TW201300131A - Prodrugs comprising an exendin linker conjugate - Google Patents

Abstract

The present invention relates to a prodrug or a pharmaceutically acceptable salt thereof comprising an exendin linker conjugate D-L, wherein D represents an exendin moiety; and -L is a non-biologically active linker moiety-L1 represents by formula (I), wherein the dashed line indicates the attachment to one of the amino groups of the exendin moiety by forming an amide bond. The invention further relates to pharmaceutical compositions comprising said prodrugs as well as their use as a medicament for treating or preventing diseases or disorders which can be treated by exendin.

Description

Precursor containing exendin linker conjugate

The present invention relates to an exendin prodrug, a pharmaceutical composition comprising the prodrug, and the use thereof as a medicament for treating or preventing a disease or disorder which can be treated with exenatide.

Exenatide-4 is a 39-amino acid peptide that is isolated from the salivary gland secretions of the highly toxic Heloderma suspectum. It has some sequence similarity to several members of the glucagon-like peptide family, and its highest homogeneity to glucagon-like peptide-1[7-36]-decalamine (GLP-1) is 53. %. Exenatide-4 acts as a GLP-1 agonist at the GLP-1 receptor and acts as a GLP-1 insulin secretagogue on isolated rat islets. Exenatide-4 is a high potency agonist and shortened exenatide-(9-39)-guanamine, which is a glucagon-like peptide 1-(7- on insulin-secreting β-cells). 36) - an antagonist of a guanamine receptor (see, for example, Biochemistry Journal 268(26): 19650-19655). Recently, Exenatide-4 ("exenatide") has been approved in the United States and Europe to improve blood glucose in patients with type 2 diabetes who are taking metformin and/or sulfonylurea but have not yet achieved adequate glycemic control. control.

Current treatment with exenatide requires frequent injections (twice a day), resulting in high plasma concentrations after injection, which are associated with nausea (see Nauck MA, Meier JJ (2005), Regul Pept. 128(2): 135-148), and associated with low trough concentrations, which cause incomplete glycemic control (see Kim D., et al. (2007), Diabetes Care. 30(6): 1487-1493). In order to overcome these problems, long-acting formulations of exenatide-4 are highly desirable.

Ideally, the peptide is formulated in a single type to provide a sustained plasma concentration in humans for at least one injection per week or more per week after administration to the human body. Several long-acting exenatides are proposed today.

To enhance the physicochemical or pharmacokinetic properties of the drug in the body, such as its half-life, the drugs can be combined with a carrier. If the drug is transiently bound to a carrier and/or a linker, such systems are generally referred to as prodrugs. According to the definition provided by IUPAC (as given at http://www.chem.qmul.ac.uk/iupac.medchem, accessed on July 22, 2009), a drug is a prodrug before the carrier is attached. It contains a temporary attachment of a given active substance to a transient carrier which produces improved physicochemical or pharmacokinetic characteristics and which can be easily removed in the body, usually by hydrolysis.

The linker used in the drug before the connection of the carrier may be transient, which means that it is equal to physiological conditions (aqueous buffer at pH 7.4, 37 ° C), non-enzymatic hydrolysis, degradable (cleavable), half life The period ranges from, for example, one hour to three months. A suitable carrier is a polymer and can be directly bound to a linker reagent or via a non-cleavable spacer.

The ligation reaction of a transient polymer via a tracer-free prodrug comprises an extended cycle time due to the attachment of the polymer and the original pharmacological recovery of the native peptide after release from the polymer conjugate. .

When a polymer-linker peptide conjugate is used, the native unaltered peptide is slowly released after administration to the patient, controlled by the release kinetics of the linker and the pharmacokinetics of the polymer carrier. Ideally, the release kinetics are independent of the appearance of enzymes such as protease or esterase in body fluids to ensure a consistent and uniform release pattern.

International Patent Application No. WO-A 2009/095479 refers to a drug comprising a drug linker conjugate, wherein the linker is covalently linked to a biologically active moiety, such as Exenatide, via a cleavable linkage. The biologically active moiety is released from the prodrug by a cyclic imine formation reaction by a cyclic imine formation reaction. An exenatide-prodrug is described, wherein the linker is predominantly L-alanine.

Still, there is still a need to develop long-lasting exenatide prodrugs with longer half-life. Accordingly, it is an object of the present invention to provide an exenatide prodrug having a longer half-life.

This is achieved by a prodrug or a pharmaceutically acceptable salt thereof, which comprises a covalent DL exenatide prodrug, wherein D represents an exenatide moiety; and -L is of formula (I) ) represents the non-biologically active linker moiety - L ¹ ,

Wherein, the dotted line refers to one of the amine groups attached to exenatide by forming a guanamine bond; R ¹ is selected from a C _1-4 alkyl group, preferably CH ₃ ; R ² , R ^2a a group independently selected from the group consisting of H and C _1-4 alkyl; wherein the L ¹ is substituted by one L ² -Z and optionally substituted, but the hydrogen in the formula (I) is marked with an asterisk Substituted by a substituent, and wherein L ² is a single chemical bond or a spacer; and Z is a hydrogel.

It has been found that the stereochemistry shown in formula (I) is the prodrug linker, that is, the amino acid in its D-form, and the amino acid in its L-form is the prodrug linker. Compared with the beneficial half-life. Furthermore, such prodrugs can provide exenatide for release from a subcutaneous depot in a structurally intact form for a period of at least 2 days of administration. Another advantage is that the structural integrity of the released exenatide can be provided by minimizing the intramolecular contact of the exenatide molecule by a well-hydrated polymer matrix and can be obtained by exenatide And the self-cracking prodrug linker between the polymer matrix is continuously released.

Therefore, it is possible to administer exenatide in a prior drug form of the present invention at a lower frequency than the currently used long-acting exenatide. Other advantages are the small peak-to-valley ratio, which greatly reduces the risk of negative conditions such as nausea and gastrointestinal complications. This helps the patient to reduce the frequency of injections while maintaining optimal control of the plasma concentration of exenatide and the resulting blood glucose.

The term "exenatide" refers to an exenatide agonist, an exenatide analog, an exenatide derivative, a truncated exenatide, and a shortened exenatide agonist. Agent, shortened exenatide derivative, shortened exenatide analog, elongated exenatide, elongated exenatide agonist, elongated exenatide derivative, elongated An exenatide analog, a GLP-1, a GLP-1 analog, or a GLP-1 derivative, such as a GLP-1 or GLP-1 analog that is in a guanidinated, shortened or elongated form. Preferably, the Exenatide is an Exenatide or Exenatide agonist of Sequence ID 1 to ID 21 (see below), and preferably Exendin-3 having Sequence ID 2 or Exendin-4 with sequence ID 1.

The term "elongation" refers to a peptide or protein which has an additional amino acid residue at its N-terminus or C-terminus or which has an internal insertion. The term also refers to the fusion of the peptide or protein to other peptides or proteins, such as, for example, GST protein, FLAG peptide, hexa-histamine peptide, protein binding to maltose.

Examples of exenatide agonists for use herein are Exendin-3 or Exendin-4 agonists, including but not limited to:

(i) Exendin-4 analogue and amidated exenatide-4 analog in which one or more amino acid residues have been comprised of different amine groups of the N-terminal variant Replaced by acid residues;

(ii) Shortening and elongation of Exendin-4 and shortening and elongation of the amidation;

(iii) shortened and elongated forms of shortened and elongated exenatide-4 and amiled, in which one or more amino acid residues have been replaced by different amino acid residues;

(iv) GLP-1 and glycosylated GLP-1;

(v) a GLP-1-analog and a glycosylated GLP-1 analog in which one or more amino acid residues have been replaced by a different amino acid residue comprising an N-terminal modification;

(vi) shortened and elongated forms of shortened and elongated GLP-1 and proline;

(vii) a shortened version of shortened GLP-1 and amiodalation in which one or more amino acid residues have been replaced by different amino acid residues;

(viii) Known substances AVE-0010/ZP-10/Lixisenatide (Sanofi-Aventis Zealand Pharma; sequence ID 21), BAY-73-7977 (Bayer), TH-0318, BIM-51077 ( Ipsen, Tejin, Roche), NN2211 (Novo Nordisk), LY315902.

Examples of the above Exenatide agonists may be those wherein the analog of Exendin-4 is selected from the group consisting of H-desPro ³⁶ - Exenatide-4-Lys ₆ -NH ₂ , H-des (Pro ^36,37 )-exenatide-4-Lys ₄ -NH ₂ and H-des(Pro ^36,37 )-exenatide-4-Lys ₅ -NH ₂ , or pharmacologically tolerable thereof salt.

Other examples of the above Exenatide agonists may be those wherein the analog of Exendin-4 is selected from the group consisting of desPro ³⁶ [Asp ²⁸ ] Exendin-4 (1-39), desPro ³⁶ [IsoAsp ²⁸ ] Exendin-4 (1-39), desPro ³⁶ [Met(O) ¹⁴ , Asp ²⁸ ] Exendin-4 (1-39), desPro ³⁶ [Met(O) ¹⁴ , IsoAsp ²⁸ ] Exenatide-4 (1-39), desPro ³⁶ [Trp(O ₂ ) ²⁵ , Asp ²⁸ ] Exenatide-2 (1-39), desPro ³⁶ [Trp(O ₂ ) ²⁵ , IsoAsp ²⁸ ] Exenatide-2 (1-39), desPro ³⁶ [Met(O) ¹⁴ Trp(O ₂ ) ²⁵ , Asp ²⁸ ] Exenatide-4 (1-39) and desPro ³⁶ [Met(O ¹⁴ Trp(O ₂ ) ²⁵ , IsoAsp ²⁸ ] Exenatide-4 (1-39), or a pharmacologically tolerable salt thereof.

Other examples of exenatide agonists described in the preceding sections may be those in which the peptide-Lys ₆ -NH ₂ system is linked to the Exendin-4 analog.

Other examples of the aforementioned Exenatide agonists may be those wherein the Exenatide-4 analog is selected from the group consisting of H-(Lys) ₆ -des Pro ³⁶ [Asp ²⁸ ] Exendin-4 ( 1-39)-Lys ₆ -NH ₂ des Asp ²⁸ Pro ³⁶ , Pro ³⁷ , Pro ³⁸ Exenatide-4(1-39)-NH ₂ , H-(Lys) ₆ -des Pro ³⁶ , Pro ³⁷ , Pro ³⁸ [Asp ²⁸ ] Exendin-4 (1-39)-NH ₂ , H-Asn-(Glu) ₅ des Pro ³⁶ , Pro ³⁷ , Pro ³⁸ [Asp ²⁸ ] Exenatide-4 (1 -39)-NH ₂ ,des Pro ³⁶ ,Pro ³⁷ ,Pro ³⁸ [Asp ²⁸ ] Exenatide-4(1-39)-(Lys) ₆ -NH ₂ ,H-(Lys) ₆ -des Pro ³⁶ , Pro ³⁷ , Pro ³⁸ [Asp ²⁸ ] Exenatide-4(1-39)-(Lys) ₆ -NH ₂ ,H-Asn-(Glu) ₅ -des Pro ³⁶ ,Pro ³⁷ ,Pro ³⁸ [Asp ²⁸ ] Exenatide-4(1-39)-(Lys) ₆ -NH ₂ ,H-(Lys) ₆ -des Pro ³⁶ [Trp(O ₂ ) ²⁵ ,Asp ²⁸ ]Exendin-4 1-39)-Lys ₆ -NH ₂ ,H-des Asp ²⁸ Pro ³⁶ ,Pro ³⁷ ,Pro ³⁸ [Trp(O ₂ ) ²⁵ ]Exendin-4(1-39)-NH ₂ ,H-( Lys) ₆ -des Pro ³⁶ , Pro ³⁷ , Pro ³⁸ [Trp(O ₂ ) ²⁵ , Asp ²⁸ ] Exenatide-4(1-39)-NH ₂ ,H-Asn-(Glu) ₅ -des Pro ³⁶ , Pro ³⁷ , Pro ³⁸ [Trp(O ₂ ) ²⁵ , Asp ²⁸ ] Exenatide-4(1-39)-NH ₂ ,des Pro ³⁶ ,Pro ³⁷ ,Pro ³⁸ [Trp(O ₂ ) ²⁵ ,Asp ²⁸ ]Exendin-4(1-39)-(Lys) ₆ -NH ₂ ,H-(Lys) ₆ -des Pro ³⁶ ,Pro ³⁷ ,Pro ³⁸ [Trp(O ₂ ) ²⁵ , Asp ²⁸ ] Exenatide-4(1-39)-(Lys) ₆ -NH ₂ ,H-Asn-(Glu) ₅ -des Pro ³⁶ ,Pro ³⁷ ,Pro ³⁸ [Trp(O ₂ ) ²⁵ , Asp ²⁸ ] Exenatide-4(1-39)-(Lys) ₆ -NH ₂ ,H-(Lys) ₆ -des Pro ³⁶ [Met(O) ¹⁴ ,Asp ²⁸ Exenatide-4(1-39)-Lys ₆ -NH ₂ , des Met(O) ¹⁴ Asp ²⁸ Pro ³⁶ , Pro ³⁷ , Pro ³⁸ Exenatide-4(1-39)-NH ₂ , H-(Lys)6-des Pro ³⁶ , Pro ³⁷ , Pro ³⁸ [Met(O) ¹⁴ , Asp ²⁸ ] Exenatide-4(1-39)-NH ₂ ,H-Asn-(Glu) ₅ - Des Pro ³⁶ , Pro ³⁷ , Pro ³⁸ [Met(O) ¹⁴ , Asp ²⁸ ] Exenatide-4(1-39)-NH ₂ , des Pro ³⁶ , Pro ³⁷ , Pro ³⁸ [Met(O) ¹⁴ , Asp ²⁸ ] Exenatide-4(1-39)-(Lys) ₆ -NH ₂ ,H-(Lys) ₆ -Pro ³⁶ ,Pro ³⁷ ,Pro ³⁸ [Met(O) ¹⁴ ,Asp ²⁸ ]Esser That peptide-4(1-39)-Lys ₆ -NH ₂ , H-Asn-(Glu) ₅ des Pro ³⁶ , Pro ³⁷ , Pro ³⁸ [Met(O) ¹⁴ , Asp ²⁸ ] Exendin-4 1-39)-(Lys) ₆ -NH ₂ ,H-(Lys) ₆ - des Pro ³⁶ [Met(O) ¹⁴ ,Trp(O ₂ ) ²⁵ ,Asp ²⁸ ]Exendin-4(1-39 )-Lys ₆ -NH ₂ ,des Asp ²⁸ Pro ³⁶ , Pro ³⁷ , Pro ³⁸ [Met(O) ¹⁴ , Trp(O ₂ ) ²⁵ ] Exenatide-4(1-39)-NH ₂ ,H-(Lys) ₆ -des Pro ³⁶ ,Pro ³⁷ ,Pro ³⁸ [Met(O) ¹⁴ , Trp(O ₂ ) ²⁵ , Asp ²⁸ ] Exenatide-4(1-39)-NH ₂ , H-Asn-(Glu) ₅ -des Pro ³⁶ , Pro ³⁷ , Pro ³⁸ [Met(O) ¹⁴ , Asp ²⁸ ] Exenatide-4(1-39)-NH ₂ , des Pro ³⁶ , Pro ³⁷ , Pro ³⁸ [Met(O) ¹⁴ , Trp(O ₂ ) ²⁵ , Asp ²⁸ Exenatide-4(1-39)-(Lys) ₆ -NH ₂ ,H-(Lys) ₆ -des Pro ³⁶ ,Pro ³⁷ ,Pro ³⁸ [Met(O) ¹⁴ ,Trp(O ₂ ) ²⁵ , Asp ²⁸ ] Exenatide-4(1-39)-(Lys) ₆ -NH ₂ ,H-Asn-(Glu) ₅ -des Pro ³⁶ ,Pro ³⁷ ,Pro ³⁸ [Met(O) ¹⁴ ,Trp (O ₂ ) ²⁵ , Asp ²⁸ ] Exenatide-4(1-39)-(Lys) ₆ -NH ₂ , or a pharmacologically tolerable salt thereof.

Another example of an Exenatide agonist as described above is Arg ³⁴ , Lys ²⁶ (N ^ε (γ-Valretinyl (N ^α -hexamethylene))) GLP-1 (7-37) [Luraglutide ( Liraglutide)] or a pharmacologically tolerable salt thereof.

The Exenatide agonist mimics the activity of Exendin-3 or Exendin-4 by binding to Essinide-3 or Exendin-4 to facilitate its use as an insulinotropic agent And by acting as a receptor for the treatment of diabetes, or by mimicking the effect of exenatide in the urine process, slowing the stomach air, inducing satiety, increasing urinary sodium excretion, and/or reducing the concentration of potassium in the urine. Binding to receptors where exenatide causes these effects.

In one embodiment, the Exenatide or Exenatide agonist having Sequence ID No: 1-22 can be used to prepare the long-acting polymeric conjugate of the present invention:

[Seq ID No: 1] Exenatide-4

HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG

PSSGAPPPS-NH2

[Seq ID No: 2] Exenatide-3

HSDGTFTSDL SKQMEEEAVR LFIEWLKNGG

PSSGAPPPS-NH2

[Seq ID No: 3]

HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG P

[Seq ID No: 4]

HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG Y

[Seq ID No: 5]

HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG

[Seq ID No: 6]

HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG-NH2

[Seq ID No: 7]

HGEGTFTSDL SKQMEEEAVR LFIEWLKN-NH2

[Seq ID No: 8]

HGEGTFTSDL SKQLEEEAVR LFIEFLKNGG

PSSGAPPPS-NH2

[Seq ID No: 9]

HGEGTFTSDL SKQLEEEAVR LFIEFLKN-NH2

[Seq ID No: 10]

HGEGTFTSDL SKQLEEEAVR LAIEFLKN-NH2

[Seq ID No: 11]

HGEGTFTSDL SKQLEEEAVR LFIEWLKNGG

PSSGAPPPS-NH2

[Seq ID No: 12]

HGDGTFTSDL SKQMEEEAVR LFIEWLKNGG

PSSGAPPPS-NH2

[Seq ID No 13] GLP-1 (7-36) guanamine

HAEGTFTSDV SSYLEGQAAK EFIAWLVKGR-NH2

[Seq ID No 14]

HSEGTFTSDV SSYLEGQAAK EFIAWLVKGR-NH2

[Seq ID No 15] GLP-1 (7-37)

HAEGTFTSDV SSYLEGQAAK EFIAWLVKGRG

[Seq ID No 16]

HAXaaGTFTSDV SSYLEGQAAK EFIAWLVKGR-NH2

Xaa=P, F, Y

[Seq ID No 17]

HXaaEGTFTSDV SSYLEGQAAK EFIAWLVKGR-NH2

Xaa=T, a-aminobutyric acid, D-Ala, V, Gly

[Seq ID No 18]

HaEGTFTSDV SSYLEGQAAK EFIAWLVKGG

[Seq ID No 19]

R-HAEGTFTSDV SSYLEGQAAK EFIAWLVKGR-NH2

R = ethyl sulfhydryl, pyroglutanyl, N-2-hydroxyphenyl fluorenyl, N-trans-3-hexenyl fluorenyl

[Seq ID No 20]

HXaaAEGTFTSDV SSYLEGQAAK EFIAWLVKGR-NH2

Xaa = 6-amino-hexane.

[Seq ID No 21] AVE-0010/ZP-10/Lisila (lixisenatide)

HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPSKKKKKK-NH2

[Seq ID No 22] Arg ³⁴ , Lys ²⁶ (N ^ε (γ-glutamyl (N ^α - hexamethylene)))

GLP-1 (7-37) [liraglutide]

HAEGTFTSDV SSYLEGQAAXaaEFIAWLVRGRG

Xaa=Lys(N ^ε (γ-glutamyl (N ^α -hexamethylene)))

Preferably, the Exenatide has sequence ID 1, ID 13, ID 15, ID 21 or ID 22.

More preferably, the Exenatide has the sequence ID 1, ID 13, or ID 21.

In one embodiment, the Exenatide is Exendin-4 having the sequence ID 1.

In another embodiment, the Exenatide is an analog having the sequence ID 13.

In another embodiment, the Exenatide is an analog having the sequence ID21.

The Exenatide and Exenatide agonist derivatives of the present invention will have any and all of the activity of the original unmodified molecule, but with a prolonged duration of action.

Exenatide linked to a non-biologically active linker is referred to as the "exenatide moiety".

"Non-bioactive linker" refers to a linker that does not exhibit the pharmacological efficacy of a drug derived from a bioactive agent.

"Protecting group" refers to a portion of its temporary chemical group that protects one molecule in a synthetic process and is chemoselective in a random chemical reaction. The protecting group for an alcohol is, for example, a benzyl group and a trityl group, and the protecting group of the amine is, for example, a tert-butoxycarbonyl group, a 9-fluorenylmethyloxycarbonyl group, and a benzyl group and a thiol group. Examples of protecting groups are 2,4,6-trimethoxybenzyl, phenylthiomethyl, acetamidomethyl, p-methoxybenzyloxycarbonyl, tert-butylthio , triphenylmethyl, 3-nitro-2-pyridylthio, 4-methyltrityl.

"Protected functional group" refers to a chemical functional group protected by a protecting group.

"Deuteration agent" means a moiety having the structure R-(C=O)- which provides a mercapto group in a deuteration reaction, optionally attached to a release group such as a mercapto chloride, N- Hydroxy amber imine, pentafluorophenol and p-nitrophenol.

"Alkyl" means a straight or branched carbon chain. Each hydrogen of the alkyl carbon may be replaced by a substituent.

"Aryl" means any substituent derived from a monocyclic or polycyclic or fused aromatic ring, including heterocycles such as phenyl, thiophene, fluorenyl, naphthyl, pyridyl, which may be optionally further Was replaced.

"Amidino" refers to a chemical functional group of the formula R-(C=O)- wherein R is alkyl or aryl.

"C1-4 alkyl" means an alkyl chain having from 1 to 4 carbon atoms, for example, as found at the end of a molecule: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl a base, a second butyl group, a tert-butyl group, or such as -CH ₂ -, -CH ₂ -CH ₂ -, -CH(CH ₃ )-, -CH ₂ -CH ₂ -CH ₂ -, -CH (C ₂ H ₅ )-, -C(CH ₃ ) ₂ -, when two moieties of the molecule are linked by an alkyl group. Each hydrogen of the C _1-4 alkyl carbon may be replaced by a substituent.

"C _1-6 alkyl" means an alkyl chain having from 1 to 6 carbon atoms, for example, as found at the end of a molecule: C _1-4 alkyl, methyl, ethyl, n-propyl, isopropyl Base, n-butyl, isobutyl, second-butyl; tert-butyl, n-pentyl, n-hexyl, or as -CH ₂ -, -CH ₂ -CH ₂ -, -CH(CH ₃ ) -, -CH ₂ -CH ₂ -CH ₂ -, -CH(C ₂ H ₅ )-, -C(CH ₃ ) ₂ -, when two moieties of the molecule are linked by an alkyl group. Each hydrogen of the C _1-6 alkyl carbon may be replaced by a substituent.

Thus, "C _1-18 alkyl" refers to an alkyl chain having from 1 to 18 carbon atoms and "C _8-18 alkyl" refers to an alkyl chain having from 8 to 18 carbon atoms. Thus, "C _1-50 alkyl" refers to an alkyl chain having from 1 to 50 carbon atoms.

"C _2-50 alkenyl" means a branched or unbranched alkenyl chain having 2 to 50 carbon atoms, for example, as found at the end of a molecule: -CH=CH ₂ , -CH=CH-CH ₃ ,- CH ₂ -CH=CH ₂ , -CH=CH-CH ₂ -CH ₃ , -CH=CH-CH=CH ₂ , or as -CH=CH-, when the two parts of the molecule are based on alkenyl Group connection. Each hydrogen of the C _2-50 alkenyl group can be replaced by a further illustrated substituent. Thus, the term "alkenyl" relates to a carbon chain containing at least one carbon-carbon double bond. Any one of them can have one or more 叁 keys.

"C _2-50 alkynyl" means a branched or unbranched alkynyl chain having 2 to 50 carbon atoms, for example, as found at the end of a molecule: -C≡CH, -CH ₂ -C≡CH, CH ₂ -CH ₂ -C≡CH, CH ₂ -C≡C-CH ₃ , or such as -C≡C-, when two parts of the molecule are linked by an alkynyl group. Each hydrogen of the C _2-50 alkynyl group can be replaced by a further illustrated substituent. Thus, the term "alkynyl" relates to a carbon chain containing at least one carbon-carbon triple bond. Any one of them can have one or more double keys.

"C _3-7 cycloalkyl" or "C _3-7 cycloalkyl ring" means a cycloalkyl chain having from 3 to 7 carbon atoms which may have at least partially saturated carbon-carbon double bonds, such as Cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl. Each hydrogen of the cycloalkyl carbon may be replaced by a substituent. The term "C _3-7 cycloalkyl" or "C _3-7 cycloalkyl ring" also includes bridged bicyclic rings such as norbornane or norbornene. Thus, "C _3-5 cycloalkyl" refers to a cycloalkyl group having from 3 to 5 carbon atoms.

Thus, "C _3-10 cycloalkyl" refers to a cycloalkyl group having from 3 to 10 carbon atoms, such as C _3-7 cycloalkyl; cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclo Hexenyl, cycloheptyl, cyclooctyl, cyclodecyl, cyclodecyl. The term "C _3-10 cycloalkyl" also includes at least partially saturated carbon mono- and - bicyclic rings.

"Halogen" means fluoro, chloro, bromo or iodo. Generally preferred halogens are fluorine or chlorine.

"4 to 7 membered heterocyclic group" or "4 to 7 membered heterocyclic ring" means a ring having 4, 5, 6 or 7 ring atoms which may contain up to the maximum number of double bonds (aromatic or non-aromatic) a family ring which is fully, partially or unsaturated) wherein at least one ring atom has up to four ring atoms selected from the group consisting of sulfur (including -S(O)-, -S(O)2-), The heteroatoms of oxygen and nitrogen (including =N(O)-) are replaced and wherein the ring is attached to the remainder of the molecule via a carbon or nitrogen atom. An example of a 4- to 7-membered heterocyclic ring is azetidin, Tantan, thitan, furan, thiophene, pyrrole, pyrroline, imidazole, imidazoline, pyrazole, pyrazoline, Azole, Oxazoline Azole Oxazoline, thiazole, thiazoline, isothiazole, isothiazoline, thiadiazole, thiadiazoline, tetrahydrofuran, tetrahydrothiophene, pyrrolidine, imidazoline, pyrazole, Azole, different Azole, thiazolidine, isothiazolidine, thiadiazolidine, alkane, pyran, dihydropyran, tetrahydropyran, imidazolidinium, pyridine, hydrazine , pyr , pyrimidine, hexahydropyridyl , hexahydropyridine, morpholine, tetrazole, triazole, triazole, tetrazolidine, diazepane, aza High hexahydropyridyl .

"9 to 11 member heterobicyclic group" or "9 to 11 member heterocyclic ring" means a heterocyclic ring system having 9 to 11 ring atoms, wherein at least one ring atom system shares two rings and it may contain up to the maximum a number of double bonds (aromatic or non-aromatic rings, which are fully, partially or unsaturated) wherein at least one ring atom and up to six ring atoms are selected from the group consisting of sulfur (including -S(O)-, -S(O)2-), replaced by a heteroatom of oxygen and nitrogen (including =N(O)-) and wherein the ring is attached to the remainder of the molecule via a carbon or nitrogen atom. Examples of 9 to 11 membered heterocyclic rings are anthracene, porphyrin, benzofuran, benzothiophene, benzo Azole Azole, benzothiazole, benzisothiazole, benzimidazole, benzimidazoline, quinoline, quinazoline, dihydroquinazoline, quinoline, dihydroquinoline, tetrahydroquinoline, decahydroquinoline , isoquinoline, decahydroisoquinoline, tetrahydroisoquinoline, dihydroisoquinoline, benzazepine , porphyrin or pteridine. The term 9 to 11 member heterocyclic ring also includes the structure of a bicyclic snail such as 1,4-two. -8-Azaspiro[4.5]decane or a bridged heterocyclic group such as 8-aza-bicyclo[3.2.1]octane.

Where the exenatide prodrug containing a compound according to formula (I) contains one or more acidic or basic groups, the invention also includes the corresponding pharmaceutical or toxicologically pharmacologically acceptable Salts, especially for their pharmaceutically acceptable salts. Accordingly, the exenatide prodrug comprising a compound of formula (I), which contains an acidic group, can be used according to the invention, for example, as an alkali metal salt, an alkaline earth metal salt or as an ammonium salt. More precise examples of such salts include sodium salts, potassium salts, calcium salts, magnesium salts or salts with ammonia or organic amines such as, for example, ethylamine, ethanolamine, triethanolamine or amino acids. An Exendin prodrug comprising a compound of formula (I) which contains one or more basic groups, ie a group which can be protonated, which can be added to an inorganic or organic acid according to the invention The type of salt is presented or used. Examples of suitable acids include hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalene disulfonic acid, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formic acid, Propionic acid, pivalic acid, diethyl acetate, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid, aminosulfonic acid, phenylpropionic acid, gluconic acid, ascorbic acid, Isonicotinic acid, citric acid, adipic acid, and other acids known to those skilled in the art. If the Exenatide prodrug comprising a compound of formula (I) contains both acidic and basic groups in the molecule, the invention also includes, in addition to the salt forms already mentioned, internal salts or betaines (zwitterions). According to conventional methods well known to those skilled in the art, the individual salts of the exenatide prodrugs comprising formula (I) can be obtained, for example, by combining them with an organic or inorganic acid or base. Contact in a solvent or dispersant, or by anion exchange or cation exchange with other salts. The invention also includes all salts of exenatide prodrugs comprising a compound of formula (I) which, due to its low physiological inclusiveness, are not suitable for direct use in pharmaceuticals and are useful, for example, as chemically reactive or pharmaceutically acceptable An intermediate in the preparation of a salt.

In order to enhance the physicochemical or pharmacokinetic properties of drugs, such as exenatide, in the body, such drugs can be conjugated to the carrier. If the drug is transiently linked to a carrier and/or linker, such systems are generally referred to as prodrugs. According to the definition provided by IUPAC (as provided by http://www.chem.qmul.ac.uk/iupac. medchem, accessed in July 22, 2009), the drug is attached to the carrier containing a given transient carrier The temporary attachment of the active substance of the group to the drug, which produces improved physicochemical or pharmacokinetic characteristics and can be easily removed in vivo, usually by hydrolysis.

The linker transient used in the drug prior to the ligation of the vector means that it is non-enzymatically hydrolytically degradable (cleavable) under physiological conditions (aqueous buffer at pH 7.4, 37 ° C). Its half-life range is, for example, 1 hour to 3 months.

The hydrogel carrier can be directly joined to the linker L or via a spacer, preferably a non-cleavable spacer. The term "exenatide hydrogel prodrug" refers to a prodrug of exenatide to a carrier, wherein the carrier is a hydrogel. The terms "hydrogel prodrug" and "pre-hydrogel attached" mean that the bioactive agent is immediately attached to the hydrogel and is synonymous in use.

"Hydrogel" can be defined as a stereo, hydrophilic or amphoteric polymeric network capable of carrying large amounts of water. The network includes homopolymers or copolymers that are insoluble due to the presence of covalent chemical or physical (ionic, hydrophobic interaction, entanglement) crosslinks. This cross-linking provides network structure and physical integrity. Hydrogels have thermodynamic compatibility with water which allows them to swell in an aqueous medium. The chains of the network are connected in such a way that there are holes and a substantial portion of the holes have a diameter between 1 nm and 1000 nm.

By "free form" of a drug is meant a drug, especially exenatide, in its unmodified, pharmacologically active form, such as after release from a polymeric conjugate.

It should be understood that the pharmacologically active form of exenatide also includes oxidative and deamidated drugs.

"Pharmaceutical", "biologically active molecule", "biologically active moiety", "biologically active agent", "active agent" are used synonymously and refer to exenatide, either as their bound form or as a free form. .

By "therapeutically effective amount" of exenatide herein is meant an amount sufficient to treat, alleviate or partially arrest the clinical manifestations and complications of a given disease. A quantity sufficient to accomplish this is defined as a "therapeutically effective amount." The effective amount for each purpose will depend on the severity of the disease or injury and the weight and general condition of the individual. It will be appreciated that routine dose testing can be performed by determining the appropriate dosage, by constructing a useful matrix and testing at different points in the matrix, all within the general skill of a trained physician.

"Stable" and "stability" means that the hydrogel conjugate is still joined and not hydrolyzed to a significant extent during the storage time mentioned and that exenatide exhibits an acceptable impure condition. When considered stable, the composition contains less than 5% of the drug in its free form.

The term "pharmaceutically acceptable" means approved by a regulatory agency such as EMEA (Europe) and/or FDA (United States) and/or any other national regulatory agency for animals, preferably humans.

"Pharmaceutical composition" or "composition" means one or more active constituents, and one or more inert constituents, as well as any product, either directly or indirectly, comprising a combination of any two or more constituents. The substance, complex or polymer is produced, or produced by the dissociation of one or more constituents, or by other forms of reaction or interaction of one or more constituents. Accordingly, the pharmaceutical composition of the present invention comprises any composition prepared by blending a compound of the present invention with a pharmaceutically acceptable excipient (pharmaceutically acceptable carrier).

By "dry composition" is meant that the exenatide hydrogel prodrug composition is in a dry form in a container. Suitable methods for drying are spray drying and freeze drying (lyophilization). The dry composition of such exenatide hydrogel prodrugs has a residual water content of up to 10%, preferably less than 5% and more preferably less than 2% (as determined by Karl Fischer). A preferred drying method is freeze drying. "Freeze-dried composition" means that the exenatide hydrogel polymer prodrug composition is first frozen and then dewatered by decompression. This term does not exclude other drying steps that occur during the manufacturing process prior to filling the composition into the final container.

"Freeze-drying" (freeze-drying) is a dehydration process characterized by freezing the composition and then reducing the surrounding pressure and, optionally, heating to sublimate the chilled water in the composition from the solid phase to the gas phase. Typically, the sublimated water is collected by desublimation.

"Recombinant" refers to a form in which a liquid is added to a dry composition to make it a liquid or suspension composition. It should be understood that the term "recombinant" is not limited to the addition of water, but refers to the addition of any liquid, including, for example, buffers or other aqueous solutions.

"Recombinant solution" refers to a liquid used to reconstitute the dry composition of the exenatide hydrogel prodrug prior to administration to a patient in need thereof.

By "container" is meant any container in which the Exenatide hydrogel prodrug composition is contained and can be stored until reconstitution.

"Buffer" or "buffer" refers to a compound that maintains the pH in the desired range. Physiologically tolerated buffers are, for example, sodium phosphate, succinate, histidine, bicarbonate, citrate and acetate, pyruvate. An antacid such as Mg(OH) ₂ or ZnCO ₃ can also be used. The cushioning force can be adjusted to meet the conditions most sensitive to pH stability.

"Excipient" means a compound that is administered with a therapeutic agent, for example, a buffer, an isotonicity adjusting agent, a preservative, a stabilizer, an anti-adsorbent, an oxidative protectant, or other adjuvant. However, in some cases, one excipient may have two or three functions.

A "cryoprotectant" is a molecule that, when combined with a protein of interest, significantly avoids or reduces the chemical and/or physical instability of the protein, usually during drying and especially during freeze-drying and then upon storage. Examples of the cryoprotectant include sugars such as sucrose or trehalose; amino acids such as arginine, glycine, glutamic acid or histidine; methylamines such as betaines; readily soluble salts such as magnesium sulfate; Alcohols such as trivalent or higher sugar alcohols such as glycerol, erythritol, glycerol, arabitol, xylitol, sorbitol, and mannitol; ethylene glycol; propylene glycol; polyethylene glycol ; pluronics; hydroxyalkyl starches, such as hydroxyethyl starch (HES), and combinations thereof.

"Surfactant" means a humectant that reduces the surface tension of a liquid.

"Iteromicity modifier" refers to a compound that minimizes the pain caused by damage to cells that differ in the osmotic pressure of the injected reservoir.

The term "stabilizer" refers to a compound used to stabilize a polymer prodrug. Stabilization is achieved by strengthening the force of stabilizing the protein, by destabilizing the denatured state, or by directly bonding the excipient to the protein.

"Anti-adsorbent" refers primarily to surfactants or other proteins or soluble polymers that are ionic or nonionic to be fully coated or adsorbed onto the inner surface of the container of the composition. The concentration and type of excipient selected will depend on the effect to be avoided, but typically forms a single layer of surfactant at the interface directly above the CMC value.

"Oxidation protectant" means an antioxidant such as ascorbic acid, tetrahydropyrimidine, glutathione, methionine, monothioglycerol, morin, polyethyleneimine (PEI), gallnuts Acid propyl ester, vitamin E, chelating agents such as citric acid, EDTA, hexaphosphate, thioglycolic acid.

"Antimicrobial" means a chemical substance that kills or inhibits the growth of microorganisms such as bacteria, molds, yeasts, protozoa and/or viruses.

By "sealing a container" is meant that the container is closed in such a manner that it is airtight such that there is no gas exchange between the outside and the inside and the contents are kept sterile.

The term "reagent" or "precursor" means an intermediate or starting material used in the manufacture of a prodrug of the invention.

The term "chemically functional group" means a carboxylic acid and a reactive derivative, an amine group, a maleimide, a thiol and a derivative, a sulfonic acid and a derivative, a carbonate and a derivative, an amine formate and a derivative thereof. , hydroxy, aldehyde, ketone, oxime, isocyanate, isothiocyanate, phosphoric acid and derivatives, phosphonic acid and derivatives, haloacetamidine, halogenated hydrocarbon, propylene oxime and other -β unsubstituted Michael acceptor, arylating agent such as aryl fluoride, hydroxylamine, disulfide such as pyridine disulfide, vinyl fluorene, vinyl ketone, diazo alkane, diazo acetyl compound, ethylene oxide , and aziridine.

If a chemically functional group is coupled to another chemically functional group, the resulting chemical structure is referred to as a "linkage." For example, the reaction of an amine group with a carboxyl group produces a guanamine linkage.

A "reactive functional group" is a chemically functional group of the backbone portion that is attached to the hyperbranched moiety.

A "functional group" is a collective term of "reactive functional group", "degradable cross-linking functional group", or "join functional group".

A "degradable, interconnected functional group" is a linkage that includes a biodegradable linkage that is considered to be linked to one of the spacer portions of the linking backbone and is considered Link to the cross-linking section. The terms "degradable cross-linking functional group", "biodegradable cross-linking functional group", "cross-linked biodegradable functional group" and "crosslinking functional group" are used synonymously.

The terms "blocking group" or "blocking group" are used synonymously and refer to a moiety that is irreversibly linked to a reactive functional group such that it cannot react with, for example, a chemical functional group.

The term "protecting group" or "protective group" refers to a moiety that is irreversibly linked to a reactive functional group such that it cannot react with, for example, a chemical functional group.

The term "crosslinkable functional group" refers to a chemical functional group that participates in the polymerization of a group and is part of a crosslinking reagent or a backbone reagent.

The term "polymerizable functional group" refers to a chemical functional group that participates in the polymerization of the bonding-type and is part of the crosslinking reagent and the backbone reagent.

A trunk portion can include a spacer portion joined to the stem portion at one end and to the cross-link portion at the other end.

The term "derivative" refers to a chemically functional group, suitably substituted with a protecting group and/or an activating group, or an activated version of a corresponding chemical functional group, which is known to those skilled in the art. . For example, the activation pattern of a carboxyl group includes, but is not limited to, an active ester such as amber sulphate, benzotriazole, nitrophenyl ester, pentafluorophenyl ester, azabenzotriazole, hydrazine A halogen, a blend or a symmetric anhydride, a mercapto imidazole.

The term "non-enzymatically cleavable linker" refers to a hydrolytically degradable linker that is not enzymatically active under physiological conditions.

"Non-bioactive linker" refers to a linker that does not exhibit the pharmacological action of a drug (D-H) derived from a biologically active moiety.

The terms "spacer", "spacer group", "spacer molecule", and "spacer portion" are used interchangeably and are used to describe the part of the hydrogel carrier of the present invention. In connection with any part of two moieties, such as a C _1-50 alkyl group, a C _2-50 alkenyl group or a C _2-50 alkynyl group, the fragment is optionally inserted by one or more groups selected from the group consisting of: -NH-,-N(C _1-4 alkyl)-, -O-, -S-, -C(O)-, -C(O)NH-, -C(O)N(C _1-4 Alkyl)-, -OC(O)-, -S(O)-, -S(O) ₂ -, 4 to 7 membered heterocyclic group, phenyl or naphthyl.

The term "terminal", "end point" or "end" refers to the position of a functional group or linkage in a molecule or moiety, wherein such a functional group can be a chemically functional group and the linkage can be degradable Or permanent connection, characterized by being located adjacent or within the connection between the two parts or at the end of the oligomeric or polymeric chain.

The phrase "in a restraint pattern" or "moiety" refers to a substructure that is part of a larger molecule. The phrase "in a restrained version" is used to simplify the reference to a portion by naming or listing reagents, the starting material or the hypothetical starting material being well known in the art, and thus "in a restrained version" means, for example, a Or a plurality of hydrogen groups (-H), or one or more activating or protecting groups are present in the reagent or the starting material is not present in the moiety.

It should be understood that all reagents and moieties comprising polymeric moieties refer to macromolecular entities known to have variations in molecular weight, chain length or degree of polymerization, or number of functional groups. Therefore, the structures shown in the main reagent, the backbone portion, the crosslinking reagent, and the crosslinking agent portion are merely representative examples.

The reagent or part can be linear or branched. If the reagent or moiety has two terminal groups, it is referred to as a linear reagent or moiety. If the reagent or moiety has more than two terminal groups, it is considered to be a branched or complex-functional reagent or moiety.

The term "poly(ethylene glycol)-based polymeric chain" or "PEG-based chain" refers to a molecular chain of an oligo- or polymer.

Preferably, the poly(ethylene glycol)-based polymeric chain is attached to the branching core, which is a linear poly(ethylene glycol) chain, one of which is connected to the branching core and the other is connected to the overrunning Branching. It will be appreciated that the PEG-based chain can be terminated or inserted by an alkyl or aryl group optionally substituted with a hetero atom and a chemical functional group.

If the term "poly(ethylene glycol)-based polymeric chain" is used in relation to a crosslinking agent, it means a crosslinking moiety or chain comprising at least 20% by weight of an ethylene glycol moiety.

The invention is described in more detail in the following sections.

The present invention relates to a prodrug or a pharmaceutically acceptable salt thereof, which comprises exenatide linker conjugate DL, wherein D represents an exenatide moiety; and -L is represented by formula (I) Non-biologically active linker moiety - L ¹ ,

Wherein, the dotted line refers to one of the amine groups attached to exenatide by forming a guanamine bond; R ¹ is selected from a C _1-4 alkyl group, preferably CH ₃ ; R ² , R ^2a Is independently selected from the group consisting of H and C _1-4 alkyl groups; wherein L ¹ is substituted by one L ² -Z and optionally substituted, but in the formula (I), it is marked with an asterisk Hydrogen is not replaced by a substituent and wherein L ² is a single chemical bond or a spacer; and Z is a hydrogel.

Preferably, in formula (I), R ² is replaced by L ² -Z.

Preferably, in the formula (I), R ¹ is CH ₂ -L ² -Z.

Preferably, L ^{1 is} not replaced.

Preferably by the exenatide or part of the system is connected to L ¹ via the N- terminal part of Exenatide nitrogen via nitrogen from lysine chains. Most preferred, the portion of exenatide L ¹ is connected to the system via the N- terminal nitrogen.

Preferred prodrugs of the invention comprise exenatide linker conjugate D-L, wherein L is represented by formula (Ia) or (Ib):

Wherein D, R ¹ , R ² , R ^2a , L ² , Z have the definitions and preferred definitions herein and wherein the L is optionally substituted, but is marked with an asterisk in formula (Ia) or (Ib) the hydrogen substituents replaced by a unsubstituted, but is preferably not further substituted with L (L ² -Z group except for the substitution has been shown in (Ia) and (Ib) by the setting).

As shown by the formula (Ia) or (Ib), one of the hydrogens of L ¹ in the formula (I) is substituted by the group L ² -Z.

Typically, L ² can be attached to L ¹ at any position in formula (I) except for the substitution of an asterisk-labeled hydrogen. Preferably, ^one of the hydrogens provided by R ¹ , R ² , R ^2a is directly or a hydrogen of a C _1-4 alkyl group or other group is replaced by L ² -Z.

Further, L ¹ in the formula (I) can be optionally substituted. Generally, any substituent can be used as long as the cleavage is not affected. However, it is preferred that L ^{1 is} not further substituted.

Preferably, one or more other optional substituents are independently selected from the group consisting of: halogen; CN; COOR ⁹ ; OR ⁹ ; C(O)R ⁹ ; C(O)N(R ⁹ R ^9a ) ;S(O) ₂ N(R ⁹ R ^9a ); S(O)N(R ⁹ R ^9a ); S(O) ₂ R ⁹ ;S(O)R ⁹ ;N(R ⁹ )S(O) ₂ N(R ^9a R ^9b ); SR ⁹ ; N(R ⁹ R ^9a ); NO ₂ ; OC(O)R ⁹ ; N(R ⁹ )C(O)R ^9a ; N(R ⁹ )S(O ₂ R ^9a ; N(R ⁹ )S(O)R ^9a ; N(R ⁹ )C(O)OR ^9a ; N(R ⁹ )C(O)N(R ^9a R ^9b ); OC(O) N(R ⁹ R ^9a ); T; C _1-50 alkyl; C _2-50 alkenyl; or C _2-50 alkynyl, wherein T; C _1-50 alkyl; C _2-50 alkenyl; The C _2-50 alkynyl group is optionally substituted by one or more R ¹⁰ which are the same or different and wherein C _1-50 alkyl; C _2-50 alkenyl; and C _2-50 alkynyl are optionally One or more selected from the group consisting of: T, -C(O)O-; -O-; -C(O)-; -C(O)N(R ¹¹ )-; -S(O ₂ N(R ¹¹ )-;-S(O)N(R ¹¹ )-;-S(O) ₂ -; -S(O)-;-N(R ¹¹ )S(O) ₂ N(R ^11a )-;-S-;-N(R ¹¹ )-;-OC(O)R ¹¹ ;-N(R ¹¹ )C(O)-;-N(R ¹¹ )S(O) ₂ -;- N(R ¹¹ )S(O)-;-N(R ¹¹ )C(O)O-;-N(R ¹¹ )C(O)N(R ^11a )-; and -OC(O)N(R ¹¹ R ^11a ); R ⁹ , R ^9a , R ^9b are independently selected from the group consisting of H; T; and C _1-50 alkyl; C _2-50 alkenyl; or C _2-50 alkynyl, wherein T; C _1-50 Alkyl; C _2-50 alkenyl; and C _2-50 alkynyl optionally substituted by one or more R ¹⁰ which are the same or different and wherein C _1-50 alkyl; C _2-50 alkenyl And a C _2-50 alkynyl group optionally inserted by one or more groups selected from the group consisting of: T, -C(O)O-; -O-; -C(O)-; -C(O) N(R ¹¹ )-;-S(O) ₂ N(R ¹¹ )-;-S(O)N(R ¹¹ )-;-S(O) ₂ -;-S(O)-;-N( R ¹¹ )S(O) ₂ N(R ^11a )-; -S-; -N(R ¹¹ )-; -OC(O)R ¹¹ ; -N(R ¹¹ )C(O)-;-N( R ¹¹ )S(O) ₂ -; -N(R ¹¹ )S(O)-; -N(R ¹¹ )C(O)O-; -N(R ¹¹ )C(O)N(R ^11a ) -; and -OC(O)N(R ¹¹ R ^11a ); T is selected from the group consisting of phenyl; naphthyl; anthracenyl; hydroquinone; tetralin; C _3-10 cycloalkyl; 4 to 7 members a heterocyclic group; or a group of 9 to 11 membered heterobicyclic groups, wherein the T system is optionally substituted by one or more R ¹⁰ which are the same or different; R ¹⁰ is halogen; CN; keto (=O) ;COOR ¹² ;OR ¹² ;C(O)R ¹² ;C(O)N(R ¹² R ¹² a);S(O) ₂ N(R ¹² R ¹² a);S(O)N(R ¹² R ¹² a);S(O) ₂ R ¹² ;S(O)R ¹² ;N(R ¹² )S(O) ₂ N(R ^12a R ^12b );SR ¹² ;N(R ¹² R ^12a );NO ₂ ;OC(O)R ¹² ;N( R ¹² )C(O)R ^12a ;N(R ¹² )S(O) ₂ R ^12a ;N(R ¹² )S(O)R ^12a ;N(R ¹² )C(O)OR ^12a ;N(R ¹² ) C(O)N(R ^12a R ^12b ); OC(O)N(R ¹² R ^12a ); or a C _1-6 alkyl group, wherein the C _1-6 alkyl group is optionally substituted by one or more halogens Substituted, which are the same or different; R ¹¹ , R ^11a , R ¹² , R ^12a , R ^12b are independently selected from the group consisting of H; or a C _1-6 alkyl group, wherein the C _1-6 alkyl group is optionally Substituted by one or more halogens, which are the same or different.

The term "inserted" refers to the insertion of a group between two carbons or between carbon and hydrogen at the end of the carbon chain.

L ² is a single chemical bond or a spacer. When L ² is a spacer, it is preferably defined as one or more substituents as defined above, but the L ² is replaced by Z.

Therefore, when L ^{2 is} not a single chemical bond, L ² -Z is COOR ⁹ ; OR ⁹ ; C(O)R ⁹ ; C(O)N(R ⁹ R ^9a ); S(O) ₂ N(R ⁹ R ^9a ); S(O)N(R ⁹ R ^9a ); S(O) ₂ R ⁹ ; S(O)R ⁹ ; N(R ⁹ )S(O) ₂ N(R ^9a R ^9b ); ⁹ ; N(R ⁹ R ^9a ); OC(O)R ⁹ ; N(R ⁹ )C(O)R ^9a ; N(R ⁹ )S(O) ₂ R ^9a ; N(R ⁹ )S(O R ^9a ; N(R ⁹ )C(O)OR ^9a ; N(R ⁹ )C(O)N(R ^9a R ^9b ); OC(O)N(R ⁹ R ^9a ); T; C _{1- a 50} alkyl group; a C _2-50 alkenyl group; or a C _2-50 alkynyl group, wherein T; a C _1-50 alkyl group; a C _2-50 alkenyl group; and a C _2-50 alkynyl group are optionally one or more Substituted by R ¹⁰ which are the same or different and wherein C _1-50 alkyl; C _2-50 alkenyl; and C _2-50 alkynyl are optionally interrupted by one or more groups selected from the group consisting of: - T-,-C(O)O-;-O-;-C(O)-;-C(O)N(R ¹¹ )-;-S(O) ₂ N(R ¹¹ )-;-S( O)N(R ¹¹ )-;-S(O) ₂ -; -S(O)-; -N(R ¹¹ )S(O) ₂ N(R ^11a )-;-S-;-N(R ¹¹ )-;-OC(O)R ¹¹ ;-N(R ¹¹ )C(O)-;-N(R ¹¹ )S(O) ₂ -; -N(R ¹¹ )S(O)-;- N(R ¹¹ )C(O)O-; -N(R ¹¹ )C(O)N(R ^11a )-; and -OC(O)N(R ¹¹ R ^11a ); R ⁹ ,R ^9a ,R ^9b is independently selected from the group consisting of: H; Z ; T; and C _1-50 alkyl; C _2-50 alkenyl; or C _2-50 alkynyl, wherein T; C _1-50 alkyl; C _2-50 alkenyl; and C _2-50 alkynyl Any optionally substituted by one or more R ¹⁰ which are the same or different and wherein C _1-50 alkyl; C _2-50 alkenyl; and C _2-50 alkynyl optionally _exemplified by one or more The following group is inserted: T, -C(O)O-; -O-; -C(O)-; -C(O)N(R ¹¹ )-; -S(O) ₂ N(R ^11a ) -; -S(O)N(R ¹¹ )-; -S(O) ₂ -; -S(O)-; -N(R ¹¹ )S(O) ₂ N(R ^11a )-;-S- ;-N(R ¹¹ )-; -OC(O)R ¹¹ ;-N(R ¹¹ )C(O)-;-N(R ¹¹ )S(O) ₂ -; -N(R ¹¹ )S( O)-;-N(R ¹¹ )C(O)O-;-N(R ¹¹ )C(O)N(R ^11a )-; and -OC(O)N(R ¹¹ R ^11a ); T system Selected from the group consisting of: phenyl; naphthyl; anthracenyl; hydroquinone; tetralin; C _3-10 cycloalkyl; 4 to 7 membered heterocyclic; or 9 to 11 membered heterobicyclic Wherein the T system is optionally substituted by one or more R ¹⁰ which are the same or different; R ¹⁰ is Z; halogen; CN; keto (=O); COOR ¹² ; OR ¹² ; C(O)R ¹² ; C(O)N(R ¹² R ^12a ); S(O) ₂ N(R ¹² R ^12a ); S(O)N(R ¹² R ^12a ); S(O) ₂ R ¹² ;S(O)R ¹² ;N(R ¹² )S(O) ₂ N(R ^12a R ^12b SR ¹² ; N(R ¹² R ^12a ); NO ₂ ; OC(O)R ¹² ; N(R ¹² )C(O)R ^12a ; N(R ¹² )S(O) ₂ R ^12a ;N( R ¹² )S(O)R ^12a ;N(R ¹² )C(O)OR ^12a ;N(R ¹² )C(O)N(R ^12a R ^12b );OC(O)N(R ¹² R ^12a ) Or a C _1-6 alkyl group, wherein the C _1-6 alkyl group is optionally substituted by one or more halogens, which are the same or different; R ¹¹ , R ^11a , R ¹² , R ^12a , R ^12b are independently selected From the group comprising: H; Z; or C _1-6 alkyl, wherein the C _1-6 alkyl group is optionally substituted by one or more halogens, which are the same or different; but R ⁹ , R ^9a , One of R ^9b , R ¹⁰ , R ¹¹ , R ^11a , R ¹² , R ^12a , and R ^12b is Z.

More preferably, L ² is a C _1-20 alkyl chain optionally interrupted by one or more groups independently selected from the group consisting of: -O-; and C(O)N(R ^3aa ); optionally a plurality of groups independently selected from the group consisting of: OH; and C(O)N(R ^3aa R ^3aaa ); and wherein R ^3aa , R ^3aaa are independently selected from the group consisting of H; and a C _1-4 alkyl group; group.

Preferably, the molecular weight of L ² ranges from 14 g/m to 750 g/mole.

Preferably, L ² is linked to Z via the following terminal group:

When L ² has such terminal groups, L ² preferably has a molecular weight calculated from 14 g/mol to 500 g/mol without the terminal group.

Preferably, the covalent linkage formed between the linker and Z is a permanent bond.

Preferably, the hydrogel Z is a biodegradable water-insoluble hydrogel based on poly(ethylene glycol) (PEG). As used herein, the term "PEG-based" means that the weight ratio of PEG chains in the hydrogel is at least 10% by weight, preferably at least 25%, based on the total weight of the hydrogel. The remainder may consist of other spacers and/or oligomers or polymers such as oligo- or poly-lysine.

Furthermore, the term "water insoluble" refers to a three dimensional spatially crosslinked molecular network that can be expanded by a hydrogel. The hydrogel, for example, suspended in an aqueous buffer of a large amount of water or physiological osmotic pressure, occupies a large amount of water, for example, up to 10-fold by weight on a weight basis, and thus is swellable but more than water removed Thereafter, the physical stability of a gel and a shape is maintained. This shape can be of any geometry and it should be understood that such individual hydrogel objects are considered to comprise a single molecule of constituents, wherein each component is linked to each other component via a chemical bond.

According to the invention, the hydrogel comprises a backbone portion which is crosslinkable by hydrolysis of a degradable bond. Preferably, the hydrogel is a PEG-based hydrogel comprising a backbone portion.

Preferably, the L ² system is connected to the backbone portion.

Preferably, the backbone portion has a molecular weight ranging from 1 kDa to 20 kDa, more preferably from 1 kDa to 15 kDa and even more preferably from 1 kDa to 10 kDa. Preferably, the backbone portion is a PEG-based one comprising one or more PEG chains.

In a hydrogel carrying an Exenatide-linker conjugate according to the invention, the backbone portion is characterized by a number of functional groups including cross-linking biodegradable functional groups and linking hydrogels a drug linker conjugate, and any encapsulating group. It means that the backbone portion is characterized by a plurality of hydrogel-attached drug linker conjugates; a functional group comprising a biodegradable cross-linking functional group; and any encapsulating group. Preferably, the total number of cross-linking biodegradable functional groups and drug linker conjugates and encapsulating groups is from 16 to 128, preferably from 20 to 100, more preferably from 24 to 80 and most preferably from 30 to 60. .

Preferably, the total number of cross-linking functional groups and the linker groups of the hydrogel-attached drug linker conjugate and backbone portion is equally divided by the number of PEG-based polymeric chains extending from the branching core. For example, if it is 32 cross-linking functional groups and a hydrogel-attached drug linker conjugate and a blocking group, the four PEG-based polymer chains extending from the core provide eight groups per line. Preferably, the branching portion is connected to the terminal of each PEG-based polymeric chain. Alternatively, eight PEG-based polymeric chains extending from the core may each provide four groups, or sixteen PEG-based polymeric chains each provide two groups. If the number of polymeric chains extending from the branching core by PEG is not allowed to be evenly distributed, it is preferred to bond the cross-linking functional group and the hydrogel-linked drug linker in each of the PEG-based polymeric chains. The deviation of the average of the total number of objects and packet groups is kept to a minimum.

In the prodrugs of the ligation vectors according to the present invention, it is desirable that almost all drug release (>90%) occurs before the release of a significant amount of the stem portion (<10%) occurs. It can be achieved by adjusting the half-life of the drug prior to the attachment of the carrier relative to the degradation kinetics of the hydrogel according to the invention.

Preferably, the backbone portion is characterized by having a branching core whereby at least three PEG-based polymeric chains are elongated. Thus, in a preferred aspect of the invention, the backbone reagent comprises a branched core whereby at least three PEG-based polymeric chains are elongated. Such branching cores may be included in a bound type of poly- or oligo alcohol, preferably pentaerythritol, tripentaerythritol, hexaglycerol, sucrose, sorbitol, fructose, mannitol, glucose, cellulose, amylose, starch, Hydroxyalkyl starch, polyvinyl alcohol, dextran, hyaluronans, or branched cores may be included in the bound form of poly- or oligoamines such as ornithine, diaminobutyric acid, tri-aminic acid, Tetra-amine acid, penta-amino acid, hexa-aminic acid, hepta-amino acid, octa-amino acid, octa-amino acid, ten-amino acid, eleven-amino acid, t-iso-acid, thirteen Amino acid, tetradecanoic acid, fifteen amino acid or oligoamine, polyethyleneimine, polyvinylamine.

Preferably, the branch core extends from 3 to 16 PEG-based polymeric chains, more preferably from four to eight. Preferred branched cores may be included in the bound form of pentaerythritol, ornithine, diaminobutyric acid, tri-aminic acid, tetra-aminic acid, penta-amino acid, hexa-amino acid, hepta-amino acid or oligoamine. Acid, low molecular weight PEI, hexaglycerol, tripentaerythritol. Preferably, the branch core extends from 3 to 16 PEG-based polymeric chains, more preferably from four to eight. Preferably, the PEG-based polymeric chain is a linear poly(ethylene glycol) chain, one end of which is attached to the branching core and the other end is attached to the hyperbranched branching moiety. It will be appreciated that the polymerized PEG-based chain may be terminated or inserted by an alkyl or aryl group optionally substituted with a hetero atom and a chemical functional group.

Preferably, the PEG-based polymeric chain is a suitably substituted poly(ethylene glycol) derivative (mainly PEG).

A preferred structure comprising a PEG-based polymeric chain extending from a branched core, which is contained in the backbone portion, is a complex arm PEG derivative. For example, see JenKem Technology, USA, for a list of products (July 28, 2009) Www.jenkemusa.com download access), 4-arm PEG derivative (pentaerythritol core), 8-arm PEG derivative (hexaglycerol core) and 8-arm PEG derivative (tripentaerythritol core). The best is 4-arm PEG amine (pentaerythritol core) and 4-arm PEG carboxyl (pentaerythritol core), 8-arm PEG amine (hexaglycerol core), 8-arm PEG carboxyl group (hexaglycerol core), 8-arm PEG amine (tripentaerythritol core) And 8-arm PEG carboxyl group (tripentaerythritol core). Preferred molecules of such complex arm PEG-derivatives in the backbone portion are from 1 kDa to 20 kDa, more preferably from 1 kDa to 15 kDa and even more preferably from 1 kDa to 10 kDa.

It will be appreciated that the terminal amine groups of the above plurality of arms are present in the backbone portion in a tethered form to further provide a crosslinking functional group and a reactive functional group of the backbone portion.

Preferably, the total number of cross-linking functional groups and reactive functional groups of the backbone portion is equally divided by the number of PEG-based polymeric chains extending from the branched core. If the number of polymeric chains extending from the branch core by PEG is not allowed to be evenly distributed, it is preferred that the deviation of the average number of cross-linking and reactive functional groups of each of the PEG-based polymeric chains is maintained until The smallest.

More preferably, the total crosslinks of the backbone moiety and the reactive functional groups are averaged by the number of polymeric chains which are predominantly PEG-extended by the branching core. For example, if there are 32 cross-linking functional groups and reactive functional groups, 4 PEG-based polymer chains extending from the branch nucleus may each provide 8 groups, preferably by branching moieties to The terminal of each PEG-based polymer chain. Alternatively, eight polymeric chains extending from the branch core by PEG may each provide four groups or 16 PEG-based polymeric chains each providing two groups.

These additional functional groups can be provided by the branching moiety. Preferably, the molecular weight of each of the branched portions is in the range of from 0.4 kDa to 4 kDa, more preferably from 0.4 kDa to 2 kDa. Preferably, each branch portion has at least 3 branches and at least 4 reactive functional groups, and up to 63 branches and 64 reactive functional groups, preferably at least 7 branches and at least 8 reactive functional groups and up to 31 branches and 32 reactive functional groups.

Examples of such preferred branching moieties include the tethered version of the tri-amino acid, tetra-aminic acid, penta-amino acid, hexa-amino acid, hepta-amino acid, octa-amino acid, octa-amino acid, Decenoic acid, eleven amino acid, dodecanoic acid, thirteen acid, tetradecanoic acid, fifteen amino acid, hexadecanolic acid, seventeen amino acid, eighteen Amino acid, 19-isoleic acid. Examples of such preferred branching moieties include the tethered version of the tri-amino acid, tetra-ammonic acid, penta-amino acid, hexa-amino acid, hepta-amino acid, and preferably the tri-amine of the bound form. Acid, penta-amino acid or hepta-amino acid, ornithine, diaminobutyric acid.

Preferably, the hydrogel carrier of the present invention is characterized in that the backbone portion has a quaternary carbon of the formula C(A-Hyp) ₄ , wherein each A is independently a poly(ethylene glycol)-based polymerization. The chain terminal is linked to the quaternary carbon by a permanent covalent bond, and the terminal end of the PEG-based polymeric chain is covalently bonded to the branched portion Hyp, and each branch portion Hyp has at least four functional groups representing the intersection. A functional group and a reactive functional group.

Preferably, each A is independently selected from the group -(CH ₂ ) _n1 (OCH ₂ CH ₂ ) _n X-, wherein n1 is 1 or 2; n is an integer from 5 to 50; and X is a covalent linkage Chemical functional groups of A and Hyp.

Preferably, A and Hyp are covalently linked by a guanamine linkage.

Preferably, the branched portion Hyp is a hyperbranched polypeptide. Preferably, the hyperbranched polypeptide is included in a bound form of lysine. Preferably, the molecular weight of each branched portion Hyp is in the range of from 0.4 kDa to 4 kDa. It should be understood that the stem portion C(A-Hyp)4 may contain the same or different branch portions Hyp and each Hyp may be independently selected. Each part of Hyp contains 5 and 32 excipients of acid, preferably at least 7 of lysine, that is, each part of Hyp contains 5 and 32 quaternic acids in bound form. Preferably, it comprises 7 lysines in a bound form. Most preferably, the Hyp contains seven off-amines.

The reaction of a polymerizable functional group with a backbone reagent, more specifically the reaction of Hyp with a polymerizable functional group of a poly(ethylene glycol)-based crosslinking reagent, produces a permanent amine bond.

Preferably, the molecular weight of C(A-Hyp)4 ranges from 1 kDa to 20 kDa, more preferably from 1 kDa to 15 kDa and even more preferably from 1 kDa to 10 kDa.

A preferred backbone portion is shown below, and the dashed line refers to a cross-linked biodegradable moiety attached to the crosslinker moiety and n is an integer from 5 to 50:

The biodegradability of the hydrogel according to the present invention is achieved by introducing a hydrolytically degradable bond.

In the context of the present invention, "hydrolyzable degradable", "biodegradable" or "hydrolyzable cleavable", "automatic cleavable", or "self-cracking", "self-cleavable", "instant" The term "state" or "temporary" means a bond and a link which, under physiological conditions (aqueous buffer at pH 7.4, 37 ° C), is non-enzymatically hydrolytically degradable or cleavable, and its half-life range is determined by a Hours to three months, including, but not limited to, aconityls, acetals, guanamines, carboxylic anhydrides, esters, imines, guanidines, maleic acid amides, orthoesters , phosphoniumamine, phosphoester, phosphonium alkyl ester, decylalkyl ester, sulfonate, aromatic urethane, combinations thereof, and the like.

If a hydrogel according to the invention is present as a degradable crosslinkable functional group, preferably the biodegradable linkage is a carboxylate, carbonate, phosphate and sulfonate and is preferably a carboxylate or carbonate ester.

Permanently attached to physiological conditions (aqueous buffer at pH 7.4, 37 ° C) is non-enzymatically hydrolyzable and has a half-life of six months or longer, such as, for example, guanamines.

In order to introduce a hydrolyzable cleavable bond into the hydrogel carrier of the present invention, the backbone portion can be directly joined by a biodegradable bond.

In one embodiment, the backbone portions of the biodegradable hydrogel carrier can be directly joined together, i.e., no crosslinker portion is required. The hyperbranched branching portions of the two backbone portions of such biodegradable hydrogels can be joined directly via crosslinking functional groups attached to the two hyperbranched branching moieties. Alternatively, the two hyperbranched branch portions of the two different backbone portions may be crosslinked by two spacer portions attached to the backbone portion and linked to the crosslinking portion on the other side, by crosslinking functionality The group is separated.

Alternatively, the backbone portions can be joined together via a crosslinker moiety, each crosslinker moiety being terminated by at least two hydrolytically degradable linkages. In addition to terminating the degradable linkage, the crosslinker moiety can further comprise a biodegradable linkage. Therefore, each end of the cross-linking moiety attached to the stem portion contains a hydrolytically degradable bond, and other biodegradable bonds may be arbitrarily present in the cross-linking moiety.

Preferably, the biodegradable hydrogel carrier comprises a backbone portion crosslinked by hydrolysis of a degradable bond and the backbone portion is linked together via a crosslinker moiety.

The biodegradable hydrogel carrier may contain one or more different types of crosslinker moieties, preferably one. The crosslinker moiety can be a linear or branched molecule and is preferably a linear molecule. In a preferred embodiment of the invention, the crosslinker moiety is attached to the backbone moiety by at least two biodegradable linkages.

Preferably, the crosslinker moiety has a molecular weight in the range of 60 Da to 5 kDa, more preferably from 0.5 kDa to 5 kDa, still more preferably from 1 kDa to 4 kDa, and even more preferably from 1 kDa to 3 kDa. In one embodiment, the crosslinker moiety comprises a polymer.

In addition to the cross-linking portion of the oligomer or polymer, a low molecular weight cross-linking moiety can be used, especially when the hydrophilic high molecular weight backbone portion is used to form the biodegradable hydrogel according to the present invention.

Preferably, the poly(ethylene glycol)-based crosslinking agent portion is a hydrocarbon chain comprising ethylene glycol units, optionally containing other chemical functional groups, wherein the poly(ethylene glycol) is the main component. The crosslinker moieties each comprise at least m ethylene glycol units, wherein m is an integer ranging from 3 to 100, preferably from 10 to 70. Preferably, the poly(ethylene glycol)-based crosslinker moiety has a molecular weight ranging from 0.5 kDa to 5 kDa.

As used with reference to the following, a cross-linking moiety or a polymeric chain mainly linked to a branching core by PEG, the term "PEG-based" means that the cross-linking moiety or the PEG-based polymeric chain comprises at least 20 Weight % ethylene glycol fraction.

In one embodiment, the single system comprising the polymeric crosslinker moiety is attached by a biodegradable linkage. The crosslinker portion of such polymers may contain up to 100 biodegradable linkages or more, depending on the molecular weight of the crosslinker moiety and the molecular weight of the monomer units. Examples of such crosslinker moieties are polymers based on poly(lactic acid) or poly(glycolic acid). It will be appreciated that such poly(lactic acid) or poly(glycolic acid) chains may be terminated or inserted by an alkyl or aryl group and that they are optionally substituted with heteroatoms and chemical functional groups.

Preferably, the cross-linking moiety is predominantly PEG, preferably represented by a single PEG-based molecular chain. Preferably, the poly(ethylene glycol)-based crosslinking agent portion is a hydrocarbon chain containing ethylene glycol units and optionally containing other chemical functional groups, wherein the poly(ethylene glycol) is mainly used. The crosslinker moieties each comprise at least m ethylene glycol units, wherein m is an integer ranging from 3 to 100, preferably from 10 to 70. Preferably, the poly(ethylene glycol)-based crosslinker moiety has a molecular weight ranging from 0.5 kDa to 5 kDa.

In a preferred embodiment of the invention, the cross-linking moiety comprises PEG which is symmetrically linked via an ester bond to two alpha, omega-aliphatic dicarboxylic acid spacers which are bonded via a permanent guanamine bond. Connected to the trunk portion of the hyperbranched branching portion to provide.

The dicarboxylic acid attached to the backbone portion and having the other side connected to the interstitial portion of the cross-linking portion contains 3 to 12 carbon atoms, preferably 5 and 8 carbon atoms, and may be one or more Substituted on the carbon atom. Preferred substituents are alkyl groups, hydroxyl groups or decylamine or substituted amine groups. The methylene group of one or more aliphatic dicarboxylic acids may be optionally substituted by O or NH or an alkyl substituted N. Preferred alkyl groups are straight or branched alkyl groups having from 1 to 6 carbon atoms.

Preferably, a permanent guanamine bond is interposed between the hyperbranched branching portion and the spacer portion, which is attached to the backbone portion and the other side is attached to the crosslinking portion.

A preferred cross-linking moiety is shown below; the dashed line refers to the cross-linked biodegradable link to the stem portion:

Where q is an integer from 5 to 50.

Preferably, the hydrogel carrier comprises a backbone portion crosslinked by hydrolysis of degradable linkages.

More preferably, the trunk portion includes a branch core of the following formula:

Wherein the dashed line refers to the rest connected to the trunk portion.

More preferably, the backbone portion contains the structure of the following formula:

Wherein n is an integer from 5 to 50 and the dashed line refers to the remainder connected to the stem portion.

Preferably, the backbone portion includes a hyperbranched portion Hyp.

More preferably, the backbone portion includes a hyperbranched portion Hyp of the following formula:

Wherein the dashed line refers to a carbon atom that is attached to the remainder of the molecule and is marked with an asterisk to refer to the S-configuration.

Preferably, the trunk portion is connected to at least one spacer of the following formula:

Wherein one of the dashed lines refers to the hyperbranched moiety Hyp and the second dashed line refers to the remainder of the molecule; and wherein m is an integer from 2 to 4.

Preferably, the stem portion is connected together via a cross-linking moiety having the following structure

Wherein q is an integer from 3 to 100, preferably from 5 to 50.

In the hydrogel prodrug of the present invention, the rate of hydrolysis of the biodegradable bond between the backbone portion and the crosslinker moiety is affected by the number and type of atoms adjacent to the carboxyl group of the PEG-ester. And ok. For example, by selecting succinic acid, adipic acid or glutaric acid in the PEG ester formation process, it can alter the biodegradable half-life of the hydrogel carrier according to the present invention.

Are preferred, L ² is connected to the system via a Z sulfosuccinimidyl acyl imine group, which in turn via a spacer system, such as oligo ethylene glycol chain connected to the backbone portion of the hydrogel. Preferably, the link of the spacer chain to the backbone portion is a permanent bond, preferably a guanamine bond.

The hydrogel biodegradation according to the present invention is achieved by introducing hydrolysis of a degradable bond.

In the cross-linking functional group, the term "hydrolyzable degradable" means in the context of the present invention a linkage which is non-enzymatic hydrolytically degradable under physiological conditions (aqueous buffer at pH 7.4, 37 ° C). Its half-life range is from one hour to three months, including, but not limited to, aconite, acetal, carboxylic anhydride, ester, imine, hydrazine, maleic acid decylamine, orthoester , phosphoniumamine, phosphoester, phosphonium alkyl ester, decylalkyl ester, sulfonate, aromatic urethane, combinations thereof, and the like. Preferred biodegradable linkages are carboxylates, carbonates, phosphoesters and sulfonates and are preferably carboxylates or carbonates. For practical purposes, it should be understood that for accelerated in vitro conditions, for example, pH 9, 37 ° C, aqueous buffer.

The permanent linkage is a non-enzymatic hydrolysis degradable under physiological conditions (aqueous buffer at pH 7.4, 37 ° C), which is a half-life of six months or longer, such as, for example, guanamine.

The degradation of the biodegradable hydrogel carrier according to the present invention is a multi-step reaction in which a number of degradable linkages are cleaved to produce degradation products which may be water soluble or water insoluble. However, the water-insoluble degradation product may further include a degradable bond, so that it may be cleaved to obtain a water-soluble degradation product. These water soluble degradation products may include one or more backbone moieties. It will be appreciated that the released backbone portion can, for example, be permanently bonded to a spacer or blocking or linking group or affinity group and/or prodrug linker degradation product and the water soluble degradation product can include degradable key.

The structure of the branch core, the PEG-based polymeric chain, the hyperbranched branching moiety and the moiety attached to the hyperbranched branching moiety may be provided by the corresponding description provided by the section covering the hydrogel carrier of the present invention. infer. It will be appreciated that the degraded structure is based on the hydrogel pattern being degraded according to the present invention.

The total amount of the backbone portion can be measured in solution after the hydrogel according to the invention is completely degraded, and during the degradation, the fraction of the soluble backbone degradation product is separated from the insoluble hydrogel according to the invention and It can be metered without interference from other soluble degradation products released from the hydrogels according to the invention. The hydrogel object according to the present invention can be separated by sedimentation or centrifugation in excess water of a physiological osmotic buffer. Centrifugation can be carried out in this manner such that the supernatant provides at least 10% by volume of the hydrogel expanded in accordance with the present invention. The soluble hydrogel degradation product remains in the aqueous supernatant after such precipitation or centrifugation steps, and the water soluble degradation product comprising one or more backbone portions can be aliquoted by the supernatant It is detected by appropriate separation and/or analysis methods.

Preferably, the water soluble degradation product can be separated from the water insoluble degradation product by filtration through a 0.45 micron filter, after which water soluble degradation products can be found in the process. The water-soluble degradation product can also be separated from the water-insoluble degradation product by a combination of centrifugation and filtration steps.

For example, the backbone portion can carry a group having UV absorption at a wavelength at which other degradation products do not have UV absorption. These selective UV-absorbing groups may be introduced to the backbone by a structural component of the backbone moiety, such as a guanamine bond or may be attached to its reactive functional group by an aromatic ring system such as a guanidine group. .

In the exenatide prodrugs of the hydrogels according to the invention, it is desirable that almost all of the exesin release (>90%) occurs in a significant amount of the main degradation products (< 10%) before the release. This can be achieved by adjusting the half-life of the exenatide prodrug attached to the hydrogel relative to the hydrogel degradation kinetics.

Preferably, the Exenatide prodrug D-L has a structure in which the L system is represented by the formula (II)

Wherein the dotted line refers to nitrogen attached to exenatide by formation of a guanamine bond, preferably N-terminal nitrogen, and Z is a hydrogel; preferably, the hydrogel in (II) is Biodegradable water-insoluble hydrogel based on poly(ethylene glycol) (PEG).

Preferably, the hydrogel of (II) consists of a backbone portion which is crosslinked by hydrolysis of a degradable bond.

More preferably, the trunk portion includes a branch core of the following formula:

Wherein the dashed line refers to the rest connected to the trunk portion.

More preferably, the backbone portion contains the structure of the following formula:

Wherein n is an integer from 5 to 50 and the dashed line refers to the remainder attached to the molecule.

Preferably, the backbone portion comprises a hyperbranched portion Hyp.

More preferably, the backbone portion comprises a hyperbranched portion Hyp of the following formula:

Wherein the dashed line refers to a carbon atom that is attached to the remainder of the molecule and is marked with an asterisk to refer to the S-configuration.

Preferably, the trunk portion is connected to at least one spacer of the following formula:

Wherein one of the dashed lines is attached to the hyperbranched moiety Hyp and the second dashed line is attached to the remainder of the molecule; and wherein m is an integer from 2 to 4.

Preferably, the trunk portion is connected to at least one spacer of the following formula:

Wherein, the dotted line marked with an asterisk refers to a bond between the hydrogel and N of the thiosuccinimide group; wherein the other dotted line means a link to Hyp; and wherein p is an integer from 0 to 10. .

Preferably, the backbone portion is joined together via a crosslinker portion having the following structure

Where q is an integer from 3 to 100.

The rate of hydrolysis of the biodegradable linkage between the backbone and the crosslinker moiety is determined by the number and type of atoms attached to the carboxy-ester carboxyl group. For example, succinic acid, adipic acid or glutaric acid, which is formed by a reaction selected from PEG esters, can change the half-life of the degrading crosslinking agent.

The exenatide prodrug of the present invention which is linked to the hydrogel can be prepared by the conventional method known in the art from the hydrogel of the present invention. Doctors in this area know that there are many ways. For example, the bioactive moiety is covalently attached thereto to the prodrug linker which is reactive with the reactive functional group of the hydrogel of the present invention, which has been partially or fully carried.

In a preferred method of preparation, the hydrogel is produced via a chemical bonding reaction. The hydrogel can be formed from two macromolecular educts containing complementary functional groups which undergo a reaction such as a condensation reaction or addition. One of these starting materials is a crosslinking reagent having at least two identical functional groups and the other starting material is a versatile backbone reagent. Suitable functional groups present on the crosslinking reagent include terminal amine groups, carboxylic acids and derivatives, maleimide and other alpha, beta unsubstituted Michael acceptors such as vinyl hydrazine, thiols, hydroxyl groups . Suitable functional groups present in the backbone reagent include, but are not limited to, amine groups, carboxylic acids and derivatives, maleimide and other alpha, beta unsubstituted Michael acceptors such as vinyl hydrazine, thiols, hydroxyl groups .

If the crosslinking reagent reactive functional group is used in a substoichiometrically manner with respect to the backbone reactive functional group, the resulting hydrogel will be a free reactive functional group containing a linker to the backbone structure. Reactive hydrogel.

Optionally, the prodrug linker can be first joined to exenatide and the resulting exenatide-prodrug linker conjugate is reacted with a reactive functional group of the hydrogel. Alternatively, after activation of one of the functional groups of the prodrug linker, the linker-hydrogel conjugate can be contacted with exenatide in a second reaction and an excess of exenatide is Exenatide is removed by filtration after attachment to the drug linker prior to binding to the hydrogel.

The preferred preparation method of the prodrug according to the present invention is as follows: the preferred starting material in the main reagent synthesis method is 4-arm PEG tetraamine or 8-arm PEG octaamine, wherein the molecular weight of the PEG reagent is in the range of 2000 to 10000 Dalton. The best is from 2000 to 5000 Da. The multi-arm PEG-derivatives thus far are coupled to the amine acid residues in sequence to form a hyperbranched backbone reagent. It will be appreciated that during the coupling step, the lysine may be partially or fully protected by a protecting group and the final backbone reagent may contain a protecting group. A preferred construction step is bis-boc lysine. Alternatively, instead of sequentially adding an lysine residue, the branched poly- oleic acid moiety is first organized and then coupled to a 4-arm PEG tetraamine or an 8-arm PEG octaamine. It is desirable to have a backbone reagent carrying 32 amine groups, thus attaching seven lysines to each arm of the 4-arm PEG or attaching five lysines to each arm of the 8-arm PEG. In another embodiment, the multi-arm PEG derivative is a tetra- or octacarboxy PEG. In this case, the branching moiety can be produced from glutaric acid or aspartic acid, and the resulting backbone reagent will carry 32 carboxyl groups. It will be appreciated that all or part of the backbone reagent's functional groups may be present in free form, either as a salt or as a linker to protect the group. It will be appreciated that for practical reasons, the number of amine acids per PEG arm in the backbone reagent will be between six and seven, more preferably about seven.

Preferred reagents are shown below:

The synthesis of the crosslinking reagent is initiated by a linear PEG chain having a molecular weight in the range of from 0.2 to 5 kDa, more preferably from 0.6 to 2 kDa, using a dicarboxylic acid such as adipic acid or glutaric acid. The half ester is esterified. A preferred protecting group in the half ester formation process is a benzyl group. The resulting bis-dicarboxylic acid PEG half ester is converted to a more reactive carboxyl compound such as mercapto chloride or an active ester such as pentafluorophenyl or N-hydroxysuccinimide, more preferably N- Hydroxyammonium imidate, of which the preferred structure is shown below.

Alternatively, the bis-dicarboxylic acid PEG half ester can be activated in the presence of a coupling agent such as DCC or HOBt or PyBOP.

In a further embodiment, the backbone reagent carrier carboxyl group and associated crosslinking reagent are selected from the group consisting of ester-containing amino-terminal PEG-chains.

The backbone reagent and the crosslinking reagent can be polymerized to form a hydrogel according to the present invention using a reverse phase emulsion polymerization. After selecting the desired chemical amount between the backbone and the crosslinker functional group, the stem and crosslinker are dissolved in DMSO and a suitable emulsifier having an appropriately selected HLB value, preferably Arlacel P135, a mechanical machine The stirrer is used to control the agitation speed to form an inverse emulsion. The polymerization is initiated by the addition of a suitable base, preferably by N, N, N', N'-tetramethylethylene diamine. After stirring for a suitable period of time, the reactants are quenched by the addition of an acid, such as acetic acid and water. The pellets were collected, washed, and fractionated by mechanical sieving according to particle size. Optionally, the protecting group can be removed at this stage.

Furthermore, such hydrogels according to the invention may be functionalized with a spacer carrying different reactive functional groups and then provided by a hydrogel. For example, the maleic imine reactive functional group can be introduced into the hydrogel by coupling a suitable heterobifunctional spacer such as Mal-PEG6-NHS to the hydrogel. The functionalized hydrogels can be further joined to an Exenatide linker reagent, carrying a reactive thiol group to the linker moiety to form a exenatide prodrug that is attached to the hydrogel according to the present invention. .

After loading the exenatide-linker conjugate to the functionalized maleimide group containing hydrogel, all remaining functional groups are encapsulated with a suitable blocker, such as thiolethanol. To avoid unwanted side effects.

In another preferred embodiment of the invention, an exenatide-linker conjugate carrying a free thiol is attached to the linker moiety and is hydrolyzed with a maleimide-functionalized hydrogel. The reaction is carried out at room temperature and 4 ° C, more preferably at room temperature, in a buffered aqueous solution of pH 5.5-8, preferably at a pH of 6.5-7.5. Immediately, the resulting drug-linker-hydrogel conjugate is treated with a low molecular weight compound comprising a thiol group, preferably a 34-500 Da thiol-containing compound, most preferably a thiol-containing ethanol. Between room temperature and a temperature of 4 ° C, more preferably a buffered aqueous solution at pH 5.5-8 at room temperature, preferably pH 6.5-7.5.

In another preferred embodiment of the invention, the exenatide-linker conjugate carrying the maleic imine group and the thiol-functionalized hydrogel according to the invention are at room temperature and The reaction is carried out at 4 ° C, more preferably at room temperature, in a buffer solution having a pH of 5.5-8, preferably pH 6.5-7.5. Immediately thereafter, the corresponding drug-linker-hydrogel conjugate is treated with a low molecular weight compound containing a maleimide group, preferably a 100-300 Da maleimide-containing compound, For example, N-ethyl-maleimide at room temperature and 4 ° C, more preferably at room temperature, in a buffered aqueous solution of pH 5.5-8, preferably at 6.5-7.5.

Another aspect of the invention is a method comprising the following steps:

(a) an aqueous suspension containing hydrolyzed microparticles functionalized with maleic imine in a buffered aqueous solution of pH 5.5-8 between room temperature and 4 ° C in a AI containing the present invention Contacting a solution of a sernapeptide-linker reagent, wherein the chemical functional group L ² * comprises a thiol group to produce an exenatide-linker-hydrogel conjugate;

(b) Optionally, the exenatide-linker-hydrogel conjugate from step (a) is used with a thiol-containing compound from 34 Da to 500 Da at room temperature and 4 ° C at pH 5.5. Treatment in a buffered aqueous solution of -8.

Another aspect of the invention is a method comprising the following steps:

(a) an aqueous suspension containing thiol-functionalized hydrogel microparticles and an exenatide-linker reagent comprising the present invention in a buffered aqueous solution of pH 5.5-8 at room temperature and 4 ° C Solution contact, wherein the chemical functional group L ² * comprises a maleic imine group to produce an exenatide-linker-hydrogel conjugate;

(b) Optionally, the exenatide-linker-hydrogel conjugate from step (a) is treated with 100 to 300 Da of the compound containing maleimide at room temperature and 4 ° C Treated in a buffered aqueous solution at pH 5.5-8.

A particularly preferred method of preparing a prodrug of the invention comprises the following steps:

(a) A compound of the formula C(A'-X ¹ ) ₄ wherein A'-X ¹ represents A and X ¹ is a suitable functional group before it adheres to the precursor of Hyp or Hyp, and the formula Hyp'-X ^{2 the} compound is reacted, wherein Hyp'-X ² represents Hyp and X ² is a suitable functional group before it adheres to the precursor of A or Hyp, and reacts with X ¹ ;

(b) optionally reacting the compound produced in step (a) in one or more further steps to produce a compound of formula C(A-Hyp) ₄ having at least four functional groups;

(c) reacting a compound having at least four functional groups produced in the step (b) with a precursor of a poly(ethylene glycol)-based crosslinking agent, wherein the crosslinking agent precursors an active ester group Producing a hydrogel in a sub-stoichiometric use compared to the total amount of reactive functional group C(A-Hyp) ₄ ;

(d) the unreacted functional group remaining in the hydrogel backbone of step (c) (representing the reactive functional group contained in the backbone of the hydrogel) and the biologically active moiety and transient prodrug linker The covalent conjugate is reacted, or the unreacted functional group is first reacted with the transient prodrug linker and then reacted with the biologically active moiety;

(e) arbitrarily encapsulating the remaining unreacted functional groups to produce a prodrug of the present invention.

In particular, the hydrogel of the exenatide prodrug of the present invention is synthesized as follows: in the agglomeration polymerization, the main reagent and the crosslinking reagent are blended in an amine/active ester ratio of 2:1 to 1.05:1. Hehe.

Both the main reagent and the crosslinking reagent are dissolved in DMSO to give a solution having a concentration of 5 to 50 g per 100 ml, preferably 7.5 to 20 g per 100 ml and most preferably 10 to 20 g per 100 ml.

In order to effect the polymerization, 2 to 10% by volume of N,N,N',N'-tetramethylethylenediamine (TMEDA) is added to the DMSO solution containing the crosslinking reagent and the main reagent and the mixture is Oscillate for 1 to 20 seconds and allow to stand. The mixture solidifies in less than 1 minute.

The hydrogels according to the present invention are preferably pulverized by mechanical means such as stirring, crushing, cutting pressing, or grinding, and arbitrarily sieving.

In the emulsion polymerization, the reaction mixture comprises a dispersed phase and a continuous phase.

In the case of dispersing the phase, the main reagent and the crosslinking reagent are mixed in an amine/active ester at a ratio of 2:1 to 1.05:1 and dissolved in DMSO to obtain a solution having a concentration of 5 to 50 g per 100 ml, preferably per 100 ml 7.5 to 20 g and most preferably 10 to 20 g per 100 ml.

The continuous phase is any solvent that is immiscible in DMSO, is not alkaline, is inert to protons and exhibits a viscosity below 10 Pa*s. Preferably, the solvent is immiscible in DMSO, is non-basic, inert to protons, exhibits a viscosity of less than 2 Pa*s and is non-toxic. More preferably, the solvent is a linear or branched saturated hydrocarbon having 5 to 10 carbon atoms. Most preferably, the solvent is n-heptane.

In order to form an emulsion of the dispersed phase in the continuous phase, an emulsifier is added to the continuous phase and then to the dispersed phase. The amount of the emulsifier is 2 to 50 mg/ml of the dispersed phase, more preferably 5 to 20 mg/ml of the dispersed phase, and most preferably 10 mg/ml of the dispersed phase.

The emulsifier has an HLB-value of from 3 to 8. Preferably, the emulsifier is a sorbitan and a fatty acid or a poly(hydroxyl fatty acid)-poly(ethylene glycol) conjugate. More preferably, the emulsifier is a poly(hydroxyl fatty acid)-polyethylene glycol conjugate having a molecular weight of linear poly(ethylene glycol) ranging from 0.5 kDa to 5 kDa and a molecular weight of poly(hydroxy-fatty acid) units. In the range from 0.5 kDa to 3 kDa at each terminal of the chain. Most preferably, the emulsifier is poly(ethylene glycol) dipolyhydroxystearic acid, Cithrol DPHS (Cithrol DPHS, former Arlacel P135, British Business Cologne International Public Company Limited).

The droplets of the dispersed phase are similar in geometry, such as Isojet, Intermig, Propeller (EKATO R) The axial flow impeller stirrer of hr- und Mischtechnik GmbH, Germany)) is produced by agitation, preferably similar to Isojet, which has a diameter of 50 to 90% of the reactor diameter. Preferably, the agitation is initiated prior to the addition of the dispersed phase. The agitator speed is set at 0.6 to 1.7 m/s. The dispersed phase is added at room temperature, and the concentration of the dispersed phase is from 2% to 70%, preferably from 5 to 50%, more preferably from 10 to 40%, and most preferably from 20 to 35% of the total reaction volume. The mixture containing the dispersed phase, the emulsifier and the continuous phase is stirred for 5 to 60 minutes and then a base is added to initiate the polymerization.

A base of 5 to 10 equivalents (referring to each of the amide linkages to be formed) is added to the mixture containing the dispersed phase and the continuous phase. The base is inert to protons, non-nucleophilic and soluble in the dispersed phase. Preferably, the base is inert to protons, non-nucleophilic, and readily soluble in both the dispersed phase and DMSO. More preferably, the base is inert to protons, non-nucleophilic, soluble in both the dispersed phase and DMSO, monoamine and non-toxic. Most preferably, the base is N, N, N', N' tetramethylethylene diamine (TMEDA). Stirring is continued for 1 to 16 hours in the presence of a base.

During the stirring, the droplets of the dispersed phase are hardened to become the crosslinked hydrogel pellets according to the present invention, which are collected and fractionated according to the size of the 75 micron and 32 micron plates on the vibrating continuous sifter. Invented hydrogel microparticles.

Another aspect of the invention is the Exenatide-linker conjugate intermediate D-L', wherein L' is of the following formula (III)

Wherein the dashed line refers to an amine group attached to the Exenatide moiety by the formation of a guanamine bond; in another aspect of the invention is the Exenatide-Linker Reagent DL*, wherein D represents Essex a peptide moiety; and L* is a non-biologically active linker reagent represented by the following formula (IV),

Wherein the dotted line refers to an amine group attached to exenatide by forming a guanamine bond; R ¹ is selected from a C _1-4 alkyl group, preferably CH ₃ ; R ² , R ^2a , Independently selected from the group consisting of H and C _1-4 alkyl groups, wherein L* is substituted by one L ² * and optionally substituted, but the asterisk-labeled hydrogen is not substituted in formula (IV) The base is substituted and wherein L ² * is a spacer which is attached to L* and comprises a chemical functional group intended to be bonded to a hydrogel.

Preferably, the R ² in formula (IV) is replaced by L ² *.

Preferably, R ² in formula (IV) is CH ₂ -L ² *.

Preferably, L* in formula (IV) is not further substituted.

Preferably, L ² * comprises a thiol group.

Preferably, L ² * comprises a maleimine group.

Preferably, L ² * is L ² -H.

The hydrogel of the prodrug of the present invention can be obtained in the form of a shaped object such as a sieve or a stent or a microparticle in the preparation method. Most preferably, the hydrogel forms microspheres which can be administered by subcutaneous or intramuscular injection through a standard syringe. These soft pellets are between 1 and 500 microns in diameter.

Preferably, the suspension is suspended in an isotonic preparation buffer having a diameter between 10 and 100 microns, more preferably between 20 and 100 microns, and the preferred diameter is between 25 and 80 microns.

Preferably, the microparticles can be administered by injection through a needle having an inner diameter of less than 0.6 mm, preferably via a needle having an inner diameter of less than 0.3 mm, more preferably a needle having an inner diameter of less than 0.225 mm, and more preferably via a needle having an inner diameter of less than 0.225 mm. A needle having an inner diameter of less than 0.175 mm, and most preferably a needle that passes through an inner diameter of less than 0.16 mm.

It should be understood that the term "administerable by injection", "injectable" or "injectable" means a combination of factors, such as application of considerable force to contain a specific temperature and a specific concentration (w/v). a piston of a syringe of a biodegradable hydrogel according to the present invention expanded in a liquid, having a needle having a given internal diameter connected to the outlet of the syringes, and being extruded by the syringe through the needle The time required for the volume of the biodegradable hydrogel according to the invention.

To provide injectability, a 1 ml volume of the Exenatide prodrug according to the invention is expanded in water to a concentration of at least 5% (w/v) and contained in a syringe holding a 4.7 mm diameter piston. It was extruded by applying a force of less than 50 Newton at room temperature for 10 seconds.

Preferably, the injectability of the Exenatide prodrug according to the present invention is achieved by swelling in water to a concentration of about 10% (w/v).

Another aspect of the invention is a method of preparing a needle-type injectable prodrug comprising the steps of:

(a) preparing the exenatide hydrogel prodrug of the present invention into a microparticle form;

(b) Screen the microparticles.

(c) Select a fraction of the drug pellets between 25 and 80 microns in diameter.

(d) suspending the globule fraction of step (c) in an aqueous buffer solution suitable for injection.

Another aspect of the invention is a needle-type injectable prodrug obtainable from the above method, wherein the needle-type injectable prodrug is injected via a needle having an inner diameter of less than 300 μm, preferably less than 225 μm via internal diameter The needle is for injection and, more preferably, can be injected via a needle having an inner diameter of less than 175 microns.

Another aspect of the invention is a pharmaceutical composition comprising a prodrug of the invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. This pharmaceutical composition is further illustrated in the following sections.

The Exenatide-Hydrogel prodrug composition can be provided as a suspension composition or as a dry composition. Preferably, the pharmaceutical composition of the Exenatide-Hydrogel prodrug is a dry composition. Suitable drying methods are, for example, spray drying and freeze drying (lyophilization). Preferably, the pharmaceutical composition of the Exenatide-Hydrogel prodrug is dried by freeze drying.

Preferably, the Exenatide hydrogel prodrug is present in the composition in an amount sufficient to provide a therapeutically effective amount of Exenatide for at least three days in a single administration. More preferably, the exenatide hydrogel prodrug is administered once a week for one week.

The Exenatide-hydrogel prodrug according to the invention contains one or more excipients.

Excipients used in parenteral compositions can be classified as buffers, isotonicity adjusters, preservatives, stabilizers, anti-adsorbents, oxidative protectants, thickeners/viscosity enhancers, or other adjuvants. . In some cases, these components may have two or three functions. The Exenatide-Hydrogel prodrug composition according to the present invention contains one or more than one excipient selected from the group consisting of:

(i) Buffer: A physiologically tolerated buffer to maintain the pH in the desired range, such as sodium phosphate, bicarbonate, succinate, histidine, citrate and acetate, sulfate, nitrate , chloride, pyruvate. Antacids such as Mg(OH) ₂ or ZnCO ₃ can also be used. Adjustable buffering capacity to match conditions most sensitive to pH stability

(ii) Isotonicity adjuster: minimizes the pain caused by cell damage due to the difference in osmotic pressure at the time of injection accumulation. Examples are glycerin and sodium chloride. The effective concentration can be measured by an osmometer using a hypothetical osmotic pressure of 285-315 mOsmol/kg serum.

(iii) Preservatives and/or Antimicrobial Agents: Multiple doses of parenteral formulations require the addition of a sufficient level of preservative to minimize the risk of infection at the time of injection and establish a corresponding regulatory requirement. Typical preservatives include m-cresol, phenol, methylparaben, hydroxyphenethyl ester, hydroxypropylpropyl ester, hydroxyphenyl butyl ester, butanol chloride, benzyl alcohol, phenyl nitrate, thiomersal, sorbic acid, sorbic acid Potassium, benzoic acid, chlorocresol, and benzalkonium chloride

(iv) Stabilizer: Stability is achieved by strengthening the protein-stabilizing force, by the instability of the denaturedstater, or by directly attaching the excipient to the protein. The stabilizer may be an amino acid such as alanine, arginine, aspartic acid, glycine, histidine, lysine, proline, sugar such as glucose, sucrose, trehalose, polyol such as glycerol, Mannitol, sorbitol, salts such as potassium phosphate, sodium sulfate, chelating agents such as EDTA, hexaphosphate, ligands such as divalent metal ions (zinc, calcium, etc.), other salts or organic molecules such as phenol derivative. In addition, oligomers or polymers such as cyclodextrin, dextran, dendrimers, PEG or PVP or protamine or HSA can be used.

(v) Anti-Adsorbent: A ionic or nonionic surfactant or other protein or soluble polymer is used to completely coat or adsorb to the inner surface of the container of the composition. For example, Pluronic F-68, PEG lauryl ether (Brij 35), polysorbate 20 and 80, dextran, polyethylene glycol, PEG-polyhistidine, BSA and HSA And gelatin. The selected excipient concentration and type are based on the effect to be avoided, but typically form a single layer of surfactant at the interface directly above the CMC value.

(vi) Lyo- and/or cryoprotectants: During lyophilization or spray drying, the excipients counteract the destabilizing effects caused by breaking hydrogen bonds and removing water. For this purpose, sugars and polyols can be used but the corresponding positive effects of surfactants, amino acids, non-aqueous solvents, and other peptides are also observed. Trehalose is particularly effective in reducing the polymerization of induced moisture and also improves the thermal stability that may result from contact of the hydrophobic groups of the protein with water. Mannitol and sucrose may also be used, either as the sole lyo/freeze protectant or in combination with one of the higher mannitols: the ratio of sucrose is known to promote the physical stability of the lyophilized cake. Mannitol can also be combined with trehalose. Trehalose can also be combined with sorbitol or used as the sole protectant with sorbitol. Starch or starch derivatives can also be used.

(vii) Oxidative protectants: antioxidants such as ascorbic acid, tetrahydropyrimidine, methionine, glutathione, monothioglycerol, moline, polyethyleneimine (PEI), propyl gallate, Vitamin E, chelating agents such as citric acid, EDTA, hexaphosphate, thioglycolic acid

(viii) Thickeners or Viscosity Enhancers: hinder the sedimentation of particles in vials and syringes to promote blending and resuspension of the particles and to make the suspension easier to inject (ie, the syringe piston requires less force). Suitable thickeners or viscosity enhancers are, for example, carbomer thickeners such as Carbopol 940, Carbopol Ultrez 10, cellulose derivatives such as hydroxypropylmethylcellulose (hypromellose, HPMC) or Ethylaminoethylcellulose (DEAE or DEAE-C), colloidal magnesium citrate (Veegum) or sodium citrate, hydroxyapatite gel, tricalcium phosphate gel, xanthan gum, carrageenan such as mountain tower ( Satia) gum UTC30, aliphatic poly(alkyd), such as poly(D,L- or L-lactic acid) (PLA) and poly(glycolic acid) (PGA) and copolymers thereof (PLGA), D, L - a terpolymer of lactic acid, glycolic acid and caprolactone, a poloxamer, a hydrophilic poly(oxyethylene) oxime segment and a hydrophobic poly(oxypropylene) oxime segment to form a poly(oxyethylene)-poly (oxypropene)-poly(oxyethylene) three-stage (eg Pluronic) ), polyether ester copolymers, such as polyethylene glycol terephthalate / polybutylene terephthalate copolymer, sucrose acetate isobutyrate (SAIB), dextran or its derivatives , dextran and PEG composition, polydimethyl methoxy oxane, collagen, chitosan, polyvinyl alcohol (PVA) and derivatives, polyalkylimine, poly (acrylamide - co- Diallyldimethylammonium (DADMA), polyvinylpyrrolidone (PVP), mucopolysaccharides (GAGs) such as dermatan sulfate, chondroitin sulfate, keratan sulfate, heparin, heparin sulfate, hyaluronic acid, ABA triterpenoid or AB segment copolymer containing a hydrophobic A-quinone such as polylactic acid (PLA) or poly(lactic-co-glycolic acid) (PLGA), and a hydrophilic B-column such as poly Ethylene glycol (PEG) or polyvinylpyrrolidone. Such a segmented copolymer and the above poloxamer may have a reversible thermal gelation behavior (liquid at room temperature to facilitate administration and gel after body temperature above the sol-gel transition temperature) Aspect).

(ix) Dispersing or diffusing agent: modifying the permeability of connective tissue by hydrolyzing a component of the extracellular matrix in the space within the cell, such as, but not limited to, hyaluronidase, glycans, which are attached to the connective The intracellular space of the tissue was found. Dispersing agents, such as, but not limited to, hyaluronic acid, temporarily reduce the viscosity of the extracellular matrix and promote the spread of the injected drug.

(x) Other adjuvants: such as wetting agents, viscosity modifiers, antibiotics, hyaluronidase. Acids and bases such as hydrochloric acid and sodium hydroxide are necessary adjuvants for pH adjustment during manufacture.

Preferably, the exenatide-hydrogel prodrug composition contains one or more than one thickener and/or viscosity modifier.

The term "excipient" preferably refers to a diluent, adjuvant, or carrier, and the therapeutic system is administered with it. Such pharmaceutical excipients can be sterile liquids, such as water and oil, including such petroleum, animal, vegetable or synthetic sources including, but not limited to, peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred excipient when the pharmaceutical composition is administered orally. When the pharmaceutical composition is administered intravenously, saline and aqueous dextrose are preferred excipients. Saline solutions and aqueous dextrose and glycerol solutions are preferred as liquid excipients in injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, white peony, silicone, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, Glycerol, propylene, ethylene glycol, water, ethanol, etc. If desired, the composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions may be in the form of solutions, suspensions, emulsions, lozenges, tablets, capsules, powders, sustained release formulations and the like. The composition is a sizing agent, which comprises a conventional adhesive and an excipient such as triglyceride. Oral formulations include standard excipients such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Suitable examples of pharmaceutical excipients are set forth in "Remington's Pharmaceutical Sciences" by E. W. Martin. Such a composition will contain a therapeutically effective amount of the therapeutic agent, preferably in a neat form, together with an appropriate amount of excipient to provide a suitable mode of administration to the patient. The formulation should conform to the form of administration.

In a typical embodiment, the pharmaceutical compositions of the present invention, whether in a dry form or in suspension or other form, may be provided as a single or multiple dose composition.

In one embodiment of the invention, the dry composition of the Exenatide-Hydrogel prodrug is provided in a single dose, meaning that the container contains a pharmaceutical dose.

Thus, in another aspect of the invention, the composition is provided as a single dose composition.

Preferably, the suspension composition is a multiple dose composition which means that it contains more than one therapeutic dose. Preferably, the plurality of dose compositions contain at least 2 doses. The multiple dose compositions of such exenatide-hydrogels can be administered to different patients in need of such treatment or to a patient, wherein the remaining dose is stored after the first dose is administered until needed.

In another aspect of the invention, the composition is contained in a container. Preferably, the container is a double tank syringe. In particular, a dry composition according to the present invention is provided in a first tank of a dual tank syringe and a reconstituted solution is provided in a second tank of the dual tank syringe.

The dry composition is reconstituted prior to administration of the dry composition of the exenatide-hydrogel prodrug to the desired patient. Recombination can be carried out in a container wherein the dry composition of the Exenatide-Hydrogel prodrug is provided, for example, in vials, syringes, dual-slot syringes, ampoules, and syringes. Recombination is carried out by adding a predetermined amount of the recombinant solution to the dry composition. The recombinant solution is a sterile liquid, such as water or a buffer, which may further contain additives such as preservatives and/or antibacterial agents. If the exenatide-hydrogel prodrug composition is provided in a single dose, the reconstituted solution may contain one or more preservatives and/or antibacterial agents. Preferably, the recombinant solution is sterile water. If the exenatide-hydrogel prodrug composition is a multiple dose composition, the recombinant solution preferably contains one or more preservatives and/or antibacterial agents, for example, benzyl alcohol and cresol.

A further aspect of the invention relates to a method of administering a recombinant exenatide hydrogel prodrug composition. The exenatide hydrogel prodrug composition can be administered by injection or infusion, including intradermal, subcutaneous, intramuscular, intravenous, intraosseous, and intraperitoneal administration.

Yet another aspect is a method of preparing a recombinant composition comprising a therapeutically effective amount of an exenatide hydrogel prodrug, and any one or more pharmaceutically acceptable excipients, wherein the Exenatide is transient Connected to the hydrogel, the method includes the following steps

• The composition of the invention is contacted with a recombinant solution.

Another aspect is a recombinant composition comprising a therapeutically effective amount of an exenatide hydrogel prodrug, and any one or more pharmaceutically acceptable excipients, wherein the Exenatide is transiently linked to The hydrogel obtained by the above method.

Another aspect of the invention is a method of preparing a dry composition of an exenatide-hydrogel prodrug. This suspension composition is made by

(i) blending the exenatide-hydrogel prodrug with one or more excipients,

(ii) transferring the amount equivalent to a single or multiple doses to a suitable container,

(iii) drying the composition in the container, and

(iV) Seal the container.

Suitable containers are vials, syringes, double-slot syringes, ampoules, and syringes.

On the other hand, it is a kit for parts. When the drug delivery device is simply a hypodermic syringe, the kit may comprise a syringe, a needle and a container containing the dry exenatide-hydrogel prodrug composition for use with the syringe and a containing A second container of the recombinant solution. In more preferred embodiments, the syringe is not a simple hypodermic syringe and thus the separate container containing the reconstituted exenatide-hydrogel prodrug is used in conjunction with the syringe The composition in the container is attached to the outlet of the syringe at the time of use. Examples of drug delivery devices include, but are not limited to, hypodermic syringes and pen-type syringes. A particularly preferred syringe is a pen-type syringe. In this case, the container is a syringe, preferably a disposable syringe.

A preferred kit of parts comprises a needle and a container containing the composition according to the invention and optionally further comprising a reconstituted solution for use with the needle. Preferably, the container is a double tank syringe.

In another aspect, the invention provides a syringe comprising an Exenuin-hydrogel prodrug composition for use in a one-shot syringe as previously described. The syringe can contain a single dose or multiple doses of exenatide.

In one embodiment of the invention, the suspension composition of the Exenatide-Hydrogel prodrug comprises not only the Exenatide-Hydrogel prodrug and one or more than one excipient, but also other It is in its free form or a biologically active agent as a prodrug. Preferably, the additional one or more bioactive agents are a prodrug, more preferably a hydrogel prodrug. Such biologically active agents include, but are not limited to, the following types of compounds:

(i) sulfonylureas, for example, chlorpropamide, methotrexate, tolbutamide, glibenclamide, glipizide, glimepiride, glibenclamide, gliclazide, etc.;

(ii) amphetamines, for example, repaglinide, nateglinide or mitiglinide;

(iii) Glucagon-like peptide-1 (GLP-1) and its analogues, glucose-insulin peptide (GIP) and its analogues, exenatide and its analogues, and dipeptidase inhibition Agent (DPPIV);

(iv) biguanides, for example, metformin;

(v) thiazolidinediones such as, for example, rosiglitazone, pioglitazone, troglitazone, isaglitazone (known as MCC-555), 2-[2-[(2R)- 4-hexyl-3,4-dihydro-3-keto-2H-1,4-benzoene 2-yl]ethoxy]-phenylacetic acid, cycloglitazone, rosiglitazone or the compound is described in WO 97/41097 by Dr. Reddy's Research Foundation, especially 5-[[4-[(3, 4-dihydro-3-methyl-4-keto-2-quinazolinylmethoxy]phenyl]methyl]-2,4-thiazolidinedione;

(vi) GW2570, etc.;

(vii) a retinoid-X receptor (RXR) modulator, such as, for example, bismurodidine, 9-cis-retinal, and the like;

(viii) other insulin sensitizers, such as, for example, INS-1, PTP-1B inhibitors, GSK3 inhibitors, glycogen phosphorylase inhibitors, fructose-1,6-bisphosphatase inhibitors, etc.;

(ix) insulin, including short-acting or intermediate-effect, and long-acting insulin, inhaled insulin, insulin derivatives, and insulin analogs, such as insulin molecules with minor differences in the sequence of the native amino acid;

(x) Small molecule mimics of insulin, including, but not limited to, L-783281, TE-17411, etc.;

(xi) Sodium-dependent glucose transport agent 1 and / or 2 (SGLT1, SGLT2) inhibitors, such as KGA-2727, T-1095, T-1095A, SGL-0010, AVE 2268, SAR 7226, SGL-5083, SGL -5085, SGL-5094, ISIS-388626, shegliflozin, dapagliflozin or remogliflozin etabonate, canagliflozin, phlorizin, etc.;

(xii) an Amylin agonist, which includes, but is not limited to, pramlintide, etc.;

(xiii) Glucagon antagonists such as AY-279955, and the like.

(xiv) Regulators of gastrointestinal hormones and gastrointestinal hormones, such as Somatostatin, Oxyntomodulin, Cholecystokinin, Incretins, Ghrelin, PYY _3-36 , and the like.

The above insulins may be independently selected from the group consisting of bovine insulin, porcine insulin, and human insulin. More preferably, the insulin is independently selected from human insulin. The insulin may be selected from unmodified insulin, more particularly selected from the group consisting of bovine insulin, porcine insulin, and human insulin.

Insulin derivatives are naturally occurring derivatives of insulin and/or insulin analogs which are obtained by chemical modification. The chemical modification can include, for example, the addition of one or more defined chemical groups to one or more amino acids.

Illustrated in EP 0 214 826, EP 0 375 437, EP 0 678 522, EP 0 885 961, EP 0 419 504, WO 92/00321, German Patent Application 102008 003 568.8 and 10 2008 003 566.1, and EP-A 0 An insulin analog of 368 187 can be part of the composition of the invention. The documents EP 0 214 826, EP 0 375 437, EP 0 678 522, EP 0 419 504, WO 92/00321, and EP-A 0 368 187 are incorporated herein by reference.

One of the preferred insulin analogs may be selected from the group consisting of Gly(A21)-Arg(B31)-Arg(B32) human insulin (insulin glargine, Lantus); Arg(A0)-His(A8)- Glu(A15)-Asp(A18)-Gly(A21)-Arg(B31)-Arg(B32) human insulin, Lys(B3)-Glu(B29) human insulin; Lys ^B28 Pro ^B29 human insulin (lyspro insulin), B28 Asp human insulin (insulin), in which the proline is in the position B28, the human insulin is replaced by Asp, Lys, Leu, Val or Ala and wherein the Lys at position B29 can be replaced by Pro

AlaB26 human insulin; des (B28-B30) human insulin; des (B27) human insulin or B29Lys (ε-tetramine), des (B30) human insulin (detemir).

Preferred insulin derivatives are selected from the group consisting of: B29-N-myristatin-des (B30) human insulin, B29-N-palm-dess (B30) human insulin, B29-N-myristyl Human insulin, B29-N-palm human insulin, B28-N-myristyl Lys ^B28 Pro ^B29 human insulin, B28-N-palm 醯-Lys ^B28 Pro ^B29 human insulin, B30-N-nutmeg-Thr ^B29 Lys ^B30 human insulin, B30-N-palm 醯-Thr ^B29 Lys ^B30 human insulin, B29-N-(N-palm 醯-Y-glutenyl)-des(B30) human insulin, B29-N-(N-stone bile Base-Y-glutenyl)-des (B30) human insulin, B29-N-(ω-carboxy sulfonium)-des (B30) human insulin, and B29-N-(ω-carboxy sulfhydryl) human insulin.

More preferred insulin derivatives are selected from the group consisting of Gly(A21)-Arg(B31)-Arg(B32) human insulin, Lys ^B28 Pro ^B29 human insulin (lyspro insulin), B28 Asp human insulin (aspart insulin), B29Lys (ε-tetrapurine), desB30 human insulin (tete insulin).

Preferably, the additional one or more bioactive agents are hydrogel prodrugs of insulin as described in WO 2011/012718 and WO 2011/012719.

In addition to antidiabetic agents, the bioactive compound can be an anti-obesity agent such as orlistat, a pancreatic lipase inhibitor that prevents disintegration and absorption of fat; or sibutramine, an appetite suppressant and a serotonin reuptake inhibitor, The brain's norepinephrine and dopamine, a growth factor that increases fat circulation (eg, growth hormone, IGF-1, growth hormone releasing factor), glucagon and auxin regulators. Other potential bioactive anti-obesity agents include, but are not limited to, appetite-inhibitors via adrenergic mechanisms such as benzethetamine, morpholine, phentermine, diethylamine ketone, mazin哚, sibutramine, phenylpropanolamine or ephedrine; appetite-inhibitors act via serotonin mechanisms, such as quinoline (quipazine), fluoxetine, sertraline, fenfluramine, or dexfenfluramine; appetite-inhibitors act via dopamine mechanisms, eg, apomorphine; appetite-inhibitors act via histamine mechanism (eg, histamine analogs, H3 receptor modulators); energy-enhancing enhancers, such as beta-3 adrenergic agonists and stimulants that do not couple protein functionalities; leptin and leptin analogs (eg, beauty) Triptorine; neuropeptide Y antagonist; melanocortin-1, 3 and 4 receptor modulators; cholecystokinin agonists; glucagon-like peptide-1 (GLP-1) analogues and the like (eg, exenatide); androgens (eg, dehydroepiandrosterone and derivatives such as reduced urinary ketone), testosterone, anabolic steroids (eg, oxymetholone), and steroid hormones; Aprotinin receptor antagonist; cytokine agent such as ciliary neurotrophic factor; amylase inhibitor; enterostatin agonist/analog; orexin/thalectin antagonist; urocortin antagonist; bombesin agonist; protein kinase A modulator; corticotropin releasing factor analog; cocaine- and amphetamine-regulation Recording the like; calcitonin gene-related peptide analogs; and fatty acid synthase inhibitors.

In other embodiments, the exenatide-hydrogel prodrug composition according to the present invention is combined with the second biological activity in such a manner that the exenatide-hydrogel prodrug is first administered to The patient in need is then administered a second compound. Alternatively, the exenatide-hydrogel composition is administered to the same patient after administration of the other compound to the desired patient.

Another aspect of the invention is a prodrug of the invention or a pharmaceutical composition of the invention for use as a pharmaceutical.

Another aspect of the invention is a prodrug of the invention or a pharmaceutical composition of the invention for use in a method of treating or preventing a disease or disorder treatable with exenatide. The composition is for use in a method for treating diseases and disorders which are treated or prevented with exenatide and exenatide agonists, for example, for the treatment and prevention of hyperglycemia and for the treatment and prevention of any type Diabetes, such as insulin-dependent diabetes mellitus, non-insulin-dependent diabetes mellitus, pre-diabetes or gestational diabetes, for the prevention and treatment of metabolic syndrome and/or obesity and/or eating disorders, insulin resistance syndrome, lowering blood lipid levels, lowering Heart risk, reduced appetite, lower body weight, etc.

Patients requiring treatment with the long-acting exenatide compositions described in the present invention are at high risk for developing complications. Thus, combinations comprising the long-acting exenatide of the present invention and a suitable biologically active compound can be used, for example, for prevention, delayed development or treatment selected from the group consisting of diseases and disorders including: hypertension (including but not limited to simple Sexually contractive hypertension and familial dyslipidemia), congestive heart failure, left ventricular hypertrophy, peripheral arterial disease, diabetic retinopathy, macular degeneration, cataract, diabetic nephropathy, glomerular sclerosis, chronic renal failure , diabetic neuropathy, X syndrome, premenstrual syndrome, coronary heart disease, angina pectoris, thrombosis, atherosclerosis, myocardial infarction, transient ischemic attack, stroke, vascular restenosis, hyperglycemia, hyperinsulinemia , hyperlipidemia, hypertriglyceridemia, insulin resistance, impaired glucose metabolism, impaired glucose tolerance, impaired fasting blood glucose, obesity, erectile dysfunction, skin and connective tissue diseases, foot ulcers And ulcerative colitis, endothelial dysfunction and impaired vascular compliance.

Prevention, delayed development or treatment may be achieved by a combination comprising a long-acting exenatide composition of the invention and at least one biologically active compound selected from the group of drugs used to treat the condition, including AT ₁ -receptor antagonist; angiotensin converting enzyme (ACE) inhibitor; renin inhibitor; beta adrenergic receptor blocker; alpha adrenergic receptor blocker; calcium channel blocker; aldosterone synthesis Enzyme inhibitors; aldosterone receptor antagonists; neutral endopeptidase (NEP) inhibitors; dual angiotensin converting enzyme/neutral endopeptidase (ACE/NEP) inhibitors; endothelin receptor antagonism Diuretic; statin; nitrate; anticoagulant; natriuretic peptide; digitalis compound; PPAR regulator.

In the case of a bioactive agent; a prodrug, especially a hydrogel prodrug containing one or more acidic or basic groups, the invention also includes such corresponding pharmaceutically or toxicologically acceptable salts , especially its pharmaceutically acceptable salts. Therefore, the acidic group-containing prodrug can be used according to the present invention, for example, an alkali metal salt, an alkaline earth metal salt or ammonium. More precise examples of such salts include sodium salts, potassium salts, calcium salts, magnesium salts or salts with ammonia or organic amines such as, for example, ethylamine, ethanolamine, triethanolamine or amino acids. A group containing one or more basic groups, i.e., protonated, may be present and may be used in accordance with the present invention in the form of addition salts with inorganic or organic acids. Examples of suitable acids include hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalene disulfonic acid, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formic acid, Propionic acid, pivalic acid, diethyl acetate, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid, aminosulfonic acid, phenylpropionic acid, gluconic acid, ascorbic acid, Isonicotinic acid, citric acid, adipic acid, and other acids known to those skilled in the art. If the prodrug contains both acidic and basic groups in the molecule, the present invention also includes internal salts or betaines (zwitterions) in addition to the aforementioned salt forms. The respective salts can be obtained by conventional methods known to those skilled in the art, for example, by contacting these with an organic or inorganic acid or base in a solvent or dispersant, or by reacting with other salts. Anion exchange or cation exchange. The present invention also encompasses all salts of the prodrugs which, due to their low physiological compatibility, are not suitable for direct use in pharmaceuticals, for example, as intermediates for chemical reactions or for the preparation of pharmaceutically acceptable salts. class.

The term "pharmaceutically acceptable" means approved for use in animals, preferably humans, by a monitoring unit such as EMEA (Europe) and/or FDA (USA) and/or any other national monitoring unit.

Still another aspect of the invention is a method of treating, controlling, delaying or preventing a mammalian patient in need of treatment in one or more conditions, comprising administering a therapeutically effective amount of a prodrug or a prodrug of the invention The pharmaceutical composition or pharmaceutically acceptable salt of the invention is administered to the patient.

Instance Substance and method

Exenatide-4 [Seq ID No: 1] synthesized by resin in Fmoc mode (loading about 0.1 mmol/g) was obtained from CASLO Laboratory Aps, Lyngby, Denmark. Lithra® [Seq ID No 21] and GLP-1 [Seq ID No 13] synthesized by resin in Fmoc mode (loading about 0.1 mmol/g) were obtained from the peptide Specialty Laboratories, Heidelberg, Germany. The branch of the peptide is completely protected and contains a free N-terminus.

The amine 4-arm PEG 5kDa was obtained from JenKem Technology, P. R., Beijing, China.

N-(3-maleimidopropyl)-21-amino-4,7,10,13,16,19-hexaoxa-nonanoic acid NHS ester (Mal-PEG6-NHS) From Celares GmbH in Berlin, Germany. 6-(S-Tritylthiol)hexanoic acid was purchased from Jusheng Peptide Co., Ltd., Strasbourg, France. If not stated, the amino acid used is in the L configuration.

All other compounds were obtained from Sigma-ALDRICH Chemie GmbH, Taufkirchen, Germany.

Fmoc's deprotection:

To remove the Fmoc protecting group, stir the resin with 2/2/96 (v/v/v) hexahydropyridine/DBU/DMF (twice, 10 minutes each) and rinse with DMF (ten times) .

RP-HPLC purification:

Unless otherwise noted, RP-HPLC was coupled to a Waters 600 HPLC System and Waters 2487 Absorbance assay on a 100 x 20 mm or 100 x 40 mm C18 ReproSil-Pur 300 ODS-35 micron column (Dr. Maisch, Ammerbuch, Germany). The device is carried out. Using a solution A (0.1% TFA in H ₂ O) and a linear gradient of solution B (0.1% TFA in acetonitrile). The HPLC fraction containing the product was concentrated and lyophilized.

Flash chromatography

Flash chromatographic separation and purification was performed on an Isolera One system from Biotage AB, Sweden, using a Biotage KP-Sil(R) hose column with n-heptane and ethyl acetate as the eluent. The product was tested at 254 nm.

In the case of a hydrogel pellet, a syringe having a polyethylene frit is used as a reaction tank or for a washing step.

Analytical Ultra Performance LC (UPLC) was performed on a Waters Acquity system equipped with a Waters BEH300 C18 column (2.1 x 50 mm, 1.7 micron particle size) coupled to an LTQ Orbitrap Discovery mass spectrometer from Thermo Scientific.

Due to the degree of polymerization of the PEG starting material, the MS of the PEG product shows a series of (CH ₂ CH ₂ O) _n moieties. For ease of understanding, only one single representative m/z signal is provided in the example.

The content of the peptide in the hydrogel: the peptide content is expressed as the peptide weight relative to the hydrogel hair weight (the sum of the maleimide-functionalized hydrogel weight and the peptide linker thiol weight) . The weight of the peptide linker thiol in the hydrogel (and then the peptide weight alone) is determined by the consumption of the peptide linker thiol during the conjugation reaction with the maleimide-functionalized hydrogel. . The consumption of the peptide linker thiol was determined by the Ellman test (Ellman, GL et al, Biochem. Pharmacol., 1961 , 7, 88-95).

Example 1 Synthesis of main reagent 1g

The main reagent 1g was synthesized from the amine 4-arm PEG5000 1a according to the following procedure:

To synthesize compound 1b , the amine 4-arm PEG 5000 1a (MW about 5200 g/mole, 5.20 g, 1.00 mmol, HCl salt) was dissolved in 20 mL DMSO (anhydrous). Boc-Lys(Boc)-OH (2.17 g, 6.25 mmol) in 5 mL DMSO (anhydrous), EDC HCl (1.15 g, 6.00 mmol), HOBt ‧ H2O (0.96 g, 6.25 mmol) , and collidine (5.20 ml, 40 mmol) were added. The reaction mixture was stirred at room temperature for 30 minutes.

The reaction mixture was diluted with 1200 ml of DCM and used with 600 mL of 0.1 NH ₂ SO ₄ (2 x), brine (1 x), 0.1 M NaOH (2 x), and 1/1 (v/v) brine/water (4 x ) Cleaning. The aqueous layer was re-extracted with 500 mL of DCM. The organic phase was dried on Na ₂ SO _4, filtered and evaporated to give 6.3 g of the crude product as a colorless oil 1b. Compound 1b was purified by RP-HPLC: yield 3.85 g (yield: 59%) of colorless glassy product 1b . MS: m/z 1294.4 = [M + 5H] ^{5 +} (MW calc. [M+5H] ⁵⁺ = 1294.6).

Compound 1c was prepared by dissolving 3.40 g of compound 1b (0.521 mmol) in 5 ml of methanol and 9 ml of 4 N HCl. The alkane was obtained by stirring at room temperature for 15 minutes. The volatiles were removed in vacuo. The product was used in the next step without further purification. MS: m/z 1151.9 = [M + 5H] ^{5 +} (MW calc. [M+5H] ⁵⁺ = 1152.0).

To synthesize compound 1d , 3.26 g of compound 1c (0.54 mmol) was dissolved in 15 ml of DMSO (anhydrous). Will contain 2.99 g of Boc-Lys(Boc)-OH (8.64 mmol) in 15 ml DMSO (anhydrous), 1.55 g EDC HCl (8.1 mmol), 1.24 g HOBt‧H ₂ O (8.1 mmol) , and 5.62 ml of collidine (43 mmol) were added. The reaction mixture was stirred at room temperature for 30 minutes.

The reaction mixture was diluted with 800 ml of DCM and used with 400 ml of 0.1 NH ₂ SO ₄ (2 x), brine (1 x), 0.1 M NaOH (2 x), and 1/1 (v/v) brine/water (4 x ) Cleaning. The aqueous layer was re-extracted with 800 mL of DCM. The organic phase was dried on Na ₂ SO _4, filtered and evaporated to give the crude product as glass-like.

The product was dissolved in DCM and taken up in cold (EtOAc) EtOAc. This process was repeated twice and the precipitate was dried in vacuo. Yield: 4.01 g (89%) of colorless glassy product 1d , which was used in the next step without further purification: MS:m/z 1405.4=[M+6H] ⁶⁺ (MW calculated [M+6H] ^{6 +} =1405.4).

Compound 1e is obtained by dissolving a solution containing compound 1d (3.96 g, 0.47 mmol) in 7 ml of methanol with 20 ml of 4 N HCl. The alkane was obtained by stirring at room temperature for 15 minutes. The volatiles were removed in vacuo. The product was used in the next step without further purification. MS: m/z 969.6 = [M + 7H] ^{7 +} (MW calc. [M+7H] ⁷⁺ = 969.7).

To synthesize compound 1f , compound 1e (3.55 g, 0.48 mmol) was dissolved in 20 mL DMSO (anhydrous). Boc-Lys(Boc)-OH (5.32 g, 15.4 mmol) in 18.8 mL DMSO (anhydrous), EDC HCl (2.76 g, 14.4 mmol), HOBt ‧ H ₂ O (2.20 g, 14.4 mmol) Ear), and 10.0 ml of collidine (76.8 mmol) was added. The reaction mixture was stirred at room temperature for 60 minutes.

The reaction mixture was diluted with 800 ml of DCM and used with 400 ml of 0.1 NH ₂ SO ₄ (2 x), brine (1 x), 0.1 M NaOH (2 x), and 1/1 (v/v) brine/water (4 x ) Cleaning. The aqueous layer was re-extracted with 800 mL of DCM. The organic phase was dried on Na ₂ SO _4, filtered and evaporated to give the crude product as a colorless oil of 1f.

The product was dissolved in DCM and taken up in cold (EtOAc) EtOAc. This step was repeated twice and the precipitate was dried in vacuo. Yield: 4.72 g (82%) of colorless glassy product 1f which was used in the next step without further purification. MS: m/z 1505.3 = [M + 8H] ^{8 +} (MW calc. [M+8H] ⁸⁺ = 1505.4).

The main reagent 1g is obtained by dissolving a solution containing compound 1f (MW about 12035 g/mole, 4.72 g, 0,39 mmol) in 20 ml of methanol with 40 ml of 4 N HCl. The alkane was obtained by stirring at room temperature for 30 minutes. The volatiles were removed in vacuo. Yield: 3.91 g (100%), glass-like product backbone reagent 1 g . MS: m/z 977.2 = [M+9H] ⁹⁺ (MW calc. [M+9H] ⁹⁺ = 977.4).

1g of other synthetic routes

For the synthesis of compound 1b , boc-Lys(boc)- was added to a suspension containing 4 arms of PEG5000 tetraamine ( 1a ) (50.0 g, 10.0 mmol) in 250 ml of iPrOH (anhydrous) at 45 °C. OSu (26.6 g, 60.0 mmol) and DIEA (20.9 mL, 120 mmol) and the mixture was stirred for 30 min.

Immediately, n-propylamine (2.48 ml, 30.0 mmol) was added. After 5 minutes, the solution was diluted with 1000 ml of MTBE and stored at -20 ° C overnight without stirring. Approximately 500 ml of the supernatant was decanted and discarded. After 300 ml of cold MTBE was added and after shaking for 1 minute, the product was collected by filtration through a glass filter and washed with 500 ml of cold MTBE. The product was dried in vacuo for 16 hours. Yield: 65.6 g (74%) 1b was a white, irregular solid. MS: m/z 937.4 = [M + 7H] ^{7 +} (MW calc. [M+7H] ⁷⁺ = 937.6).

Compound 1c was obtained by stirring Compound 1b (48.8 g, 7.44 mmol) from the above procedure in 156 ml of 2-propanol at 40 °C. A mixture containing 196 ml of 2-propanol and 78.3 ml of acetamidine chloride was added with stirring over 1-2 minutes. The solution was stirred at 40 ° C for 30 minutes and cooled to -30 ° C overnight without stirring. 100 ml of cold MTBE was added, and the suspension was shaken for 1 minute and cooled at -30 ° C for 1 hour. The product was collected by filtration through a glass filter and washed with 200 mL cold MTBE. The product was dried in vacuo for 16 hours. Yield: 38.9 g (86%) 1c as a white powder MS: m/z 960.1 = [M+6H] ⁶⁺ (MW = [M+6H] ⁶⁺ = 960.2).

To synthesize compound 1d , boc-Lys(boc)-OSu (16.7 g, 37.7 mmol) and DIPEA (13.1 ml, 75.4 mmol) were added to a 1c containing the above steps (19.0) at 45 °C. Grams, 3.14 mmoles in a suspension of 80 mL of 2-propanol and the mixture was stirred at 45 ° C for 30 minutes. Immediately, n-propylamine (1.56 ml, 18.9 mmol) was added. After 5 minutes, the solution was precipitated with 600 ml of cold MTBE and centrifuged (3000 min -1,1 min). The precipitate was dried in vacuo for 1 hour and dissolved in 400 mL THF. 200 ml of diethyl ether were added and the product was cooled to -30 ° C for 16 hours without stirring. The suspension was filtered through a glass filter and washed with 300 mL cold MTBE. The product was dried in vacuo for 16 hours. Yield: 21.0 g (80%) 1d as a white solid MS: m/z: </ RTI></RTI></RTI></RTI></RTI></RTI> [M+6H] ⁶⁺ (MW calc. [M+6H] ⁶⁺ = 1405.4).

Compound 1e was obtained by dissolving Compound 1d (15.6 g, 1.86 mmol) from the above procedure in 3 N MeOH (EtOAc (EtOAc) 200 ml of MeOH and 700 ml of iPrOH were added and the mixture was stored at -30 ° C for 2 hours. To complete the crystallization, 100 ml of MTBE was added and the suspension was stored at -30 ° C overnight. 250 ml of cold MTBE was added and the suspension was shaken for 1 minute and filtered through a glass filter and washed with 100 mL cold MTBE. The product was dried in vacuo. Yield: 13.2 g (96%) 1e as a white powder MS: m/z 679.1=[M+10H] ¹⁰⁺ (MW calc. [M+10H] ¹⁰⁺ = 679.1).

To synthesize compound 1f , boc-Lys(boc)-OSu (11.9 g, 26.8 mmol) and DIPEA (9.34 ml, 53.6 mmol) were added to a 1e (8.22) from the above steps at 45 °C. Grams, 1.12 mmoles in 165 ml of 2-propanol suspension and the mixture was stirred for 30 minutes. Immediately, n-propylamine (1.47 ml, 17.9 mmol) was added. After 5 minutes, the solution was cooled to -18 °C for 2 hours, then 165 ml of cold MTBE was added and the suspension was shaken for 1 minute and filtered through a glass filter. Immediately, the filter cake was rinsed with 4 x 200 mL cold MTBE/iPrOH 4:1 and 1 x 200 mL cold MTBE. The product was dried in vacuo for 16 hours. Yield: 12.8 g, MW (90%) 1f as a pale yellow smear solid MS: m/z 1505.3=[M+8H] ⁸⁺ (MW calculated [M+8H] ⁸⁺ = 1505.4).

1 g of the obtained main reagent was obtained by dissolving in 30 ml of MeOH containing 4-arm PEG5kDa (-LysLys ₂ Lys ₄ (boc) ₈ ) ₄ ( 1f ) (15.5 g, 1.29 mmol) and cooling to 0 ° C. . 4 N HCl in two The alkane (120 mL, 480 mmol, cooled to 0 °C) was added over 3 min and the ice bath was removed. After 20 minutes, 3 N HCl was added in methanol (200 mL, 600 m.m., cooled to 0 ° C) over 15 min and the solution was stirred at room temperature for 10 min. The product solution was precipitated with 480 ml of cold MTBE and centrifuged at 3000 rpm for 1 minute. The precipitate was dried in vacuo for 1 hour and redissolved in 90 mL MeOH, which was then taken up with &lt;RTI ID=0.0&gt;&gt; 1 g of the product was dried in vacuo. Yield: 11.5 g (89%) in pale yellow flakes. MS: m/z 1104.9 = [M + 8H] ^{8 +} (MW calc. [M+8H] ⁸⁺ = 1104.9).

Example 2 Synthesis of crosslinking reagent 2d

The crosslinking reagent 2d was prepared from monobenzyl adipate (UK, Arthur R. et al., J. Med. Chem., 1990 , 33(1), 344-347) and PEG2000 according to the following diagram:

A solution of PEG 2000 (2a) (11.0 g, 5.5 mmol) and benzyl adipate half ester (4.8 g, 20.6 mmol) in DCM (90.0 mL) was cooled to 0. Dicyclohexylcarbodiimide (4.47 g, 21.7 mmol) was added followed by a catalytic amount of DMAP (5 mg) and the solution was stirred and allowed to reach room temperature overnight (12 h). The flask was stored at +4 ° C for 5 hours. The solid was filtered and the solvent was completely removed by distillation in vacuo. The residue was dissolved in 1000 ml of 1/1 (v/v) diethyl ether/ethyl acetate and stored at room temperature for 2 hours to form a small amount of a flaky solid. The solid was removed by filtration through a brine pad. The solution was stored in a tight flask at -30 ° C for 12 hours until the crystallization was complete. The crystalline product was filtered through a frit and washed with cold diethyl ether (-30 ° C). The filter cake was dried in vacuo. Yield: 11.6 g (86%) of 2b of a colorless solid. The product was used in the next step without further purification. MS: m/z 813.1 = [M + 3H] ^{3 +} (MW calculated [M+3H] ³⁺ = 813.3)

In a 500 ml glass autoclave, PEG2000-bis-adipate-bis-benzyl ester 2b (13.3 g, 5.5 mmol) was dissolved in ethyl acetate (180 ml) and 10% palladium wood Carbon (0.4 g) was added. The solution was hydrogenated at 6 bar at 40 ° C until the hydrogen ceased to be consumed (5-12 hours). Filtration method via Celite salt pad (Celite The catalyst was removed and the solvent was evaporated in vacuo. Yield: 12.3 g (quantitative) 2c was a pale yellow oil. The product was used in the next step without further purification. MS: m/z 753.1 = [M + 3H] ^{3 +} (MW calculated [M+3H] ³⁺ = 753.2)

Will contain PEG2000-bis-adipate half ester 2c (9.43 g, 4.18 mmol), N-hydroxysuccinimide (1.92 g, 16.7 mmol) and dicyclohexylcarbodiimide (3.44 g, A solution of 16.7 mmoles in 75 mL of DCM (anhydrous) was stirred at room temperature overnight. The reaction mixture was cooled to 0 ° C and the precipitate was filtered. The DCM was evaporated and the residue was crystallized from THF. Yield: 8.73 g (85%) of cross-linking reagent 2d as colorless solid MS: m/z 817.8 = [M+3H] ³⁺ (MW calc. [M+3H] ³⁺ = 817.9 g/m).

Example 3 Preparation of hydrogel beads (3) containing free amine groups

A solution containing 1200 mg of 1 g and 3840 mg of 2d in 28.6 ml of DMSO was added to a solution containing 425 mg of Arlacel P135 (British Cologne International Co., Ltd.) in 100 ml of heptane. The mixture was stirred with a screw mixer at 650 rpm at 25 ° C for 10 minutes to form a suspension in a 250 ml reactor equipped with a baffle. 4.3 ml of TMEDA was added for polymerization. After 2 hours, the stirring speed was lowered to 400 rpm and the mixture was stirred for another 16 hours. 6.6 ml of acetic acid was added and then, after 10 minutes, 50 ml of water and 50 liters of a saturated aqueous solution of sodium chloride were added. After 5 minutes, the stirrer was stopped and the aqueous phase was drained.

The water-hydrogel suspension was wet sieved on 75, 50, 40, 32 and 20 micron mesh steel screens when fractionating the pellet size. It will remain at 32, 40 and 50 micron sieve fractions were collected and the pellet washed three times with water, washed with ethanol, and 10 times at 0.1 mbar for 16 hours to give 3 as a white powder of.

The content of the amine group in the hydrogel is achieved by coupling the fmoc-amino acid to the free amine group of the hydrogel and then as Gude, M., J. Ryf, et al. (2002) Letters in Peptide Science 9 (4): Measured by the fmoc-measure method described in 203-206.

The amine group content of the different batches of 3 was determined to be between 0.11 and 0.16 mmol/g.

Example 4 Preparation of maleic imine-functionalized hydrogel beads (4) and measurement of maleimide substitution

The hydrogel pellets 3 were pre-washed with 99/1 (v/v) DMSO/DIPEA, washed with DMSO and containing Mal-PEG6-NHS (2.0 eq relative to the theoretical amount of amine groups in the hydrogel) ) Incubate in DMSO for 45 minutes. The pellet 4 was washed 5 times with DMSO and washed 5 times with pH 3.0 succinate (20 mM, 1 mM EDTA, 0.01% Tween-20). The samples were washed 3 times with pH 6.0 sodium phosphate (50 mM, 50 mM ethanolamine, 0.01% Tween-20) and incubated for 1 hour at room temperature in the same buffer. Thereafter, the pellet was washed 5 times with sodium succinate (20 mM, 1 mM EDTA, 0.01% Tween-20) at pH 3.0.

To determine the maleimide content, the entire hydrogel pellet 4 was rinsed with water and ethanol each time. The samples were lyophilized and weighed. Another portion of the hydrogel pellet 4 was reacted with an excess of thiolethanol (in 50 mM sodium phosphate buffer, 30 minutes at room temperature) and passed through an Ellman test (Ellman, GL et al, Biochem. Pharmacol . , 1961 , 7, 88-95) Determination of the consumption of thiol-ethanol. The content of maleimide was determined to be between 0.10 and 0.13 millimoles per gram of dry hydrogel.

Example 5 Synthesis of linker reagents 5c

The linker reagent 5c was synthesized according to the following scheme:

Synthesis of linker reagent intermediate 5a :

m-Methoxytrityl chloride (3 g, 9.71 mmol) was dissolved in DCM (20 mL) and added dropwise to EtOAc (EtOAc, EtOAc, EtOAc ) in the solution. After 2 hours, the solution was poured into diethyl ether (300 mL) and washed 3 times with 30/1 (v / v) brine / 0.1 M NaOH solution (50 ml each time) and once with brine (50 ml) . The organic phase was dried on Na ₂ SO ₄ and the volatiles were removed under reduced pressure. Mmt-protected intermediate (3.18 g, 9.56 mmol) was used in the next step without further purification.

The Mmt-protected intermediate (3.18 g, 9.56 mmol) was dissolved in anhydrous DCM (30 mL). Add 6-(S-tritylhydroxythio)hexanoic acid (4.48 g, 11.47 mmol), PyBOP (5.67 g, 11.47 mmol) and DIPEA (5.0 mL, 28.68 mmol) and The mixture was stirred at room temperature for 30 minutes. The solution was diluted with diethyl ether (250 mL) and washed three times with 30/1 (v/v) brine / 0.1 M NaOH solution (50 mL each) and washed once with brine (50 mL). The organic phase was dried on Na ₂ SO ₄ and the volatiles were removed under reduced pressure. 5a was purified by flash chromatography. Yield: 5.69 g (8.09 mmol). MS: m/z 705.4 = [M+H] ⁺ (MW = 705.0).

Synthesis of linker reagent intermediate 5b :

Containing 5a (3.19 g, 4.53 mmol) was added BH in dry THF (50 ml) solution of _{3 ‧THF} (1 M solution, 8.5 mL, 8.5 mmol) and the solution was stirred at room temperature for 16 hours. Further BH ₃ ‧ THF (1 M solution, 14 mL, 14 mmol) was added and stirred at room temperature for 16 h. The reaction was quenched by the addition of methanol (8.5 mL). N,N-Dimethyl-ethylenediamine (3 mL, 27.2 mmol) was added and the solution was heated to reflux for 3 h. The reaction mixture was cooled to room temperature and was then be diluted with ethyl acetate (300 mL), washed with saturated aqueous ₂ CO ₃ Na (2 x 100 mL) and saturated aqueous NaHCO ₃ (2 x 100 mL). The organic phase was dried on Na ₂ SO ₄ and the volatiles were removed under reduced pressure to give the crude amine intermediate (3.22 g).

The amine intermediate (3.22 g) was dissolved in DCM (5 mL). Boc ₂ O (2.97 g, 13.69 mmol) was dissolved in DCM (5 mL) and DIPEA (3.95 mL, 22.65 mmol) was added and the mixture was stirred at room temperature for 30 min. The mixture was purified by flash chromatography to give crude Boc- and Mmt-protected intermediate (3.00 g). MS: m / z 791.4 = [M + H] ⁺ , 519.3 = [M-M.sup.+H] ⁺ (MW = 791.1).

0.4 M aqueous HCl (48 mL) was added to a solution containing a mixture of Boc- and Mmt-protected intermediate in acetonitrile (45 mL). The mixture was diluted with acetonitrile (10 mL) and stirred at room temperature for 1 hour. Immediately, the pH of the reaction mixture was adjusted to 5.5 by the addition of a 5 M NaOH solution. The acetonitrile was removed under reduced pressure and the aqueous was extracted with DCM (4 <RTIgt; The combined organic phases in the Na ₂ SO ₄ dried, and the volatiles were removed under reduced pressure. The crude 5b was used in the next step without further purification. Yield: 2.52 g (3.19 mmol). MS: m/z 519.3 = [M+H] ⁺ (MW calc. = 519.8 g/m).

Synthesis of linker reagent 5c:

Intermediate 5b (985 mg, 1.9 mmol) and p-nitrophenyl chloroformate (330 mg, 2.5 mmol) were dissolved in dry THF (10 mL). DIPEA (0.653 mL, 3.7 mmol) was added and the mixture was stirred at room temperature for 2 h. The solution was acidified by the addition of acetic acid (1 mL). 5c was purified by RP-HPLC. Yield: 776 mg, (1.13 mmol). MS m/z 706.3 = [M+Na] ⁺ (MW = 706.3).

Example 6 Synthesis of Exenatide Linker Reagent 6d

Exenatide linker reagent 6d was synthesized according to the following procedure:

Synthesis of Exenatide Linker Reagent Intermediate 6a:

A completely protected N-terminal endothelin-containing exenatide-4 (2.00 g, 0.2 mmol, load about 0.1 mmol/g) on the resin was transferred to 20 ml with filter material. In the syringe. 8 ml of anhydrous DMF was introduced into the syringe and the syringe was shaken (600 rpm) for 15 minutes to pre-expand the resin. The solvent was discarded and one contained Fmoc-D-alanine-OH (187 mg, 0.6 mol), PyBOP (312 mg, 0.6 mmol), and DIPEA (174 μL, 1.0 mmol) in anhydrous A solution of DMF (4 ml) was introduced into the syringe. The syringe was shaken at room temperature and 600 rpm for 60 minutes. The solution was drained and the resin was washed 10 times with DMF.

Fmoc-deprotection is carried out according to "MaterialS and Methods".

Synthesis of Exenatide Linker Reagent Intermediate 6b :

A solution containing 5c (137 mg, 0.4 mmol) in dry DMF (3 mL) was added to Resin 6a (0.2 mmol), followed by one containing DIPEA (80 μL, 0.46 mmol) A solution of anhydrous DMF (4.5 mL) was added and the reaction mixture was shaken (600 rpm) at 22 ° C for 15 hours.

The resin was washed 10 times with DMF and 10 times with DCM and dried in vacuo.

Synthesis of Exenatide Linker Reagent Intermediate 6c :

3-Nitro-2-pyridine-sulfenyl chloride (48 mg, 0.25 mmol) was filled into a syringe containing 6b (0.05 mmol, 0.5 g). Anhydrous DCM (4 mL) was introduced into a syringe and the mixture was shaken at room temperature. After 2 hours, the solution was discarded and the resin was washed 14 times with DCM and dried in vacuo.

Synthesis of Exenatide Linker Reagent Intermediate 6d:

In a round bottom flask, o-cresol (1.5 ml), thioanisole (1.5 ml), DTT (1.125 g), TES (1.125 ml), and water (1.5 ml) were dissolved in TFA (37.5 ml) in. 6c (0.15 mmol, 1.5 g) was added to the stirred (250-350 rpm) solution at room temperature to give a homogeneous suspension. Stirring was continued for 45 minutes. The solution was separated from the resin pellet by filtration, and the pellet was washed twice with TFA (2 ml each time) and the washing solution was combined with the filtrate. The TFA was removed from the combined solution in a stream of nitrogen.

The crude 6d was precipitated from a concentrated solution (ca. 10 mL) by diethyl ether (30 mL) and vigorously shaken. After centrifugation (2 minutes, 5000 rpm), the supernatant was discarded and the precipitate was washed twice with diethyl ether (20 mL each time).

The dried precipitate was dissolved in a solution containing TCEP (114 mg, 0.39 mmol) in 30 mL of 1/19 (v/v) acetonitrile/water containing 0.01% TFA (v/v). The mixture was incubated for 15 hours at room temperature. The 6d was purified by illustration in the Materials and Methods used in the RP-HPLC 150 x 30 mm Waters XBridge ^TM BEH300 C18 10 micron column and 40 ml / minute flow rate.

A mixture of up to 12 ml is loaded on the column. The elution was carried out using a linear gradient of 5% to 30% solvent B (5 minutes) followed by a linear gradient (40 minutes) from 30% to 35% solvent B. Fractions containing product 6d were concentrated and lyophilized. Purity: 86% (215 nm) Yield: 85.2 mg (19.2 micromoles starting from 2.00 grams of resin). MS m/z 1486.7 = [M+3H] ³⁺ , (MW calculated = 4460.0 g/m).

Example 7 Synthesis of Exenatide Linker Reagent 7

Exenatide linker reagent 7 is as described in Exenatide Linker Reagents 6a-6d . The free N-terminal exenatide-4 (336 mg, completely protected on the resin) 34 micromolar was synthesized starting but using Fmoc-L-alanine-OH instead of Fmoc-D-alanine-OH. The reagents were metered accordingly to obtain the same ratios as used in 6a-6d. Yield: 13.4 mg MS: m/z 1487.4 = [M+3H] ³⁺ (MW calculated = 4460.0)

Example 8 Ai Synthesis of Serenide Linker Hydrogel 8

242.5 mg of maleimide-functionalized hydrogel 4 (25.0 micromolar quinone imine group), such as succinate buffer (20 mM, 1 mM EDTA, 0.01%) suspended at pH 3.0 Tween-20) is filled into a syringe containing filter material. The hydrogel was washed 10 times with 1/1 (v/v) acetonitrile/water containing 0.1% TFA (v/v). A solution containing exenatide linker reagent 6d (122.7 mg, 27.5 micromoles) in 1/1 (v/v) acetonitrile/water plus 0.1% TFA (3.7 mL) was taken and shaken at room temperature for 2 min. A balanced suspension is obtained. 334 microliters of phosphate buffer (pH 7.4, 0.5 M) was added and the syringe was agitated (600 rpm) for 15 minutes at room temperature. The consumption of mercaptans was monitored by the Ellman test. The hydrogel was washed 10 times with 1/1 (v/v) acetonitrile/water containing 0.1% TFA (v/v).

Hydrogenthioethanol (47 μL) was dissolved in 1/1 (v/v) acetonitrile/water + 0.1% TFA (3 mL) and phosphate buffer (0.5 mL, pH 7.4, 0.5 M). The solution was taken out to the syringe and the sample was stirred (600 rpm) for 1 hour at room temperature. The solution was discarded and the hydrogel was washed 10 times with 1/1 (v/v) acetonitrile/water + 0.1% TFA. The hydrogel was then washed 10 times with succinate buffer (10 mM succinate, 46 g/L mannitol, 0.05% Tween-20, adjusted to pH 5.0 with Tris) and stored at 4 °C.

The exenatide content in the Exenatide linker hydrogel was determined according to Materials and Methods.

An exenatide was obtained in an amount of 30% by weight.

Example 9 Release kinetics in vitro

Transfer the entire portion of Exendin Linker Hydrogel 8 (0.5 mg exenatide) to a syringe containing filter material and use pH 7.4 phosphate buffer (60 mM, 3 mM EDTA, 0.01% Tween) -20) Wash 5 times. The hydrogel was suspended in the same buffer and incubated at 37 °C. At the time point (one to seven days after the incubation period), the supernatant was exchanged and the released exenatide was quantified by RP-HPLC of 215 nm. The UV-signals associated with the released exenatide were pooled and plotted against the incubation period.

The curve fitting soft system is used to estimate the relevant release half-life.

The first-order release kinetics with a half-life of 45 days was obtained (see Figure 1 ).

Example 10 Synthesis of Exenatide Linker Hydrogel 10

Exenatide linker hydrogel 10 is synthesized as described in exenatide linker hydrogel 8 but synthesized with exenatide linker thiol 7 instead of exenatide linker thiol 6d . .

The exenatide content in the Exenatide linker hydrogel was determined according to Materials and Methods. An exenatide was obtained in an amount of 30.5% by weight.

Example 11 Synthesis of lixisenatide linker reagent 11 d

Composite icon:

Synthesis of lixisenatide linker intermediate 11a :

A fully protected, free N-terminal lixisenatide (300 mg, loading of about 0.1 mmol/g) on the resin was transferred to a 5 ml syringe equipped with filter material. 4 ml of anhydrous DMF was introduced into the syringe and the syringe was shaken (600 rpm) for 15 minutes to pre-expand the resin. The solvent was discarded and will contain Fmoc-D-alanine-OH (28 mg, 90 micromoles), PyBOP (47 mg, 90 micromoles), and DIPEA (26 microliters, 150 micromoles) in anhydrous A solution of DMF (2 ml) was introduced into the syringe. The syringe was shaken at room temperature and 600 rpm for 60 minutes. The solution was drained and the resin was washed 10 times with DMF.

Fmoc-deprotection is carried out according to "Materials and Methods".

Synthesis of lixisenatide linker reagent intermediate 11b :

A solution containing 5c (41 mg, 60 micromoles) in dry DMF (1.5 mL) was added to Resin 11a (30 micromoles) followed by DIPEA (13 microliters, 75 micromoles) and The homogenized reaction mixture was shaken (600 rpm) at 22 °C for 22 hours.

The resin was washed 10 times with DMF and dried in vacuo.

Synthesis of lixisenatide linker intermediate 11c :

3-Nitro-2-pyridine-sulfenyl chloride (38 mg, 0.20 mmol) was added to a syringe equipped with a filter containing 11b . Anhydrous DCM (2 mL) was introduced into a syringe and the mixture was shaken at room temperature (600 rpm). After 3.5 hours, the solution was discarded and the resin was washed 15 times with DCM and dried in vacuo.

Synthesis of lixisenatide linker reagent 11d :

In a 50 ml-Falcon tube, NBu ₄ Br (2.9 mg), thioanisole (58.3 μl), DTT (170 mg), TES (170 μL), and water (113.3 μl) were dissolved in TFA (5.83 ml). At room temperature, 11c (30 micromoles) was added to the stirred (200 rpm) solution to obtain a homogeneous suspension. Stirring was continued for 1 hour. The pellet was filtered and washed twice with TFA (1 mL each time). The cleaning solution is combined with the filtrate.

The crude 11d was precipitated from the filtrate (about 10 mL) by adding cold diethyl ether (-18 ° C, 40 mL) and vigorously shaking. The suspension was cooled again at -18 ° C for 15 minutes and centrifuged (2 minutes, 5000 rpm). The supernatant was discarded and the precipitate was washed twice with diethyl ether (20 mL each time) and dried under reduced pressure. The precipitate was dissolved in a solution containing TCEP (27 mg, 0.94 micromolar) in 2.5 ml of 1/1 (v/v) acetonitrile/water containing 0.01% TFA (v/v). The mixture was incubated at room temperature for 1.5 hours. 20 ml of water was added and 11d was subjected to a linear gradient from 5% to 30% solvent B (5 minutes) followed by a linear gradient from 30% to 35% solvent B (40 minutes) by RP-HPLC in two flows Purified. Using 150 x 30 mm Waters XBridge ^TM BEH300 C18 10 micron column and 40 ml / minute flow rate. The fractions containing product 11d were concentrated and lyophilized. Yield 6.1 mg MS: m/z 1284.3 = [M+4H] ⁴⁺ (MW = 5131.9).

Example 12 Synthesis of lixisenatide linker hydrogel 12

The lixisenatide conjugate hydrogel 12 line was synthesized as described in the exenatide linker hydrogel 8 but using lixisenatide to link the thiol 11d instead of the exenatide linker thiol 6d .

The content of lixisenatide in the ligature-linked hydrogel is determined according to Materials and Methods. A 32.4% lixisenatide content was obtained.

Example 13 Synthesis of lixisenatide linker reagent 13

The lixisenatide linker 13 is synthesized as described in the ligatorium linker reagent 11a-11d , which is used to support the fully protected N-terminal lixisenatide (335 mg) on the resin. , 34 micromolar starting, instead of using Fmoc-D-alanine-OH. The reagents were metered accordingly to give the same ratios as used in 11a-11d . Yield 7.3 mg MS: m/z 1283.9 = [M+4H] ⁴⁺ (MW = 5131.9).

Example 14 Synthesis of lixisenatide linker hydrogel 14

The lixisenatide linker hydrogel 14 series, as described in the exenatide linker hydrogel 8 , was synthesized using lixisenatide for the linker thiol 13 instead of the exenatide linker thiol. 6d .

The lixisenatide content of the lixisenatide linker hydrogel is determined according to Materials and Methods. A lixisenatide content of 34.5% by weight was obtained.

Synthesis of GLP-1 Linker Reagent 15

The GLP-1 linker reagent 15 is synthesized as described in the lixisenatide linker reagent 11a-11d , but is a completely protected N-terminal GLP-1 (258 mg, 30 μm) Moer) starts with exenatide instead of resin. The reagents were metered accordingly to give the same ratios as used in 11a-11d . Yield 5.0 mg MS: m/z 1191.4 = [M+3H] ³⁺ (MW = 3571.1).

Example 16 Synthesis of GLP-1 Linker Hydrogel 16

The GLP-1 linker hydrogel 16 was synthesized as described in Exetinide Linker Hydrogel 8 , but using the GLP-1 linker thiol 15 instead of the Exenatide linker thiol 6d .

The GLP-1 content in the GLP-1 linker hydrogel was determined according to Materials and Methods. GLP-1 was obtained in an amount of 26.3% by weight.

Example 17 Release kinetics in vitro

Exenatide at pH 7.4,37 ℃ released by the half-life of the hydrogel 10, release half-life lixisenatide reason hydrogel of 12 and 14, and GLP-1 released from the hydrogel 16 The half-life phase was determined as described in Example 9. The release kinetics of compounds 8 , 10 , 12 , 14 and 16 are shown in Figure 1.

Example 18 Synthesis of linker reagent 18e

The linker reagent 18e was synthesized according to the following scheme :

The synthesis of the linker reagent intermediate 18b was carried out under a nitrogen pressure. A solution containing amine 18a (1.69 g, 4.5 mmol, prepared as described in WO-A 2009/133137) in 30 ml of THF (dry, m. sift) was cooled to 0 °C. Butyl chloroformate (630 microliters, 4.95 millimolar) was added to 3 ml of THF (dry, m. sieve) and DIPEA (980 microliters, 5.63 millimoles). The mixture was stirred at 0 °C for 10 minutes, cooled and the mixture was stirred at room temperature for 20 min. 1 M LiAlH ₄ was added in THF (9 mL, 9 mmol) and the mixture was refluxed for 1.5 hr. The reaction was quenched by the slow addition of methanol (11 mL) and 100 mL of saturated Na/K tartrate. The mixture was extracted with ethyl acetate, the organic layer was dried over Na ₂ SO ₄ and the solvent was evaporated under reduced pressure. The crude product (1.97 g) was used in the next step without further purification. MS: m/z 390.2 = [M+H] ⁺ (MW = 389.6).

The crude product 18b (1.97 g), N-(bromoethyl)-indenimide (1.43 g, 5.63 mmol) and K ₂ CO ₃ (1.24 g, 9.0 mmol) were taken in 120 ml of acetonitrile. The solution was refluxed for 6 hours. 60 ml of a saturated NaHCO ₃ solution was added and the mixture was extracted three times with ethyl acetate. The combined organic phases were dried (Na ₂ SO _4), and the solvent removed under reduced pressure. The phthalocyanine (PEI) 18c on silica with heptane (containing 0.02% NEt ₃₎ and increasing the amount of ethyl acetate (containing 0.02% NEt ₃₎ were purified as eluents. Yield: 0.82 g (1.46 mmol) MS: m/z 563.3 = [M+H] ⁺ (MW = 562.8).

The quinone imine 18c (819 mg, 1.46 mmol) was dissolved in 35 ml of ethanol and hydrazine hydrate (176 μl, 3.64 mmol) was added. The mixture was refluxed for 3 hours. The precipitate was filtered off. The solvent was removed under reduced pressure and the residue was taken up in 15 mL dichloromethane. The precipitate was filtered off and the dichloromethane was removed under reduced pressure. The residue was purified by RP HPLC. The HPLC fractions were concentrated by the addition of NaHCO ₃ to adjust to pH 7 and extracted several times with dichloromethane. The combined organics were dried (Na ₂ SO ₄₎ and the solvent removed under reduced pressure to give the amine 18d. Yield: 579 mg (1.34 mmol) MS: m/z 433.3 = [M+H] ⁺ (MW = 432.7).

p-Nitrophenyl chloroformate (483 mg, 2.40 mmol) was dissolved in 10 mL dichloromethane (dry, m.). A solution containing amine 18d (1.00 g, 2.31 mmol) in 5 mL dichloromethane (dry, m.) and &lt The methylene chloride was removed under reduced pressure, the residue was acidified with acetic acid and purified by RP-HPLC to yield p-nitrophenyl carbamic acid ester 18e . Yield: 339 mg (0.57 mmol) MS: m/z 598.3 = [M+H] ⁺ (MW = 597.8).

G Synthesis of LP-1 Linker Reagent 19

The GLP-1 linker reagent 19 is synthesized as described in the GLP-1 linker reagent 15 except that the linker reagent 18e is used in place of the linker reagent 5c, and the free N-terminal is completely protected by a branch on the resin. Start with GLP-1 (150 mg, 16.5 micromoles). The reagents were metered accordingly to give the same ratios as used in 11a-11d . Yield: 1.33 mg MS: m/z 1196.0 = [M+3H] ³⁺ (MW = 3585.1).

Example 20 Synthesis of GLP-1 Linker Hydrogel 20

The GLP-1 linker hydrogel 20 was synthesized as described in Exetinide Linker Hydrogel 8 , but the GLP-1 linker thiol 19 was used in place of the Exenatide linker thiol 6d .

Example 21 Release kinetics in vitro

The half-life phase of GLP-1 released from hydrogel 20 was measured as in Example 9 at pH 7.4, 37 °C.

abbreviation:

AcOH acetic acid

AcOEt ethyl acetate

Bn benzyl

Boc tert-butoxycarbonyl

DBU 1,3-diazabicyclo[5.4.0]undecene

DCC N,N-di-hexylcarbodiimide

DCM dichloromethane

DIPEA diisopropylethylamine

DMAP dimethylamino-pyridine

DMF N,N-dimethylformamide

DMSO dimethyl hydrazine

DTT DL dithiothreitol

EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

EDTA ethylene diamine tetraacetic acid

Eq chemical equivalent

EtOH ethanol

Fmoc 9-fluorenylmethoxycarbonyl

HPLC High Performance Liquid Chromatography

HOBt N-hydroxybenzotriazole

iPrOH 2-propanol

LCMS mass spectrometry-coupled liquid chromatography

Mal 3-Malayiminepropyl

Mal-PEG6-NHS N-(3-maleimidopropyl)-21-amino-4,7,10,13,16,19-hexaoxa-nonanoic acid NHS ester

Me methyl

MeOH methanol

Mmt 4-methoxytrityl

MS mass spectrometry / mass spectrometry

MTBE methyl tert-butyl ether

MW molecular weight

NHS N-hydroxy amber imine

PEG poly(ethylene glycol)

PyBOP benzotriazol-1-yl-oxy-tri-pyrrolidine-ruthenium hexafluorophosphate

Phth quinone imine

RP-HPLC reverse phase high performance liquid chromatography

Rpm revolutions per minute

RT room temperature

SEC molecular screening chromatographic separation

TCEP tris(2-carboxyethyl)phosphine hydrochloride

TES triethyl decane

TFA trifluoroacetic acid

THF tetrahydrofuran

TMEDA N,N,N ' ,N ' -tetramethylethylenediamine

Tris tris(hydroxymethyl)aminomethane

Trt triphenylmethyl, trityl

UPLC Super Performance Liquid Chromatography

v volume

<110> Sanofi-Aventis Deutschland GmbH

<120> Prescription containing exenatide linker conjugate

<130> DE2010/304S

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Figure 1 shows the release kinetics of compounds 8, 10, 12, 14 and 16 at pH 7.4, 37 °C.

Claims (22)

A prodrug or a pharmaceutically acceptable salt thereof, comprising an exendin linker conjugate DL, wherein D represents an Exenatide moiety; and -L is a non-symbol represented by Formula (I) Biologically active linker moiety - L ¹ , Wherein, the dotted line refers to one of the amine groups attached to exenatide by forming a guanamine bond; R ¹ is selected from a C _1-4 alkyl group; and R ² , R ^2a are independently selected from H and a group of a group consisting of C _1-4 alkyl groups, wherein the L ¹ group is substituted by one L ² -Z and optionally further substituted, but the hydrogen marked with an asterisk in the formula (I) is not replaced by a substituent And wherein L ² is a single chemical bond or a spacer; and Z is a hydrogel. For example, before the application of the first paragraph of the patent scope, L ^{1 is} not further substituted. For example, the drug of claim 1 or 2, wherein R ¹ is -CH ₃ -. The prodrug of any one of claims 1 to 3, wherein L ² is a C _1-20 alkyl chain, which is optionally one or more independently selected from -O-; and C(O)N (R3aa) of the inserted group; any one or more groups independently selected from OH; and substituted with C (O) N (R3aaR3aaa) of the group; and wherein R3aa, R3aaa based independently selected from H; C ₁ and a group of -C ₄ alkyl groups. The prodrug of any one of claims 1 to 4, wherein L ² is linked to Z via a terminal group selected from the group consisting of For example, before applying for any of the patent scopes 1 to 5, where L is represented by formula (Ia) Wherein the dotted line refers to nitrogen attached to exenatide by forming a guanamine bond; and wherein L ² is a single chemical bond or a spacer, and Z is a hydrogel. For example, before applying for any of the first to sixth patents, L is represented by formula (II). Wherein the dotted line refers to nitrogen attached to exenatide by formation of a guanamine bond and Z is a hydrogel. The prodrug of any one of claims 1 to 7 wherein the hydrogel is a PEG comprising a hydrogel comprising a backbone portion. For example, before applying for the patent scope, the main part contains the branch core of the following formula: The dotted line refers to the remaining portion connected to the trunk portion. For example, before applying for the patent scope, the main part contains the structure of the following formula: Wherein n is an integer from 5 to 50 and the dashed line refers to the remainder of the molecule. A pharmaceutical preparation according to any one of claims 8 to 10, wherein the trunk portion comprises a hyperbranched portion Hyp of the following formula: Wherein the dashed line refers to the remainder of the molecule; and the carbon atom labeled with an asterisk refers to the S-configuration. The pharmaceutical preparation according to any one of claims 8 to 11, wherein the trunk portion comprises at least one spacer of the following formula: Wherein one of the dotted lines refers to the hyperbranched moiety Hyp and the second dashed line refers to the remainder of the molecule; and wherein m is an integer from 2 to 4. The pharmaceutical preparation according to any one of claims 8 to 12, wherein the trunk portion comprises at least one spacer of the following formula: Wherein the dotted line marked with an asterisk refers to a bond between the hydrogel and N of the thiosuccinimide group in the fifth aspect of the patent application; wherein another dotted line means a link to Hyp; and wherein p It is an integer from 0 to 10. The pharmaceutical preparation according to any one of claims 8 to 13, wherein the trunk portion is connected via a crosslinking agent portion comprising the following structure Where q is an integer from 3 to 100. A pharmaceutical composition comprising a prodrug or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 14 and at least one pharmaceutically acceptable excipient. For example, the pharmaceutical composition of any one of claims 1 to 14 or the pharmaceutical composition of claim 15 of the patent application is used as a pharmaceutical. A pharmaceutical composition according to any one of claims 1 to 14 or a pharmaceutical composition according to claim 15 for treating or preventing a disease or disorder which can be treated with exenatide. An exenatide-linker reagent DL*, wherein D represents an Exenatide moiety; and -L* is a non-biologically active linker reagent represented by Formula (IV), Wherein the dotted line refers to one of the amine groups attached to exenatide by forming a guanamine bond; R ¹ is selected from a C _1-4 alkyl group; and R ² , R ^2a are independently selected from H and a group of a group consisting of C ₁ -C ₄ alkyl groups, wherein the L* system is substituted by one L ² * and optionally further substituted, but the hydrogen marked with an asterisk in the formula (IV) is not substituted Substituted and wherein L ² * is a spacer attached to L* and comprises a chemical functional group for bonding to the hydrogel. An Exenatide-linker conjugate intermediate D-L', wherein L' is of the following formula (III) Wherein the dashed line refers to one of the amine groups attached to Exenatide by the formation of a guanamine bond. A method for preparing a prodrug according to any one of claims 1 to 14, which comprises the following step (a) comprising a horse in a buffered aqueous solution having a pH of 5.5-8 between room temperature and a temperature of 4 ° C An aqueous suspension of the hydrazide-functionalized hydrogel microparticles is contacted with a solution comprising an Exenatide linker reagent of claim 17 wherein the chemical functional group L ² * comprises a thiol group , producing an exenatide-linker-hydrogel conjugate; (b) optionally, the exenatide-linker-hydrogel conjugate from step (a) at room temperature and 4 ° C The temperature is treated with a 34-500 Da thiol-containing compound in a buffered aqueous solution at pH 5.5-8. A method for preparing a prodrug according to any one of claims 1 to 14, which comprises the following steps (a) comprising a thiol in a buffered aqueous solution of pH 5.5-8 at room temperature and 4 ° C; An aqueous suspension of the functionalized hydrogel microparticles is contacted with a solution comprising an Exenatide-linker reagent of claim 18, wherein the chemical functional group L ² * comprises a maleimide group , producing an exenatide-linker-hydrogel conjugate; (b) optionally, the exenatide-linker-hydrogel conjugate from step (a) at room temperature and 4 ° C The temperature is treated with 100 to 300 Da of the compound containing maleimide in a buffered aqueous solution of pH 5.5-8. A method of preparing a needle-shaped injectable prodrug, comprising the steps of: (a) preparing a prodrug in the form of a microparticle according to any one of claims 1 to 14; (b) sieving the microparticle; (c) The prodrug fraction having a diameter between 25 and 80 microns is selected; (d) the pellet portion of step (c) is suspended in an aqueous buffer solution suitable for injection.