using trypsin and AspN digestion followed by LCMS analysis of the digests before and after reduction of the disulfide bonds with DTT. LCMS (general methods): 22092 Da.

[0107] ii) Albumin binder 4-(1H-tetrazol-16-yl-hexadecanoylsulfamoyl)butanoyl-Ado-γGlu-γGlu-Ado-N ε (C(O)CH 2 I) Preparation of Lys-OH (formula 5a): [ka] [ka] [ka] (Formula 3a).

[0108] First, the corresponding bromoacetamide (Formula 4a) was synthesized on a solid support at a 1 mM scale according to Scheme 1 using standard Fmoc-peptide chemistry on an ABI433 synthesizer. The peptide was assembled on Fmoc-Lys(MTT)-Wang resin using Fmoc-Ado-OH and Fmoc-Glu-OtBu protected amino acids. 4-(16-1H-Tetrazol-5-yl-hexadecanoylsulfamoyl)butyric acid was manually coupled overnight at 2 equivalents using DIC / NHS in DCM / NMP. The resin was then treated with 50 mL of DCM / TFA / TIS / water (94:2:2:2) in a flow-through configuration until the yellow color disappeared, followed by washing with DIPEA / DMF and neutralization. The resin was treated with a solution of bromoacetic acid (4 mM) in DCM / NMP (1:1), which was then activated with a mixture of NHS and DIC (both 1 mM). The resulting mixture was filtered and then combined with an additional 1 mM DIPEA. After 1 h, the reaction was complete. The resin was treated with 80 mL of TFA / TIS / water (95:2, 5:2, 5) for 1 h. Evaporated with a stream of N, precipitated by the addition of EtO, washed with EtO, and dried. The crude product was purified by preparative HPLC (two runs) with a gradient of 30–80% 0.1% TFA / MeCN to 0.1% TFA in water. Fractions were collected and lyophilized with approximately 50% MeCN to give formula 4a. TOF-MS: mass 1272.52 (M+1).

[0109] 4-(1H-tetrazol-16-yl-hexadecanoylsulfamoyl)butanoyl-Ado-γGlu-γGlu-Ado-N ε (C(O)CHCl)Lys-OH (formula 3a) can be prepared by the same method using chloroacetic acid instead of bromoacetic acid in the last step. [ka]

[0110] In the final step, bromoacetamide (formula 4a) was converted to the corresponding iodoacetamide (formula 5a) by halogen exchange as follows: Formula 4a was suspended in acetone and 5 equivalents of sodium iodide was added. The mixture was heated to reflux for 1 hour. The reaction was then cooled on an ice bath for 15 minutes. The solvent was decanted, and the remaining oil solidified when washed with water. The solid material was collected and dried in vacuo for 24 hours, yielding 45% conversion to iodoacetamide Formula 5a.

[0111] Iodoacetamide (formula 5a) can be prepared in a similar manner starting from chloroacetamide (formula 3a).

[0112] iii) Preparation of GH receptor antagonists using albumin binders at Cys101 (compound 1, formula 1a): Cystamine-protected [H18D, H21N, L101C, G120R, R167N, K168A, D171S, K172R, E174S, I179T]-hGH (21.3 mg) in 3.5 ml of PBS containing 1 mM cystamine, obtained as described above, was buffer-exchanged into 20 mM triethanolamine, 0.1 M NaCl, pH 8.5 buffer using Zeba Spin Desalting Columns (catalog number: 89893, Thermo Scientific, 7 kDa cutoff). A 10 mM solution of bis(p-sulfonatophenyl)phenylphosphine dihydrate dipotassium salt in 0.1 M NaCl, pH 8.5 was prepared, and 2 ml of this solution (corresponding to 20 equivalents) was added to the cystamine-protected polypeptide solution. The mixture was stirred at room temperature for 1 hour. The reduction of Cys101 mixed disulfide was confirmed by LCMS (General Methods). The solution was again buffer-exchanged into 20 mM triethanolamine, 0.1 M NaCl, pH 8.5 buffer using Zeba Spin Desalting Columns (Cat. No.: 89893, Thermo Scientific, 7 kDa cutoff). To the buffer-exchanged solution, the albumin binder, Formula 5a, prepared under ii) above, was added as a dry powder (6.5 mg, 5 equivalents).

[0113] The clear solution was then incubated at room temperature for 3 hours. Completion of the alkylation was confirmed by LCMS (General Methods). The reaction mixture was diluted to 25 ml with 20 mM triethanolamine, pH 8.5, containing 10% ethylene glycol (Buffer A). The solution was loaded onto a HiLoad 26 / 10 A Sepharose HP column. Unbound material was eluted with two bed volumes of Buffer A. The column was then eluted with a gradient of 0 to 40% Buffer B (20 mM triethanolamine, 1 M NaCl, 10% ethylene glycol, pH 8.5). All runs were performed at 15°C with a flow rate of 4 ml / min, and the eluate was monitored at 280 nm. Fractions containing pure material were collected and concentrated by ultrafiltration (Amicon 3KDa filter) followed by centrifugation at 14,000 rcf for 2 x 20 min. The concentrated solution was buffer exchanged into 10 mM NH4HCO3 using a 7 kDa Zeba Spin Desalting column and lyophilized to give a white powder (10.9 mg). Yield (alkylation step only) is 10.9 mg (50%). LCMS (electrospray): m / z = 23206 Da.

[0114] Example 2: GH receptor affinity by surface plasmon resonance (SPR) The purpose of this example was to determine the affinity of compounds of the present invention compared to pegvisomant. SPR experiments were performed at 25°C using Biacore T200 and Biacore 8K instruments (Cytiva). Binding kinetics were determined using an Avi-tagged biotinylated version of the extracellular domain of the growth hormone receptor (hGHR-ECD) containing hGHR residues 19-264.

[0115] For biotinylated hGHR-ECD, we used the BiotinCAPture kit (Cytiva, 28920233), which allows reversible capture of biotinylated ligands. The chip surface was prepared by overnight rehydration in HBS-EP buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v / v Surfactant P20) in the instrument using standby flow. Prior to the experiment, the chip surface was conditioned using regeneration solution (3 parts regeneration stock 1 (8 M guanidine-HCl) and 1 part regeneration stock 2 (1 M NaOH)), followed by three consecutive initiation cycles using HBS-EP buffer as the analyte. Each cycle consisted of the following steps: The following procedures were performed: 1) 50 μg / mL CAPture reagent (included in the Biotion CAPture kit) in HBS-EP buffer (flow rate: 2 μl / min, contact time: 300 s), 2) biotinylated ligand capture (flow rate: 10 μl / min, contact time: 180 s), 3) analyte (compound 1a, compound 2, somatropin, and pegvisomant) injection (flow rate: 30 μl / min, contact time: 120 s, dissociation time: 800 s), and 4) regeneration using regeneration solution (flow rate: 10 μl / min, contact time: 60 s). All analytes were diluted in HBS-EP buffer and applied to the ligand-coated chip at concentrations of 100, 20, 4, 0.8, 0.16, 0.032, and 0 nM.

[0116] Data analysis was performed using Biacore 8K evaluation software. Kinetic fitting was performed after baseline subtraction, and curves were globally fitted using a 1:1 Langmuir binding model. D The value is the number of matched associations (k a ) and dissociation constant (k d The results are shown in Table 1. [Table 1]

[0117] As can be seen in Table 1, both Compound 1a and Compound 2 have higher affinity for the hGHR-ECD compared to pegvisomant.

[0118] Example 3: GHR Binding Affinity Determined by Isothermal Titration Calorimetry (ITC) The purpose of the example was to determine affinity by measuring the thermodynamic interactions between binding partners (receptor / ligand) in solution both in the presence and absence of HSA.

[0119] Compound 1a, pegvisomant, and fatty acid-free human serum albumin (HSA) (Sigma) were dialyzed overnight into 4.38 mM histidine, 2.05 mM NaCl, pH 7.1 buffer using a Slide-A-Lyzer™ dialysis cassette. All proteins were subjected to size-exclusion chromatography with multi-angle light scattering (SEC-MALS) analysis to determine concentrations before and after ascending concentration and after the ITC experiment. SEC-MALS was performed on an Agilent 1200 Series HPLC system (Agilent, Darmstadt, Germany) using a TSK G3000 SWXL column (Tosoh Bioscience, Tokyo). The column was maintained at 20°C and equilibrated in 122 mM NaHPO, 78 mM NaHPO, 300 mM NaCl, 4% 2-propanol, pH 6.8. Detection was performed by UV absorption at 280 nm, refractive index (RI), and multi-angle laser light scattering (MALS). The RI and MALS detectors were Optilab T-rEX (Wyatt) and MiniDawn Treos (LS) (Wyatt Technology, Santa Barbara, CA, USA), respectively. Data were evaluated using Chemstation B.03.01 and Astra Ver. 6.1.1.17.

[0120] ITC experiments were performed at 37°C using a PEAQ-ITC calorimeter (Malvern, UK). Sample cells (300 μL) contained 10 μM compound 1a or pegvisomant in the presence of HSA (0, 1, 5, or 10 mg / mL). The syringe contained 100 μM hGHR-ECD (ligand). A thermal equilibration step was followed by a 60-second delay, after which an initial 0.4 μL injection of hGHR-ECD was administered, followed by 25 1.5 μL injections at 120-second intervals and a final 1.1 μL injection. The stirring speed was 750 rpm, and the reference power was held constant at 9.64 μcal / s. Data processing was performed using a one-site fitting model with fitted offset correction using PEAQ-ITC analysis software (Malvern Instruments). The results are shown in Table 2. [Table 2]

[0121] N is the bonding stoichiometry and K D is the dissociation constant, ΔH is the change in enthalpy, ΔG is the change in Gibb free energy, ΔS is the change in entropy, and T is the temperature.

[0122] K of compound 1a D is found to be lower than pegvisomant, and therefore compound 1a also exhibits higher affinity in the ITC assay. It can also be seen that for both compound 1a and pegvisomant, the affinity does not change significantly in the presence of HSA compared to 0 mg / mL HSA.

[0123] Example 4: In vitro human growth hormone receptor activity assay The purpose of this example was to determine the activity of selected compounds with respect to whether they act as agonists or antagonists at the hGHR.

[0124] To evaluate the in vitro activity of human growth hormone receptor (hGHR) agonists or antagonists, they were tested for their ability to induce STAT3 phosphorylation in reporter cells expressing hGHR. To generate reporter cell lines, BHK-21 cells (ATCC® CCL-10™) were stably transfected with a reporter plasmid encoding a STAT3 response element linked to luciferase and an expression plasmid encoding hGHR. Single-cell clones were isolated for the reporter cell lines. When reporter cells were exposed to an hGHR agonist, hGHR phosphorylates the transcription factor STAT3. Phosphorylated STAT3 activates transcription at the STAT3 response element, resulting in the production of luciferase. Luciferase can convert luciferin to oxyluciferin, a process that produces bioluminescence. Luciferase activity can be quantified by detecting luminescence upon cell lysis and the addition of a luciferase substrate. Thus, adding an hGHR agonist followed by a detection reagent to reporter cells causes the production of luminescence in a dose-dependent manner. This procedure is hereafter referred to as the agonist mode. Similarly, adding an hGHR antagonist followed by the addition of an hGHR agonist and a detection reagent causes the production of luminescence in an inverse dose-dependent manner. This procedure is referred to as the antagonist mode. By testing several different concentrations of hGHR agonist or antagonist, the EC 50 and IC in antagonist mode 50 The half-maximal effective concentration, referred to as the maximal effective concentration (HAE), can be determined. These measurements are reported for all compounds tested. In all in vitro hGHR activity experiments, somatropin was used as a positive control for hGHR antagonism, and pegvisomant was used as a positive control for hGHR antagonism.

[0125] To perform the assay, BHK21 / GHR / STAT3-RE clone 9 cells were thawed and suspended in growth medium (DMEM (Gibco 10091-148) containing 10% FBS (Gibco 31966-021) and 1% P / S (Lonza DE17-602E)). Cells were then seeded overnight at 20,000 cells / well in 25 μL / well in white opaque 384-well plates (Greiner 781080). The following day, test compounds were added to the assay medium (1% OVA (Sigma A5505), 0.01% Tween 20 (Roche The compounds were serially diluted in phenol red-free DMEM containing 1% P / S (33766700) and 1% P / S. The cell plates were emptied, and 20 μL / well of diluted test compound was added to the cells in quadruplicate. For the agonist mode, 5 μL / well of assay medium was immediately added to half of the cell plate. For the antagonist mode, after 30 minutes of incubation at 37°C and 5% CO2, 5 μL / well of 5 nM somatropin was added to the other half of the cell plate. After an additional 4 hours of incubation at 37°C and 5% CO2, 12.5 μL / well of detection reagent (Steady-GLO, Promega E2550) was added to all cell plates, and the plates were incubated in the dark at room temperature for 15 minutes. Luminescence was then detected using an EnVision Multimode Plate Reader (PerkinElmer). EC values ​​for each test compound were then measured. 50 and IC 50 To determine the α, a four-parameter logistic regression was performed on the raw data for each test compound using the Python package SciPy optimize. Each test compound was tested in at least two independent experiments. Reported values ​​are the mean ± standard error across all experiments. Results are shown in Table 3. [Table 3]

[0126] The agonist data show that, as expected, somatropin is an agonist at the hGHR. Additionally, it can be seen that neither pegvisomant, compound 1a, nor compound 2 exhibit measurable agonist activity. In the antagonist mode, it can be seen that pegvisomant, compound 1a, and compound 2 all exhibit antagonist activity, but compound 1a and compound 2 exhibit improved antagonist activity compared to the known antagonist pegvisomant.

[0127] Example 5: BAF-3 GHR Assay to Determine GHR Activity The purpose of this example was to determine the activity of selected compounds with respect to whether they act as agonists or antagonists at the hGHR.

[0128] BAF-3 cells (a murine pro-B lymphoid cell line derived from bone marrow) were originally dependent on IL-3 for growth and survival. IL-3 activates JAK-2 and STAT, the same mediators activated by GH, upon stimulation. After transfection with the human growth hormone receptor, the cell line became growth hormone-dependent. This clone can be used to evaluate the effects of different growth hormone preparations on the survival of BAF-3GHR. (Culture medium: RPMI 1640 + GlutaMAX TM +FBS+hGH).

[0129] BAF-3GHR cells were grown in starvation medium (culture medium without growth hormone) for 24 hours at 37° C., 5% CO. The cells were then used to test either growth hormone receptor agonists or antagonists.

[0130] In the agonist mode, cells were washed, resuspended in starvation medium, and plated. 10 μL of test compound or control (human growth hormone in the agonist mode) at different concentrations was added to the cells, and the plate was incubated at 37°C, 5% CO for 68 hours.

[0131] In the antagonist mode, cells were washed, resuspended in starvation medium, and plated. 10 μL of 2 nM growth hormone solution was added to the cells, followed by 10 μL of different concentrations of test compound or control (pegvisomant). The plate was then incubated at 37°C, 5% CO2 for 68 hours.

[0132] AlamarBlue® was added to each well and the cells were incubated for an additional 4 hours. AlamarBlue® is a redox indicator that is reduced by reactions that occur naturally in cellular metabolism, thus providing an indirect measure of viable cell number.

[0133] The metabolic activity of the cells was measured using a fluorescence plate reader. Fluorescence was measured in a plate reader with shaking at excitation of 544 nm and emission of 590 nm, followed by readings of 9-point pr. wells in a cross pattern. Activity (the amount of compound that stimulates / inhibits cells by 50%) was calculated from the concentration-response curve.

[0134] Three compounds, pegvisomant, compound 2, and compound 1a, were tested in the assay in the agonist mode with somatropin as the positive control, and the results are shown in Table 4.

[0135] Pegvisomant, Compound 2, and Compound 1a were tested in the antagonist assay mode, with pegvisomant as the positive control. The results are shown in Table 4. [Table 4]

[0136] The data in Table 4, in agonist mode, give an estimated EC 50 confirms the agonist effect of somatropin, but no concentration responses are obtained for the other test compounds, indicating that none of them exhibit agonist properties in the current assay.

[0137] I C 50The values ​​(antagonist mode) were, as expected, pegvisomant with an IC of 205 nM. 50 The results show that compound 2 has antagonistic properties in the presence of somatropin. Compound 2 was also able to inhibit BAF cell proliferation in the presence of somatropin, with an antagonistic potency 147 times higher than that of pegvisomant, while compound 1a had an antagonistic potency 11 times higher than that of pegvisomant. Thus, it is confirmed that both compound 1a and compound 2 have improved antagonistic potency compared to pegvisomant.

[0138] Example 6: Rat PK Data - Compound 1a The purpose of this example was to investigate the pharmacokinetic properties of compound 1a in Sprague-Dawley rats after single-dose intravenous and subcutaneous administration.

[0139] Twenty-four male Sprague-Dawley rats weighing approximately 250 g were included in the study. The animals were allowed one week to acclimate before entering the study. During the experiment, the animals were housed and handled according to standard procedures at the Novo Nordisk Animal Department and had free access to food and water. All rats were weighed before the start of the study and on the day of dosing, just before dosing. The animals were divided into two treatment groups: one treated intravenously and one treated subcutaneously.

[0140] Compound 1a was dissolved to a final concentration of 450 nmol / L in a standard buffer consisting of 8.05 mM dibasic sodium phosphate, 1.96 mM dibasic potassium phosphate, 140 mM sodium chloride, and 0.007% polysorbate 20 at pH 7.4.

[0141] At the start of the experiment, all animals received a single dose of 180 nmol / kg of Compound 1a either as a single intravenous bolus injection in the tail vein (intravenous treatment group) or as a single subcutaneous injection in the neck (subcutaneous treatment group).

[0142] Blood samples were taken from the tail vein of all animals. 0.1 ml of blood was collected according to the following schedule: Intravenous treatment group: pre-dose, 0.08, 0.5, 1, 2, 4, 8, 18, 24, 30, 48, 72, 96, 168, and 240 hours post-dose. Subcutaneous treatment group: pre-dose, 0.25, 0.5, 1, 2, 4, 8, 18, 24, 30, 48, 72, 96, 168, and 240 hours post-dose.

[0143] Blood sampling was performed as a sparse sampling schedule, with both treatment groups subdivided into four groups of three rats, and blood was collected from three animals at each time point per treatment group, ensuring that each animal was bled three to four times during the entire study.

[0144] At each sampling time point, 0.1 ml (8-10 drops) of blood was collected from the tail vein and transferred to an Eppendorf tube containing 8 mM EDTA. After gentle mixing, the blood sample was kept on ice until centrifuged at 4000 g for 5 minutes at +5°C for 10 minutes. After centrifugation, the plasma fraction was split into two fractions and analyzed for test compound and IGF-1 concentrations as described below.

[0145] Plasma analysis of IGF-1 in rat plasma: Samples were analyzed for rat IGF-1 content using an in-house developed homogeneous luminescent oxygen channeling immunoassay (LOCI). Prior to performing the analysis, samples were pretreated to release IGF-1 from its binding proteins in plasma. During the assay, concentration-dependent bead-analyte-immunocomplexes were generated, resulting in light output measured on a Perkin Elmer Envision reader. The assay used (anti-rat IGF-1) mAb 1212-0362-conjugated receptor beads and biotinylated mAb 1212-0307 (also raised against rat IGF-1) with generic streptavidin-coated donor beads. The lower limit of quantitation (LLOQ) was 20 ng / ml.

[0146] Plasma analysis of compound 1a in rat plasma: Samples were analyzed for compound 1a content using an in-house developed homogeneous luminescent oxygen channeling immunoassay (LOCI). During the assay, a concentration-dependent bead-analyte-immunocomplex was generated, resulting in light output measured on a Perkin Elmer Envision reader. The assay used growth hormone binding protein-conjugated receptor beads and biotinylated pAb 20GS10 (raised against human growth hormone) along with generic streptavidin-coated donor beads. The lower limit of quantitation (LLOQ) is 50 pM.

[0147] The data from the analysis are shown in Table 5. [Table 5]

[0148] The plasma concentration-time profiles of compound 1a after intravenous and subcutaneous administration are shown in Figure 1 and demonstrate that absorption of the subcutaneous dose was complete 24–48 h after administration and that compound 1a did not exhibit absorption-limiting elimination.

[0149] Plasma concentration-time data were analyzed by non-compartmental pharmacokinetics using Phoenix WinNonlin 6.4 (Pharsight Corporation). Calculations were performed using the mean concentration-time values ​​from three animals at each time point. Estimated pharmacokinetic parameters are shown in Table 6 below. [Table 6]

[0150] Table 6 shows the main pharmacokinetic parameters of compound 1a. C0 is the concentration of compound 1a immediately after intravenous injection. t max is the maximum plasma concentration of compound 1a after subcutaneous administration, estimated at 7 hours. max is the maximum concentration of compound 1a in the blood, estimated to be 3210 nmol / L and 552 nmol / L after intravenous and subcutaneous administration, respectively. AUC lastand AUC are the areas under the curve at the last data point, extrapolated to infinity, respectively, and are measures of total drug exposure over time. The AUC values ​​from subcutaneous and intravenous administration can be used to calculate absolute bioavailability (F). Clearance is calculated from dose / AUC. V z is the volume of distribution, and t 1 / 2 is the terminal half-life of compound 1a after the absorption phase is complete. MRT is the mean residence time of compound 1a, which corresponds to the average time compound 1a is in the body, and therefore the prolonged action resulting from subcutaneous administration.

[0151] Example 7: Dog PK Data - Compound 1a The purpose of the example was to investigate the pharmacokinetic properties of compound 1a and to measure IGF-1 concentrations in beagle dogs after single-dose intravenous administration at two doses: 10 mg / kg and 3 mg / kg.

[0152] Nine male beagle dogs, weighing approximately 11.5 kg on average, were divided into three groups: vehicle, 3 mg / kg Compound 1a, and 10 mg / kg Compound 1a, n=3 per group. The dose volume was 0.3 ml / kg for each dog at a given concentration of injection solution. All animals were fasted for approximately 20 hours before the experiment. After intravenous bolus injection, serial blood samples were collected up to 504 hours after administration for the determination of Compound 1a and circulating IGF-1. The detailed blood collection time points were -5 minutes (before administration), 5, 15, 30, 45 minutes, 1, 1.5, 2, 4, 7, 10, 24, 30, 48, 72, 120, 168, 216, 264, 312, 360, 432, and 504 hours.

[0153] For each time point, 1.2 ml of blood was collected into EDTA-coated tubes, followed by gentle inversion 3-4 times. Blood samples were kept on ice until centrifugation (4 min, 4°C, 4000 rpm).

[0154] 100 μl x 2 plasma was immediately transferred to labeled Micronic tubes and kept at -20°C until assayed. Another 200 μl back-up sample set is kept for possible retesting (-20°C or -80°C).

[0155] Plasma analysis of IGF-1 in dog plasma: The concentration of IGF-1 in canine plasma samples was determined using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Mediagnost, product number E20). Calibrators and controls were prepared by reconstituting "CAL" and "control" in 500 μL of sample buffer (SB). The dynamic range of the assay was 2-50 ng / mL. The lower limit of quantitation (LLOQ) of the assay was 0.091 ng / mL. Controls were diluted 1:20 in sample buffer PP (BUF SB). Canine plasma samples were diluted 1:15 in SB. The dilution procedure was performed on an apricot automated liquid handling system. 80 μL of antibody conjugate "Ab" was transferred to a 96-well assay plate. 20 μL of diluted sample / calibrator / control was applied to the assay plate and incubated at room temperature (RT) for 1 h with shaking at 350 rpm. The assay plate was washed five times with wash buffer (WP). 100 μL of enzyme conjugate (CONJ) was applied to the assay plate and incubated at room temperature for 1 hour with shaking at 350 rpm. The assay plate was washed 5 times with WP. 100 μL of substrate solution (SUBST) was applied to the assay plate and incubated at room temperature in the dark for 15 minutes. 100 μL of stop solution (SL) was added to the assay plate. The plate was read on a Tecan plate reader at 450 nm. The standard weight was 1 / signal. 2 Calibrator curves were generated by fitting the signals and concentrations to a five-parameter logistic model with . The concentrations of control and IGF-1 were calculated on the calibrator curve and corrected for dilution. Control recoveries are within 80%-120%. Sample dilution recoveries are within 80%-120%. The coefficient of variation (CV) between replicates is within ±10%.

[0156] Data from the analysis are shown in Table 7. [Table 7]

[0157] Plasma analysis of compound 1a in dog plasma : The concentration of compound 1a in dog plasma samples was determined using a luminescence oxygen channeling immunoassay (LOCI). Calibrators and controls were prepared by manually spiking the analyte into dog plasma. The dynamic range of the assay was 4.9–20,000 pM. The lower limit of quantification (LLOQ) of the assay was 4 pM. Three controls were 3,000 pM, 100 pM, and 4 pM, respectively. Dog plasma sample dilutions were performed using a Bravo automated liquid handling system. 2 μL of diluted sample / calibrator / control was applied with 3 μL of sample buffer in a 384-well LOCI plate and incubated overnight at 22 °C with mAb (NNC1212-0000-0575)-conjugated acceptor beads and biotinylated growth hormone binding protein. 30 μL of streptavidin-coated donor beads (67 μg / mL) was added to each well and incubated at 20 °C for 30 min. Plates were read in an Ensight plate reader at 21-22°C using a filter with a bandwidth of 520-645 nm after excitation with a 680 nm laser. The total measurement time per well was 210 ms, including a 70 ms excitation time. The normal weight was 1 / signal. 2 Calibrator curves were generated by fitting the signals and concentrations to a five-parameter logistic model with . The concentrations of the control and compound 1a were calculated on the calibrator curve and corrected for dilution. The recovery rates of the controls were within 80%-120%. The dilution recoveries of the samples were within 80%-120%. The coefficient of variation (CV) between replicates was within ±10%.

[0158] The data from the analysis are shown in Table 8. [Table 8]

[0159] Plasma concentration-time data were analyzed by non-compartmental pharmacokinetics using Phoenix WinNonlin 6.4 (Pharsight Corporation). Calculations were performed using the mean concentration-time values ​​from three animals at each time point. Estimated pharmacokinetic parameters are shown in Table 9 below. [Table 9]

[0160] Table 9 shows the main pharmacokinetic parameters of Compound 1a. C0 is the concentration of Compound 1a immediately after intravenous injection. AUClast and AUC are the areas under the curve at the last data point, extrapolated to infinity, respectively, and are measures of total drug exposure over time. Clearance is calculated as dose / AUC. Vz is the volume of distribution, and t 1 / 2 is the terminal half-life of compound 1a. MRT is the mean residence time of compound 1a, which corresponds to the average time that compound 1a is in the body.

[0161] Example 8: In vitro binding of compound 1a to proteins in plasma from mice, rats, rabbits, dogs, and humans The objective was to investigate the binding between compound 1a in free solution and plasma proteins using equilibrium shift assay techniques. Pharmacokinetic analysis was performed to determine the percentage of unbound compound 1a in plasma.

[0162] Fatty acid-free human serum albumin (HSA) was immobilized on Mini-Leak beads according to the procedure described by Kurtzhals et al. (1, 2). Briefly, the storage medium was removed from the Sepharose beads by aspiration through a filter. The beads were then washed with three volumes of isotonic sodium chloride. To one gram of Sepharose beads, 2 ml of 5% (w / v) HSA in Milli-Q water and 2 ml of 30% (w / v) PEG20000 in 0.3 M HCO3 were added. The solution was mixed overnight on a Hula-Shaker. The assay medium was then removed by aspiration through a filter, and the beads were washed with 20 ml of isotonic sodium chloride. The beads were then treated with 0.2 M ethanolamine for 5 hours. The beads were then washed with 12 ml of phosphate buffer (pH 11, three times) and 12 ml of glycine buffer (pH 3, three times). The beads were stored in PBS buffer (pH 7.2) at a concentration of 200 mg of beads per ml. The concentration of immobilized HSA was determined according to the procedure reported by Kurtzhals et al. (1).

[0163] The drug (D) binds to the immobilized protein (P imm ) and soluble protein (P sol ), two binding reactions can occur:

number

[0164] If the concentration of drug is much lower than the concentration of protein, the protein is expected to bind only one molecule of drug. At equilibrium, the association constant for the two binding reactions is:

number

[0165] K a,sol Reconstructing K a,sol can be put into the equation to give the following formula:

number

[0166] The concentration of immobilized HSA was determined by the DP sol / DP imm can be estimated by plotting K a,sol =K a,imm Assuming that, the slope of the linear plot is 1 / [HSA imm ].

[0167] The concentration of immobilized HSA can be determined by incubating a fixed amount of beads with various concentrations of soluble HSA. Here, 15.5 mg of beads were incubated with five different concentrations of soluble HSA (final concentrations of 2.5, 5, 10, 20, and 40 μM) in PBS buffer (pH 7.2) and 250 nM of a reference compound at room temperature for 2 hours. After incubation, the samples were centrifuged at 750 g for 10 minutes. The samples were then precipitated and subsequently analyzed by LC-MS.

[0168] The fraction of unbound and bound HSA (f u / f b The linear correlation between δ (i.e., δ) and the concentration of soluble HSA can be plotted, with the slope used to calculate the concentration of immobilized HSA per mg Mini-Leak beads as follows:

number

[0169] The average of 12 experiments was used to obtain a concentration of 0.51 nmol of immobilized HSA per mg of beads. This value was used as the K d,HSAimm and was used to calculate the unbound fraction in plasma.

[0170] The bond between the drug (A) and the protein (P) can be explained as follows.

number

[0171] Due to the law of conservation of mass, the binding between a drug (A) and a protein (P) can be described by a defined dissociation constant.

number

[0172] At equilibrium, the concentrations of drug (A) and drug-protein (AP) can be described as follows:

number

[0173] Therefore, we can rearrange the equation for the dissociation constant:

number

[0174] By this formula, the affinity for immobilized HSA is calculated as the ratio of bound to unbound compound (f b / f u ) can be calculated by plotting

[0175] Experimentally, this was performed by incubating six different concentrations of immobilized HSA with 250 nM compound 1a in PBS buffer (pH 7.2), as well as two reference incubations without immobilized HSA. Additionally, a final concentration of 0.01% P188 was used to prevent adsorption to the vessel. Samples were incubated for 2 hours at 37°C with mechanical shaking on a KingFisher robot (Thermo Fisher Scientific) using a 1% OVA-coated deep-well KingFisher plate and tip comb. After incubation, the samples were centrifuged at 750 g for 10 minutes to pellet the beads. Prior to sample preparation, one volume of supernatant was diluted with one volume of blank plasma. The mixtures were assayed for compound 1a by LC-MS.

[0176] The unbound and bound fractions were calculated using the following formula:

number

[0177] The average of the concentrations in the two reference incubations is f b was used to calculate f b / f u Plotting α yields a straight line, where the slope (α) can be converted to a dissociation constant by the following formula:

number

[0178] K d,HSAimm Each determination of is based on eight data points. In each calculation, the reference data point and one of the other data points can be excluded from the calculation.

[0179] The free fraction can be described as follows:

number

[0180] By this formula, the free fraction upon incubation with plasma and immobilized HSA can be expressed as:

number

[0181] The free concentration (A) is

number

[0182] The total binding concentration (e.g., bound to beads (AB) and bound to plasma (AP)) is defined as follows:

number

[0183] After removing the beads, the concentrations in the supernatant (APA) are the bound and free concentrations (A) of plasma proteins (AP):

number

[0184] The concentration of AP can be defined as follows:

number

[0185] During the ceremony, JPEG0007823234000096.jpg5170 is the ratio of compound bound to plasma proteins ([AP]) to the total amount bound to plasma proteins and immobilized HSA ([AB]). This ratio can be expressed as:

number

[0186] The concentration in the supernatant (APA) can be reconstituted.

number

[0187] The above equation can then be rearranged:

number

[0188] The pseudo-dissociation constant for a plasma protein can be estimated using the value that gives the smallest sum of squared residuals between the measured APA value and the theoretically calculated APA value.

[0189] Using an arbitrary concentration of 600 μM as the plasma protein concentration, the unbound fraction in plasma can be calculated using the following formula:

number

[0190] Experimentally, a final concentration of 250 nM of compound 1a in PBS buffer was incubated with various concentrations of immobilized HSA, 0.01% P188, and three different concentrations of plasma (final dilutions of plasma from each species are listed in Table 10). [Table 10]

[0191] Two reference solutions were prepared for each plasma dilution. Samples were incubated for 2 hours at 37°C with mechanical shaking on a KingFisher robot (Thermo Fisher Scientific) using a 1% OVA-coated deep-well KingFisher plate and tip comb. After incubation, samples were centrifuged at 750g for 10 minutes to remove the beads. Prior to sample quantification, one volume of supernatant was diluted with one volume of LYD porcine plasma to match the matrix. The mixture was assayed for compound 1a.

[0192] The calculation of the pseudo dissociation constant for a plasma protein is based on 15 data points (5 data points at each plasma dilution and 3 data points with each plasma dilution). For each individual plasma dilution, the reference data point and one of the other data points can be excluded from the calculation.

[0193] Sample analysis by LOCI All samples were analyzed for compound 1a using a homogeneous bead-based sandwich immunoassay technique, luminescent oxygen channeling immunoassay (LOCI), also known as AlphaLISA. The measured signal depends on the proximity of two types of beads. The donor beads were coated with streptavidin, while the acceptor beads in this case were conjugated with a soluble growth hormone binding protein that recognizes the hGH receptor-binding portion of the molecule. The second binding partner in the sandwich is a biotinylated monoclonal antibody (0195-0000-0127) that recognizes a different epitope on the growth hormone analog. During the assay, the three reactants combined with the analyte to form a bead-aggregated immune complex. Irradiation of the complex with a laser releases singlet oxygen from the donor beads, which is guided to the acceptor beads and induces chemiluminescence that is measured. The amount of light produced is proportional to the concentration of the hGH analog. A standard curve was generated in 50% / 50% LYD porcine plasma / PBS, giving a lower limit of quantitation of 50 pM for the compound 1a analog.

[0194] Immobilized HSA(K d,HSAimm Experiments to determine the binding affinity of compound 1a to ATP were performed and analyzed in parallel with each plasma protein binding experiment. The results of 13 individual experiments that met the criteria are shown in Table 11, with an average K d,HSAimm is used to calculate the average K d,HSAimm The value is 1.69 x 10 -6 M was calculated and used in all subsequent calculations for plasma protein binding experiments. [Table 11]

[0195] Determinations of the unbound fraction (fu) in plasma protein binding experiments in plasma from mice, rats, rabbits, dogs, and humans are listed individually and as mean values ​​in Table 12. The mean unbound fraction (as a percentage), fu, for compound 1a was 1.42% in mouse plasma, 0.62% in rat plasma, 0.41% in rabbit plasma, 0.22% in dog plasma, and 0.08% in human plasma. [Table 12]

[0196] conclusion In vitro plasma protein binding of compound 1a was determined in plasma from mice, rats, rabbits, dogs, and humans. The fraction of unbound compound 1a in plasma ranged from 0.08 to 1.42%, with the lowest unbound fraction in plasma following the order: human < dog < rabbit < rat < mouse.

[0197] Reference materials 1.Kurtzhals, P., Havelund, S., Jonassen, I., Kiehr, B., Larsen, UD, Ribel, U., and Markussen, J. (1995) Albumin binding of insulins acylated with fatty acids: characterization of the ligand-protein interaction and correlation between binding affinity and timing of the insulin effect in vivo. Biochem. J. 312 (3), 725-731. 2.Kurtzhals, P., Havelund, S., Jonassen, I., Kiehr, B., Ribel, U., and Markussen, J. (1996) Albumin binding and time action of acylated insulins in various species. J. Pharm. Sci. 85(3), 304-308.

[0198] Example 9: In vitro growth hormone receptor (GHR)-mediated internalization of compound 1a, recombinant human growth hormone (hGH), and pegvisomant The objective was to investigate the internalization of compound 1a, hGH, and pegvisomant using human GHR-expressing baby hamster kidney fibroblasts (BHK21).

[0199] Quantitative in vitro internalization studies of compound 1a, hGH, and the GHR antagonist pegvisomant were performed using human GHR-expressing baby hamster kidney fibroblasts (BHK21). Compound 1a, hGH, and pegvisomant were conjugated with Alexa Fluor 647 dye to track internalization. Labeling was performed in 50 mM Hepes buffer, pH 7.5, using Alexa Fluor 647 NHS from Life Technologies. Labeling was performed randomly on available epsilon-lysine amines. Monolabeled compounds were then isolated using ion-exchange chromatography, and the buffer was exchanged into PBS using Zeba Spin Desalting Columns (catalog number: 89893, Thermo Scientific, 7 kDa cutoff). The degree of labeling was determined by LCMS (hGH, compound 1a) or fluorometry (pegvisomant). The degree of labeling was 1.0, 1.0, and 0.7 for hGH, Compound 1a, and pegvisomant, respectively.

[0200] Cell lines and culture conditions Baby hamster kidney cells (BHK21) overexpressing the human growth hormone receptor (BHK21 / hGHR), previously used in in-house functional compound screening activities, were utilized for the internalization assay. hGHR overexpression is essential for achieving higher levels of internalization. BHK21 / hGH cells were generated using the pcDNA3,1+(neo) plasmid containing the hGHR sequence. Additionally, a plasmid encoding Stat3-luciferase (puromycin resistance) was transfected into the cells for functional screening purposes unrelated to the assay described herein. Transfected cells were selected using 600 μg / ml G418 (Gibco, #10131-027) and 1 μg / ml puromycin (Gibco, #11138-03) to generate stable pools, which were further used for selection of monoclonal cell lines.

[0201] For maintenance, cells were cultured in DMEM supplemented with Glutamax (Gibco #31966-021), 10% FBS (Thermo Fisher #10091148), 1% penicillin-streptomycin (Gibco #15140-122), 600 μg / ml G418, and 1 μg / ml puromycin at 37°C and 5% CO. For assays, cells were seeded in culture medium with a reduced FBS content of 1% and without the selection markers G418 and puromycin.

[0202] Internal migration 20,000 cells were seeded in assay medium in a 96-well PhenoPlatae™ (Revvity #6055302). For time-course experiments, one plate was prepared for each time point: 10, 20, 30, 60, 90, 120, 180, 240, and 1440 min. Cells were incubated overnight at 37°C and 5% CO2. The following day, cells were treated with compounds, and internalization was monitored using an Operetta CLS High Content Analysis System (Revvity). Compounds were titrated in cell seeding medium supplemented with 1% BSA (Miltenyi Biotech, #130-091-476) instead of 1% FBS. Titrations ranged from 10 to 0.0045 nM. Cell plates were incubated according to the indicated time points, briefly washed with 4% PFA in PBS (Ampliqon #AMPQ43226), and then fixed with 4% PFA in PBS for 20 minutes. Cells were washed three times with PBS and stored at 4°C until staining. For nuclear staining, 1:10,000 Hoechst 33342 (Invitrogen #H3570) in PBS was applied. After 15 minutes of treatment, cells were washed with PBS (Gibco #14040-091). Cells were captured by confocal imaging.

[0203] Confocal imaging Internalization was assessed using Operetta Harmony software. Cells were captured using a 40x water objective and the Hoechst 33342 and Alexa Fluor 647 channels. Nine fields per well were imaged and analyzed. The analysis sequence was as follows: find the nuclei using Hoechst 33342, find the cytoplasm using the nuclei to identify cells, adjust the cytoplasmic area and nuclei intensity and area to select the population, find the spots, and adjust the spot intensity and area to select the population. This analysis sequence resulted in a "relative spot intensity average per well." Further analysis was performed in GraphPad Prism 9.0.1. To characterize compound internalization, a nonlinear regression single-phase association fit was applied to the resulting data. Although compounds were tested at different concentrations, only data from a 1 nM compound concentration were used to determine half-lives and maximum values.

[0204] Cells were fixed at predetermined time points from 0 to 4 h, and fluorescence was determined for each combination of time point and concentration level.

[0205] Results showed that all three compounds were internalized by the GHR at concentrations ranging from 0.0045 to 10 nM. The internalization rate was highest for hGH, followed by similar but lower rates for compound 1a and pegvisomant. Internalization was reduced in the presence of unlabeled competitors.

[0206] Addition of unlabeled compound 1a and pegvisomant to cells with labeled hGH demonstrated a displacement effect of both compound 1a and pegvisomant on the GHR, suggesting that uptake is specific for the receptor.

[0207] The displacement effect of hGH was higher at 4 hours compared to 24 hours after addition of compound 1a and pegvisomant, and the displacement effect of somapsitan persisted for 24 hours.

[0208] conclusion Compound 1a undergoes receptor-mediated internalization by the GHR at a slower rate than human GH but a faster rate than pegvisomant. Compound 1a is more easily displaced from the GHR by pegvisomant compared to human GH.

[0209] Example 10: Synthesis of additional albumin binders Similarly, the following bromoacetamide-derived albumin binders were made using solid-phase methods similar to those described in US 8779109 B2, US 9895417 B2, or Example 1. The bromoacetamides can be used directly for conjugation to reduced free cysteine ​​residues on proteins (as described in Example 44 of US 8779109 B2 or Example 5.1 of US 9895417 B2). Alternatively, they can be converted to iodoacetates as described in Example 1.ii and used for conjugation to free cysteine ​​residues on proteins as described in Example 1.iii.

[0210] Br-Formula 6a: (8S, 31S)-1-Bromo-2,10,19,28,33-pentaoxo-12,15,21,24-tetraoxa-3,9,18,27,32-pentaazanonatetracontane-8,31,49-tricarboxylic acid [ka]

[0211] This compound was prepared on solid support using standard Fmoc-peptide chemistry on an ABI433 synthesizer. The peptide was assembled on Fmoc-Lys(Mtt)-Wang resin using Fmoc-Ado-OH, Fmoc-Glu-OtBu, and octadecanedioic acid mono-tert-butyl ester. Coupling was performed using 5-chloro-1-((dimethylamino)(dimethyliminio)methyl)-1H-benzo[d][1,2,3]triazole 3-oxide tetrafluoroborate (TCTU, 1 equivalent) and DIPEA (2 equivalents) in DMF. The Mtt group was removed by treatment with 80% 1,1,1,3,3,3-hexafluoropropan-2-ol (HPIF) in dichloromethane, and the final coupling of bromoacetic acid was performed with N,N'-diisopropylcarbodiimide (DIC). The compound was cleaved from the resin using trifluoroacetic acid in DCM (2:1). The resin was filtered off and washed with dichloromethane. The solvent was evaporated and acetonitrile was added to the residue. The white precipitate was filtered, washed with acetonitrile and diethyl ether, and dried under vacuum to give the title compound as a white powder. H NMR spectrum (300 MHz, AcOD-d, dH): 4.68 (dd, J = 8.3 and 5.0 Hz, 1H); 4.61 (dd, J = 8.9 and 4.7 Hz, 1H); 4.17 (s, 2H); 4.12 (s, 2H); 3.96 (s, 2H); 3.78-3.60 (m, 12H); 3.58-3.40 (m, 4H); 3.31 (t, J = 6.8 Hz, 2H); 2.45 (t, J = 7.3 Hz, 2H); 2.39-2.30 (m, 4H); 2.29-2.17 (m, 1H); 2.16-2.06 (m, 1H); 2.02-1.92 (m, 1H); 1.90-1.75 (m, 1H); 1.69-1.55 (m, 6H); 1.52-1.41 (m, 2H); 1.30 (s, 24H). LC-MS: Rt=2.82 min. LC-MS m / z:984.5 (M+H)+.

[0212] Br-Formula 7a: 15-{(S)-1-carboxy-3-[2-(2-{[2-(2-{[2-(2-bromoacetylamino)ethylcarbamoyl]methoxy}ethoxy)ethylcarbamoyl]methoxy}ethoxy)ethylcarbamoyl]propylcarbamoyl}pentadecanoic acid [ka]

[0213] This compound was prepared as described in Example 4.1 of US 9895417 B2.

[0214] Br-Formula 8a: 17-{(S)-1-carboxy-3-[2-(2-{[2-(2-{[2-(2-bromoacetylamino)ethylcarbamoyl]methoxy}ethoxy)ethylcarbamoyl]methoxy}ethoxy)ethylcarbamoyl]propylcarbamoyl}heptadecanoic acid [ka]

[0215] This compound was prepared as described in Example 4.5 of US 9895417 B2.

[0216] Br-Formula 9a: 19-{(S)-1-carboxy-3-[2-(2-{[2-(2-{[2-(2-bromoacetylamino)ethylcarbamoyl]methoxy}ethoxy)ethylcarbamoyl]methoxy}ethoxy)ethylcarbamoyl]propylcarbamoyl}nonadecanoic acid [ka]

[0217] This compound was prepared as described in Example 4.4 of US 9895417 B2.

[0218] Br-Chemical Formula 10a: (10S,15S,20S,25S)-1-Bromo-2,7,12,17,22,27-hexaoxo-3,6,11,16,21,26-hexaazatritetracontane-10,15,20,25,43-pentacarboxylic acid [ka]

[0219] This compound was prepared on solid support using standard Fmoc-peptide chemistry on an ABI433 synthesizer. The peptide was assembled on 2-chlorotrityl chloride resin using Fmoc-Glu-OtBu and octadecanedioic acid mono-tert-butyl ester, generally following protocol US 9895417 B2, Example 4.1. 1H NMR spectrum (300MHz, AcOD-d4, dH): 4.69-4.57 (m, 4H); 3.95 (s, 2H); 3.50-3.31 (m, 4H); 2.57-2.21 (m, 16H); 1.71-1.55 (m, 4H); 1.30 (bs, 24H). LC-MS Rt=2.45 min. LC-MS m / z: 994.0 (M+H)+.

[0220] Example 11: Synthesis and Characterization of Additional GH Receptor Antagonists (GHRAs) Compounds 3-19 were prepared essentially using the procedures outlined in Example 1 by the following general steps. i) GHRA peptide backbone expression in E. coli to obtain Cys101 cystamine protected mixed disulfide. ii) Synthesis of the iodoacetamide albumin binding moiety. iii) Deprotection of the Cys101 mixed disulfide and nucleophilic substitution with iodoacetamide.

[0221] Details of each step are shown in Example 1 using appropriate chemicals that will be apparent based on the structure of the target GHRA; alternatively, the compounds can be obtained using procedures essentially as described in WO 2011 / 089255 (see pages 132, lines 17 to 137, Example 44, page 161, line 5 to page 164, line 11). Characterization data are provided in Table 13. [Table 13]

[0222] Example 12: GH receptor affinity by surface plasmon resonance (SPR) for additional GHRAs The purpose of this example was to determine the affinity of compounds of the present invention compared to pegvisomant. SPR experiments were performed at 25°C using Biacore T200 and Biacore 8K instruments (Cytiva). Binding kinetics were determined using an Avi-tagged biotinylated version of the extracellular domain of the growth hormone receptor (hGHR-ECD), containing hGHR residues 19-264, and the Fc-conjugated dimeric hGHR-ECD-Fc construct.

[0223] For biotinylated hGHR-ECD, we used the BiotinCAPture kit (Cytiva, 28920233), which allows reversible capture of biotinylated ligands. The chip surface was prepared by rehydrating it in HBS-EP buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v / v Surfactant P20) in the instrument using standby flow. Prior to the experiment, the chip surface was conditioned using regeneration solution (3 parts regeneration stock 1 (8 M guanidine-HCl) and 1 part regeneration stock 2 (1 M NaOH)), followed by three consecutive initiation cycles using HBS-EP buffer as the analyte. Each cycle consisted of the following steps: The following procedures were performed: 1) 50 μg / mL CAPture reagent (included in the Biotion CAPture kit) in HBS-EP buffer (flow rate: 2 μl / min, contact time: 300 s), 2) biotinylated ligand capture (flow rate: 10 μl / min, contact time: 180 s), 3) analyte (compound 1a, compound 2, somatropin, and pegvisomant) injection (flow rate: 30 μl / min, contact time: 240 s, dissociation time: 1200 s), and 4) regeneration using regeneration solution (flow rate: 10 μl / min, contact time: 60 s). All analytes were diluted in HBS-EP buffer and applied to the ligand-coated chip at concentrations of 100, 20, 4, 0.8, 0.16, 0.032, and 0 nM.

[0224] For dimeric hGHR-ECD-Fc, SensorChip Protein A (Cytiva, 29127555) was used. The chip surface was prepared by rehydration in HBS-EP buffer in the instrument using standby flow. Prior to the experiment, the chip surface was conditioned using regeneration solution 2 (10 mM glycine-HCl pH 1.5, Cytiva, BR100354), followed by three consecutive initiation cycles using buffer as the analyte. Each cycle consisted of the following steps: 1) ligand capture (flow rate: 10 μl / min, contact time: 30 s); 2) analyte injection (flow rate: 50 μl / min, contact time: 240 s, dissociation time: 1200 s); and 3) regeneration using regeneration solution 2 (flow rate: 30 μl / min, contact time: 30 s). All analytes were diluted in HBS-EP buffer and applied to the ligand-coated chip at concentrations of 100, 20, 4, 0.8, 0.16, 0.032, and 0 nM.

[0225] All dilutions and experiments were repeated in HBS-EP buffer containing 1% bovine serum albumin (BSA, Sigma-Aldrich: A4503).

[0226] Data analysis was performed using Biacore 8K evaluation software. Kinetic fitting was performed after baseline subtraction, and curves were globally fitted using a 1:1 Langmuir binding model. D The value is the number of matched associations (k a ) and dissociation constant (k d The results are shown in Table 14. [Table 14] [Table 15] [Table 16] [Table 17]

[0227] As seen in Tables 14-17, both Compound 1a and Compound 2 have higher affinity for the hGHR-ECD compared to pegvisomant. The extension of the pegvisomant molecule is based on PEGylation. PEGylation negatively impacts receptor binding affinity for the growth hormone receptor, resulting in slower association and dissociation rates.

[0228] Example 13: Steady-state GHR binding affinity by isothermal titration calorimetry (ITC) for additional GHRAs The purpose of the example was to determine affinity by measuring the thermodynamic interactions between binding partners (receptor / ligand) in solution both in the presence and absence of HSA.

[0229] Compound 1a, pegvisomant, and fatty acid-free human serum albumin (HSA) (Sigma) were dialyzed overnight into 4.38 mM histidine, 2.05 mM NaCl, pH 7.1 buffer using a Slide-A-Lyzer™ dialysis cassette. All proteins were subjected to size-exclusion chromatography with multi-angle light scattering (SEC-MALS) analysis to determine concentrations before and after ascending concentration and after the ITC experiment. SEC-MALS was performed on an Agilent 1200 Series HPLC system (Agilent, Darmstadt, Germany) using a TSK G3000 SWXL column (Tosoh Bioscience, Tokyo). The column was maintained at 20°C and equilibrated in 122 mM NaHPO, 78 mM NaHPO, 300 mM NaCl, 4% 2-propanol, pH 6.8. Detection was performed by UV absorption at 280 nm, refractive index (RI), and multi-angle laser light scattering (MALS). The RI and MALS detectors were Optilab T-rEX (Wyatt) and MiniDawn Treos (LS) (Wyatt Technology, Santa Barbara, CA, USA), respectively. Data were evaluated using Chemstation B.03.01 and Astra Ver. 6.1.1.17.

[0230] ITC experiments were performed at 37°C using a PEAQ-ITC calorimeter (Malvern, UK). Sample cells (300 μL) contained 10 μM compound 1a or pegvisomant in the presence of HSA (0, 1, 5, or 10 mg / mL). The syringe contained 100 μM hGHR-ECD (ligand). A thermal equilibration step was followed by a 60-second delay, after which an initial 0.4 μL injection of hGHR-ECD was administered, followed by 25 1.5 μL injections at 120-second intervals and a final 1.1 μL injection. The stirring speed was 750 rpm, and the reference power was held constant at 9.64 μcal / s. Data processing was performed using a one-site fitting model with fitted offset correction using PEAQ-ITC analysis software (Malvern Instruments). The results are shown in Table 18. [Table 18]

[0231] N is the bonding stoichiometry and K D is the dissociation constant, ΔH is the change in enthalpy, ΔG is the change in Gibb free energy, ΔS is the change in entropy, and T is the temperature. K of compound 1a D is found to be lower than pegvisomant, and therefore compound 1a also exhibits higher affinity in the ITC assay. It can also be seen that for both compound 1a and pegvisomant, the affinity does not change significantly in the presence of HSA compared to 0 mg / mL HSA.

[0232] Example 14: Testing of GHRA in the STAT3 assay A series of hGH antagonists were tested in the antagonist mode (N=1) with and without human albumin in the medium. The data are summarized in Table 19. [Table 19]

[0233] conclusion This example demonstrates that compounds of the present disclosure, particularly compounds 1a, 3a-19a, exhibit promising GHRA properties for use in clinical practice.

[0234] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those skilled in the art. It is therefore to be understood that the appended claims are intended to cover all such modifications and changes that fall within the true spirit of the invention.

Claims

1. A long-acting growth hormone receptor antagonist, a. At least the following amino acid substitutions compared to human growth hormone (hGH) (SEQ ID NO: 1): L101C, G120R / G120K a growth hormone variant comprising: b. an albumin binding moiety configured for binding to albumin, e.g., binding to albumin with a dissociation constant (Kd) of less than 1 μM, having a molecular weight of 3 kDa or less, and covalently attached to the growth hormone variant via the sulfur residue of the cysteine ​​side chain at position 101 of the growth hormone variant; long-acting growth hormone receptor antagonists, including the growth hormone variant consists of [H18D, H21N, L101C, G120R, R167N, K168A, D171S, K172R, E174S, I179T]-hGH (SEQ ID NO: 2), and the albumin binding moiety has the formula 2: 【Chemistry 1】 or a pharmaceutically acceptable salt thereof.

2. Compound 1 (chemical formula 1): 【Chemistry 2】 2. The long-acting growth hormone receptor antagonist of claim 1, wherein

3. Compound 1a (chemical formula 1a): 【Transformation 3】 2. The long-acting growth hormone receptor antagonist of claim 1, wherein

4. A pharmaceutical composition comprising the long-acting growth hormone receptor antagonist of any one of claims 1 to 3 and a pharmaceutically acceptable excipient.

5. 5. The pharmaceutical composition of claim 4, wherein the long-acting growth hormone receptor antagonist is Compound 1 or Compound 1a.

6. A pharmaceutical comprising the long-acting growth hormone receptor antagonist according to any one of claims 1 to 3.

7. A pharmaceutical composition for use in the treatment or prevention of acromegaly or gigantism, comprising the long-acting growth hormone receptor antagonist of any one of claims 1 to 3.

8. The following amino acid modifications compared to hGH: H18D, H21N, L101C, G120R, R167N, K168A, D171S, K172R, E174S, and I179T (GHv-1); H18D, H21N, L101C, G120R, N149D, N152D, R167N, K168A, D171S, K172R, E174S, and I179T (GHv-2), or H18D, H21N, L101C, G120R, N149D, N152D, R167N, D171S, E174S, and I179T (GHv-3) 1. A vector comprising a nucleic acid encoding a human growth hormone variant comprising:

9. A host cell comprising the vector of claim 8.

10. 9. A method for preparing a human growth hormone variant, comprising culturing a host cell containing the vector of claim 8, wherein the human growth hormone variant is SEQ ID NO: 2.

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