prodrugs comprising an exendin ligand conjugate

专利摘要:
PROPHARMACES UNDERSTANDING AN EXENDINA BINDING CONJUGATE. The present invention relates to a prodrug or a pharmaceutically acceptable salt thereof comprising an exendin ligand conjugate D-L, wherein D represents an exendin moiety; and -L is a non-biologically active linker portion -L ^ 1 ^ represented by formula (I), in which the dotted line indicates the bond to one of the amino groups of the exendia forming an amide bond. The invention also relates to pharmaceutical compositions comprising said prodrugs as well as their use as a drug for the treatment or prevention of diseases or disorders that can be treated by exendin.
公开号:BR112013006340A2
申请号:R112013006340-8
申请日:2011-09-16
公开日:2020-08-04
发明作者:Felix Cleemann；Ulrich Hersel；Torben Lessmann；Harald Rau
申请人:Sanofi-Aventis Deutschland Gmbh；
IPC主号:

专利说明:

PAD AAA ATA EEE AA APT TP AAA A AA AE EA AA AAA AA Ao A o EE A o A aa A RARA DRA RR MR Frog "! REUSE: 1/97, Invention Patent Description for" UNDERSTANDING PROFILES AN EXEN- DINE BINDING CONJUGATE ". E The present invention relates to exendin prodrugs, pharmaceutical compositions comprising said prodrugs as well as their use as a drug for the treatment or prevention of diseases or disorders that can be treated by exendina.
Exendina4 is an amino acid peptide 39, isolated from the salivary secretions of the poisonous Gila monster (Heloderma suspectum). It has some sequence similarity to several members of the glucagon-like peptide family, with the highest homology of 53% ”for peptide 1 similar to glucagon [7-36] -amide (GLP-1). A + exendin4 acts as a GLP-1 agonist on the GLP-1 receptor and transports insulin secretagogue action similar to GLP-1 in islets - isolated derate.
Exendin-4 is a high potency agonist and truncated exendin-f (9-39) -amide an antagonist at the glucagon-like peptide 1 receptor (7-36) -amide of beta cells secreting insulin. (see, for example, J.
Biol.
Chem. 268 (26): 19650-19655). Exendin-4 ("performed") was recently approved in the United States and the European Union to improve glycemic control in patients with type 2 diabetes taking metformin and / or sulfonylurea, but without adequate glycemic control obtained.
Current exenatide therapy requires frequent injections (twice daily), resulting in elevated plasma levels after injection, which are correlated with nausea (see, Nauck M.
A., Meier J.
J. (2005), Regul Pept. 128 (2): 135-148), and with lower vat concentrations, inducing incomplete glycemic control (see, Kim D., et al. (2007), Diabetes Care. 30 (6): 1487 -1493). To overcome these problems, a longer-acting formulation for exendin4 is highly desirable.
Ideally, the peptide is formulated in a model that provides an extended plasma level in a human for at least a week after application to a human body resulting in a frequency
- 2/97 injection at once weekly or longer. Several long-acting ex- perts have been proposed. To enhance the physicochemical or pharmacokinetic properties of a drug in vivo, such as its half-life, that drug can be combined with a vehicle. If the drug is transiently linked to a € / vehicle or a linker, such systems are commonly referred to as vehicle linked prodrugs. According to the definitions provided by IU-PAC (as provided at http: /MWwww.chem.qmul.ac.uk/iupac.medchem, terminated on 22 July 22, 2009), a vehicle-linked prodrug it is a prodrug that contains a temporary binding of a certain active substance with a transient carrier group that produces properties: improved physico-chemical or pharmacokinetic and that can be easily removed in vivo, usually by hydrolytic cleavage.
The linkers employed in such vehicle-linked prodrugs can be transient, meaning that they are hydrolytically degradable non-enzymatically (cleavable) under physiological conditions (aqueous buffer at pH 7.4, 37ºC) with half-lives ranging from, for example example, one hour to three months. Suitable vehicles are polymers and can be directly attached to the binder or by means of a non-cleavable spacer.
The conjugation of transient polymer through non-trace prodrug linkers combines the advantages of extended circulation periods due to polymer binding and the recovery of the native pharmacology of the native peptide after release of the polymer conjugate.
Using polymer linker peptide conjugates, native unaltered peptide is slowly released after application to a patient, only determined by ligand release kinetics and pharmacokinetics of the polymer vehicle. Ideally, release kinetics should be independent of the presence of enzymes such as proteases or esterases in body fluids to ensure a consistent and homogeneous release pattern.
International Patent Application WO-A 2009/095479 relates to prodrugs that comprise drug linker conjugates, where the linker is covalently linked by means of a cleavable link in one and 3/97 | biologically active portion, such as exendin. The biologically active portion is released from the prodrug upon activation of cyclization by the formation of cyclic imide. An exendin prodrug is described in which the linker is based on L-alanine.
Still, there remains a need to develop long-acting exendin drugs with longer half-lives. Therefore, an objective of that of the present invention is to provide exendin prodrugs with longer half-lives. This is achieved by a prodrug or a pharmaceutically-acceptable salt thereof comprising a covalent exendin prodrug of formula D-L, where * D represents an exendin moiety; e, c -L is a non-biologically active linker portion -L 'represented by formula (1), Ro o "e R e" eoq, where the dashed line indicates the link to one of the amino groups of exendin forming an amide bond; R 'is selected from C, alkyl, preferably CH; R , Rº are independently selected from the group consisting of H and C1.4 alkyl; where L 'is substituted with a Lº-Z and optionally also substituted, provided that the hydrogens marked with the asterisks in formula (1) are not replaced by a substituent and in which L it is a simple chemical bond or a spacer; and, Z is a hydrogel.
It was found that prodrug linkers based on the stereochemistry shown in formula (1), that is, with an amino acid in its D form, have an advantageous half-life in comparison with such prodrug linkers based on amino acids in their form L. In addition,
. 4/97 such prodrugs can provide exendin release from a subcutaneous deposit in structurally intact form for periods of time of at least 2 days between administrations. As another advantage, the structural integrity of the released exendin can be provided by a matrix — a well-hydrated polymer minimizing the intermolecular contact of exendin molecules and prolonged release can be allowed through a self-cleaving prodrug ligand between the exendin and the polymer matrix.
Thus, it should be possible to administer exendin in the form of a prodrug of the present invention less frequently than current long-acting exendins. Other advantages should be a small peak to minimum ratio, which greatly reduces the risk of adverse events, such as nausea and gastro intestinal complications. This can help | patients reduce the frequency of injections at the same time. being able to maintain optimal control of exendin plasma levels and consequently blood glucose.
The term "exendin" refers to an exendin agonist, an exendin analogue, an exendin derivative, a truncated exendin, a truncated exendin agonist, a truncated exendin derivative, a truncated exendin analog, a extended exendin, an extended exendin agonist, an extended exendin derivative, an extended exendin analogue, GLP-1, a GLP-1 analogue, or a GLP-1 derivative, such as GLP-1 or GLP-1 analog in amidated, truncated or extended form. Preferably, exendin is an exendin or an exendin agonist of sequence ID 1 to ID 21 (see below), and most preferred is exendin-3 having sequence ID 2 or exendin-4 having the sequence [ID 1.
The term "extended" refers to peptides or proteins that have additional amino acid residues at their N-terminus or C-terminus or that have internal inserts. The term also refers to the fusions of said peptides or proteins, such as, for example, GST protein, FLAG peptide, hexa-his peptide, maltose binding protein.
Examples of exendin agonists as used here are now
- 5/97 nists of exendin-3 or exendinH including, but not limited to: (1) exendin4 analogs and amidated exendin-4 analogs, in which sequences of one or more amino acid residues have been replaced by different residues of amino acid including modifications of terminh (ii) truncated and extended forms of exendin4 and truncated and extended forms that are amidated; (iii) truncated and extended exendin-4 and truncated and stretched forms that are amidated, in which sequences of one or more amino acid residues have been replaced by different amino acid residues; (iv) LPG- | and LPG- | amidate; Fr (v) GLP- analogs | and amidated GLP-1 analogs, wherein sequences of one or more amino acid residues have been replaced by E different amino acid residues including N-terminal modifications; (vi) LPG-I | truncated and extended and truncated and extended forms that are amidated; (vii) GLP- | truncated and truncated forms that are amidated, in which sequences of one or more amino acid residues have been replaced by different amino acid residues; (viii) the already 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 exendin agonists as described above may be those in which an exendin4 analogue is selected from a group comprising H-desPro * -exendin-4-Lyss-NH ,, H-des (Pro *** ”) - exendin-4-Lys, -NH; and H-des (Pro ** ”) - exendin-4-Lyss-NH ,, or a pharmacologically acceptable salt thereof.
Other examples of exendin agonists as described above may be those in which an exendin-4 analogue is selected from
- 6/97 a group comprising desPro ** [Asp Jexendina-4 (1-39), desPro * [IsoAsp Jexendina-4 (1-39), desPro ** [Met (0) "*, Asp Jexendina -4 (1-39), desPro ** [Met (O) "*, IsoAsp * Jexendina-4 (1-39), desPro * [Trp (O2) *, Asp Jexendina-2 (1-39), desPro * [Trp (O2) *, IsoAsp Jexendina-2 (1-39), | desPro ** [Met (O) * Trp (02) *, Asp ”Jexendina-4 (1-39) and desPro * [Met (O) * Trp (O2) *, IsoAsp Jexendina-4 (1-39) , or a pharmacologically acceptable salt thereof. Other examples of exendin agonists as described in the preceding paragraph may be those in which the peptide -Lyss-NH, is linked to the C terminals of exendin-4 analogs. : Other examples of exendin agonists as described above may be those in which an exendin-4 analogue is selected from a group comprising H- (Lys) s- des Pro º [Asp ] Jexendina-4 (1- 39) -Lyss-NH> 2 des Asp * Pro ”, Pro ”, Proass exendina-4 (1-39) -NH ,, H- (Lys) s- des Pro , Pro ”, Pro º [ Asp Jexendina-4 (1-39) -NH>, H-Asn- (Glu) s of Pro * º, Pro ””, Pro [Asp Jexendina-4 (1-39) - NH>, des Pro ”, Pro” ”, Pro º [Asp] Jexendina-4 (1-39) - (Lys) s-NH>, H- (Lys) s- des Pro ”, Pro ”, Pro º [Asp Jexendina-4 (1-39) - (Lys) s- NH>, H-Asn- (Glu) s- des Pro * º, Pro ”, Pro º [Asp * Jexendina-4 (1-39) - (Lys) s-NH>, H- (Lys) s-des Pro º [Trp (O2) *, Asp Jexendin-4 (1-39) -Lyse-NH>, H- des Asp Pro, Pro ”, Pro º [Trp (O2) Jexendina-4 (1-39) - NH), H- (Lys) s- des Pro, Pro”, Pro º [Trp (02) , Asp Jexendin-4 (1- 39) -NH ,, H-Asn- (Glu) s- des Pro ”, Pro”, Pro º [Trp (O02) *, Asp Jexendina- A A A
4 (1-39) -NH>, des Pro *, Pro ””, Pro [Trp (O2) *, Asp Jexendin-4 (1-39) - (Lys) s- NH>, H- (Lys) s- des Pro ”, Pro” ”, Pro [Trp (O2) ”, Asp Jexendina-4 (1- 39) (Lys) s-NHo, H-Asn- (Glu) s- des Pro”, Pro ””, Pro º [Trp (O2) *, Asp ”Jexendina- 4 (1-39) - (Lys) s-NH>, H- (Lys) s- des Pro ** [Met (O)" *, Asp Jexendina-4 (1-39) -Lyss -NHo,] des Met (O) '* Asp * Pro **, Pro ”, Pro º exendina-4 (1-39) -NH, H- (Lys) s- des Pro *, Pro *”, Pro º [Met (0) "*, Asp” Jexendina-4 (1- 39) -NH ,, - H-Asn- (Glu) s- des Pro *, Pro ””, Pro º [Met (0 ) "*, Asp *] Exendina- 4 (1-39) -NH>," des Pro ”, Pro”, Pro ”[Met (0)" *, Asp Jexendina-4 (1-39) - (Lys ) s- NH, H- (Lys) s- from Pro º, Pro ”, Pro º [Met (O)" *, Asp Jexendina-4 (1- 39) -Lyss-NH ,, 'H-Asn - (Glu) s from Pro *, Pro ”, Pro [Met (0) '*, Asp *] Exendin- 4 (1-39) - (Lys) s-NH>, H- (Lys) s-des Pro º [Met (O) "*, Trp ( O02) , Asp Lexendin-4 (1-39) - Lyses-NH>, des Asp Pro ", Pro" ", Pro * º [Met (O0) '*, Trp (O2) º Jexendina-4 ( 1- 39) -NH>, H (Lys) s- from Proº ** Pro ”, Proº * [Met (O), Trp (O2)%, Asp“ Jexendina-4 (1-39) -NH>, H -Asn- (Glu) s- des Pro *, Pro ”, Pro º [Met (0)" *, Asp] exendina- 4 (1-39) -NH>, des Pro *, Pro ””, Pro [Met (0) "*, Trp (O2)%, Asp Lexendin-4 (1- 39) - (Lys) s-NH, H- (Lys) - des Proº * Pro", Proºº [Met (O) , Trp (02), Asp Jexendina-4 (1-39) - (Lys) s-NH>, H-Asn- (Glu) s- des Pro * º, Pro ””, Pro º [Met (O0 ) "*, Trp (O2)%, Asp *]
- 8/97 exendin-4 (1-39) - (Lys) s-NH ,, or a pharmacologically acceptable salt thereof. Another example of an exendin agonist as described above is Arg ”, Lys (N “(y-glutamil (Nº-hexadecanoil))) GLP-1 (7-37) [liraglutida] ouum; salfarmacologically acceptable of the same. Exendin agonists mimic the activities of exendin-3 or exendin-4 by binding to the receptor (s) in which exendin-3 or exendin4 exerts its actions that are beneficial as insulinotropics and in treatment 1 of diabetes mellitus or imitating the effects of exendin on urinary flow are decreased, decreasing gastric emptying, inducing satiety, increasing urinary sodium excretion and / or decreasing urinary potassium concentration, by binding to the receptor (s) where exendin causes these effects. In one embodiment, the exendin or exendin agonists with A ID # * sequences: 1 to 22 can be used to prepare the long-acting polymeric conjugates of the invention: f [Seg ID #: 1] Exendin-4 HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPS- NH2 [Sec ID No.: 2] Exendina-3 HSDGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPS- NH2 [Seq ID No.: 3)
HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG P [Sec ID No.: 4)
HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG Y [Sec ID #: 5] '
HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG [Sec ID No.: 6] HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG-NH2 [Sec ID No.: 7] HGEGTFTSDL SKQMEEEAVR LFIEWLKN-NH2 [Seq ID No.: 8)
k 9/97 HGEGTFTSDL SKQLEEEAVR LFIEFLKNGG PSSGAPPPS- NH2 [Sec ID no .: 9]] HGEGTFTSDL SKQLEEEAVR LFIEFLKN-NH2 [Mon ID no: 10] HGEGTFTSDL SKQLEEEAVR LAIEFLKN-1 ID No.: 12) HGDGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPS-fu NH2 [Sec ID No. 13] GLP-I (7-36) amide; HAEGTFTSDV SSYLEGQAAK EFIAWLVKGR-NH2 [Sec ID No. 14] HSEGTFTSDV SSYLEGQAAK EFIAWLVKGR-NH2 [Sec ID No. 15] GLP- (7-37)
HAEGTFTSDV SSYLEGQAAK EFIAWLVKGRG [Sec ID nº 16] HAXaaGTFTSDV SSYLEGQAAK EFIAWLVKGR-NH2 Xaa = P, F, Y [Seg ID nº 17] HXaaEGTFTSDV SSYLEGQAAK EFIAWLVKGR-NH2 ID # 18) HaEGTFTSDV SSYLEGQAAK EFIAWLVKGG [Mon ID # 19] R-HAEGTFTSDV SSYLEGQAAK EFIAWLVKGR-NH2 R = acetyl, pyroglutamyl, N-2-hydroxybenzyl, N-trans-3-] hexenocyte [Seg ID # 20] NH2
. 10/97 Xaa = 6-aminohexanoyl.
[Seg ID nº 21] AVE-0010 / ZP-10 / Lixisenatida
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPSKKKK KK-NH2 [Sec ID No. 22] Arg ”, Lys” (Nº (y-glutamil (Nº-hexadecanoil))) GLP-1 (7-37) [liraglutida] HAEGTFTSDVXYLEGQ glutamyl (Na-hexadecanoyl))) Preferably, the exendin having the sequence ID 1, ID 13, ID 15, I1D21ouliD 22. More preferably, the exendin having the sequence ID 1, ID 13. or ID 21. In one embodiment, exendin is exendin-4 having the sequence ID 1. In another embodiment, exendin is an analog having the sequence ID 13.
In another modality, exendin is an analogue having the sequence ID 21.
The exendin and exendin agonist derivatives of the invention will exert any and all activities exhibited by the unmodified origin molecule, but with a prolonged action.
The binding of exendin to a non-biologically active linker is referred to as the "exendin moiety".
"Non-biologically active linker" means a linker that does not show the pharmacological effects of the drug derived from the biologically active agent.
"Protecting groups" refers to a moiety that temporarily protects a chemical functional group of a molecule during the site to obtain chemoselectivity in subsequent chemical reactions. Protective groups for alcohols are, for example, benzyl and trityl, protective groups for amines are, for example, tert-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl and benzyl and for examples of protective groups
. 11/97 of thiols are 2,4,6-trimethoxibenzyl, phenylthiomethyl, acetamidomethyl, p-methoxybenzyloxycarbonyl, tert-butylthio, triphenylmethyl, 3-nitro-2-pyridylthio, 4-methyltriethyl. "Protected functional groups" means a chemical functional group protected by a protection group. "Acylating agent" means a portion of the structure R- (C = 0O) -; providing the acyl group in an acylation reaction, optionally connected to an leaving group, such as acid chloride, succinimized N-hydroxy, pentafluorfenol and para-nitrophenol. "Alkyl" means a straight-chain or branched carbon chain. Each hydrogen of an alkyl carbon can be replaced by a substituent r. "Aryl" refers to any substituent derived from a fused monocyclic or polycyclic alpha ring, including heterocyclic rings, for example, phenyl, thiophene, indolyl, naphthyl, pyridyl, which can optionally also be substituted.
"Acyl" means a functional chemical group of the structure R- (C = O) -, where R is an alkyl or aryl.
"C14 alkyl" means an alkyl chain having 1 to 4 carbon atoms, for example, if present at the end of a molecule: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert- butyl, or for example, -CH32, -CH2-CH2-, -CH (CH3) -, -CH2-CH2-CH2-, -CH (C2Hs) -, - C (CH3) 2-, when two portions of a molecule are linked by the alkyl group. Each hydrogen of a carbon of C, alkyl may be substituted by a substituent.
"C1.6 alkyl" means an alkyl chain having 1 to 6 carbon atoms, for example, if present at the end of a molecule: Cy, alkyl, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec -butyl; tert-butyl, n-pentyl, n-hexyl, or for example, -CH72-, -CH2-CH2-, -CH (CH3) -, - - CHCH2-CHz-, -CH (C2aHs) -, -C ( CH3) 2-, when two portions of a molecule are linked by the alkyl group. Each hydrogen of a C16 alkyl carbon can be replaced by a substituent.
-. Accordingly, "C,., 5 alkyl" means an alkyl chain having 1 to 18 carbon atoms and "Cz., 16 alkyl" means an alkyl chain having 8 to 18 carbon atoms. Consequently, "C1-509 alquita" means an alkyl chain having 1 to 50 carbon atoms.
"C2.50 alkenyl" means a branched or unbranched alkenyl chain having from 2 to 50 carbon atoms, for example, if present at the end of a molecule: -CH = CH2, -CH = CH-CH3, -CH27-CH = CH ,, - Ê CH = CH-CH7-CH3, -CH = CH-CH = CH7, or for example, -CH = CH-, when two portions of a molecule are linked by the alkenyl group. Each hydrogen of a carbon of C> .55 alkenyl can be replaced with a substitute as also specified. Consequently, the term "alkenyl". refers to a carbon chain with at least one carbon-carbon double bond. Optionally, one or more triple bonds may occur.
"Ca.50 alkyl" means a branched or unbranched chain having 2 to 50 carbon atoms, for example, if present at the end of a molecule: -C = CH, -CH2-C = CH, CH-CH2-C = CH, CH7-C = C-CH3, or for example, -C = C- when two portions of a molecule are joined by the alkynyl group. Each hydrogen of a carbon of C>. 55 alkynyl can be substituted by a substituent as also specified. Consequently, the term "alkynyl" refers to a carbon chain with at least one carbon-carbon triple bond. Optionally, one or more double bonds can occur.
"Ca cycloalkyl" or "C37 cycloalkyl ring" means an alkylcyclic chain having from 3 to 7 carbon atoms, which can have double carbon-carbon bonds being at least partially saturated, for example, cyclopropyl, cyclobutyl, cyclopentyl , cyclohexyl, cyclohexenyl, cycloheptine. Each hydrogen of a cycloalkyl carbon can be replaced by a substituent. The term "C3; cycloalkyl" or "C3.7 cycloalkyl ring" also includes bridged bicycles such as norbonane or norbonene. Consequently, "C3 5 cycloalkyl" means a cycloalkyl having 3 to 5 carbon atoms.
NNE y 13/97 Consequently, "C3.19 cycloalkyl" means a cyclic alkyl having 3 to 10 carbon atoms, for example, C3-7 cycloalkyl; cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexyl, cycloeptyl, cyclooctyl, cyclononyl, cyclodecyl. The term "C3., 9 cycloalkyl" also includes at least - less carbomono - and - partially saturated bicycles. "Halogen" means fluorine, chlorine, bromine or iodine. It is generally preferred that halogen is fluorine or chlorine.
"4- to 7-membered heterocycly" or "4- to 7-membered heterocycle" means a ring with 4, 5, 6 or 7 ring atoms that can contain up to the maximum number of double bonds (aromatic or non-aromatic ring that is totally, partially or unsaturated) in which at least one atom of b. ring up to 4 ring atoms are replaced by a hetero atom selected from the group consisting of sulfur (including -S (O) -, -S (O0)> -), oxygen and nitrogen '(including = N (O) - ) and where the ring is attached to the rest of the molecule through a carbon or nitrogen atom. Examples for a 4- to 7-membered heterocycle are azetidine, oxethane, tietan, furan, thiophene, pyrrole, pyrroline, imidazole, imidazoline, pyrazole, pyrazoline, oxazole, oxazoline, isoxazole, isoxazoline, thiazole, thiazoline, isothiazole, isothiazoline, thiadiazole, thiadiazoline, tetrahydrofuran, tetrahydrothiophene, pyrrolidine, imidazolidine, pyrazolidine, oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, thiadiazolidine, sulfolane, pira- no, pyridimidine, pyridolid, pyridolid, pyridine, pyridazine, pyridolid, piperazine, piperidine, morpholine, tetrazole, triazole, triazolidine, tetrazolidine, diazepan, azepine or homopiperazine. "9 to 11 membered heterobicyclyl" or "9 to 11 membered heterocycle" means a two-ring heterocyclic system with 9 to 11 ring atoms, where at least one ring atom is shared by both rings and which can contain up to the maximum number of double bonds (aromatic or non-aromatic ring that is totally, partially or unsaturated) in which at least one ring atom up to 6 ring atoms! is replaced by a heteroatom selected from the group consisting of sulfur (including -S (O) -, -S (0) 2-), oxygen and nitrogen (including = N (O) -) and in which the ring is connected to the rest of the molecule by means of a carbon atom or ni- "IÁ 52
. 14/97 trogen.
Examples for a 9 to 11 membered heterocycle are indole, indole, benzofuran, benzothiophene, benzoxazole, benzisoxazole, benzothiazole, benzisothiazole, benzimidazole, benzimidazoline, quinoline, quinazoline, dihydroquinazoline, quinoline, dihydroquinoline, hydroquinoline - lino isoquinolihaa - decahydroisoquinoline, tetrahydroisoquinoline, - dihydroisoquinoline, benzazepine, purine or pteridine.
The term 9 to 11-membered heterocycle also includes two-ring spiro structures such as 1,4-dioxa- 8-azaspiro [4.5] decane or bridged heterocycles such as 8-aza-bicyclo [3.2.1] octane.
In the case that exendin prodrugs comprising the compounds according to formula (1) contain one or more acidic or basic groups, the invention also comprises their corresponding pharmaceutically or toxicologically acceptable salts, in particular their pharmaceutically usable salts. .
In this way, exendin prodrugs comprising- comprising the compounds of formula (1) which contain acidic groups can be used according to the invention, for example, as alkali metal salts, alkaline earth metal salts or as salts of 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.
Exendin prodrugs comprising the compounds of formula (1) that contain one or more basic groups, that is, groups that can be protonated, can be present and can be used according to the invention in the form of their addition salts with inorganic or organic acids.
Examples for suitable acids include hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acids, Ooxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formic acid, propionic acid, pivalic acid, diethylacetic acid, malonic acid, succinic acid, pyramic acid, fumaric acid, malic acid, malic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid, and other acids known to
'15/97 person skilled in the art. If the exendin prodrugs comprising the compounds of formula (1) contain both acidic and basic groups in the molecule, the invention also includes, in addition to the salt forms mentioned, internal salts or betaines (zZwitterions). The respective salts of a- —cord with the exendin prodrugs comprising the formula (I) can be obtained by customary methods which are also known to the person skilled in the art, for example by contacting them with an organic or inorganic acid or base in a solvent or dispersant, or by anion exchange or cation exchange with other salts. The present invention also includes all salts of exendin prodrugs comprising the compounds of the formula (1) which, due to low physiological compatibility, are not directly suitable for use in pharmaceutical products, but which can be used, for example, as intermediates for chemical reactions "or for the preparation of pharmaceutically acceptable salts.
To enhance the physicochemical or pharmacokinetic properties of a drug, such as exendin, in vivo, such a drug can be conjugated to a vehicle. If the drug is transiently bound to a vehicle and / or a linker, such systems will be commonly referred to as vehicle bound prodrugs. According to the definitions provided by IUPAC (as provided at http://www.chem.qmul.ac.uk/iupac.medchem, accessed on 22 July 2009), a vehicle-linked prodrug is a prodrug that contains a temporary binding of a given active substance with a transient carrier group that produces improved physico-chemical or pharmacokinetic properties and can be easily removed in vivo, usually by hydrolytic cleavage.
The binders employed in such vehicle-linked prodrugs are transient, meaning that they are hydrolytically non-enzymatically degradable (cleavable) under physiological conditions (aqueous buffer at pH 7.4, 37ºC) with half-lives ranging, for example, from one hour to three months.
Hydrogel vehicles can be directly coupled to the L linker or by means of a spacer, preferably a non-spacer
. 16/97 cleavable. The term "exendin hydrogel prodrug" refers to prodrugs linked by exendin vehicle, wherein the vehicle is a hydrogel. The terms "hydrogel prodrug" and "hydrogel-bound prodrug" refer to the prodrugs of biologically active agents transiently linked to a hydrogel are used interchangeably.
A "hydrogel" can be defined as a three-dimensional polymeric, hydrophilic or amphiphilic network capable of absorbing large amounts of water. The networks are composed of homopolymers or copolymers, they are insoluble due to the presence of chemical or physical covalent crosslinks (ionic, hydrophobic interactions, entanglements). The cross-links provides, the network structure and physical integrity. Hydrogels exhibit compatibility. thermodynamic quality with water that allows them to swell in aqueous media. The chains of the network are connected in a model that the pores exist and that the substantial fraction of these pores are between 1 nm and 1000 nm.
"Free form" of a drug refers to a drug, specifically exendin, in its unmodified, pharmacologically active form, such as after being released from a polymer conjugate.
It is understood that the pharmacologically active form of exendin also includes oxidized and deamidated drug.
The terms "drug", "biologically active molecule", "biologically active portion", "biologically active agent", "active agent", are used synonymously and refer to exendin, in its bond or free form.
A "therapeutically effective amount" of exendin as used here means an amount sufficient to cure, alleviate or partially interrupt the clinical manifestations of a given disease and its complications. A suitable amount to accomplish this is defined as a "therapeutically effective amount". Effective amounts for each “purpose” will depend on the severity of the illness or injury as well as the individual's weight and general condition. It will be understood that the determination of a dosage can be obtained using an experimentation routine, by building MOMO) DN to O -
r 17/97 a matrix of values and testing different points in the matrix, which is fully included in the ordinary experiences of a trained doctor. "Stable" and "stability" mean that within the indicated storage period the hydrogel conjugates remain conjugated and do not hydrolyze to a substantial extent and exhibit an acceptable impurity profile in relation to exendin.
To be 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 the FDA (United States) and / or any national regulatory agency for use in animals, preferably in humans. - "Pharmaceutical composition" or "composition" means one or more active ingredients, and one or more inert ingredients, as well as any product that results, directly or indirectly, from combining, complementing or aggregating any two or more of the ingredients, or dissociation of one or more of the ingredients, or other types of reactions or interactions of one or more of the ingredients.
Accordingly, the pharmaceutical compositions of the present invention encompass any composition made by mixing a compound of the present invention and a pharmaceutically acceptable excipient (pharmaceutically acceptable carrier). "Dry composition" means that an exendin hydrogel prodrug composition is supplied in a dry form in a container.
Such suitable drying methods are spray drying and lyophilization (freeze drying). Such dry exendin hydrogel prodrug composition has a residual water content of a maximum of 10%, preferably less than 5% and more preferably less than 2% (determined according to Karl Fischer). The preferred method of drying is lyophilization. "Lyophilized composition" means that an exendin hydrogel polymer prodrug composition was first frozen and subsequently subjected to water reduction by means of reduced pressure.
This terminology does not exclude additional drying steps that occur in the manufacturing process before loading the composition into the final container.
. 18/97 "Freeze drying" (freeze drying) is a dehydration process, characterized by freezing a composition and then reducing the surrounding pressure and, optionally, adding heat to allow the frozen water in the composition to sublimate directly from the solid phase for gaseous. Typically, sublimated water is collected by sublimation. "Reconstitution" means the addition of a liquid to a dry composition to bring it into the form of a liquid composition or suspension. 7 It is understood that the term "reconstitution" is not limited to the addition of water, but refers to the addition of any liquid, including, for example, buffers or other aqueous solutions.
- "Reconstitution solution" refers to the liquid used to replenish the dry composition of an exendin hydrogel prodrug prior to administration to a patient in need of it.
"Container" means any container in which an exendin hydrogel prodrug composition is comprised and can be stored until reconstitution.
"Buffer" or "buffering agent" refers to chemical compounds that maintain the pH in a desired range. Physiologically tolerated buffers are, for example, sodium phosphate, succinate, histidine, bicarbonate, citrate and acetate, pyruvate. Antacids such as Mg (OH); or ZnCO; can also be used. The buffering capacity can be adjusted to match the conditions most sensitive to pH stability.
"Excipients" refers to compounds administered together as a therapeutic agent, for example, buffering agents, isotonicity modifiers, preservatives, stabilizers, anti-absorption agents, oxidation protection agents, or other auxiliary agents. However, in some cases, an excipient may have dual or triple functions.
A "lyoprotectant" is a molecule that, when combined with] a protein of interest, significantly prevents or reduces the chemical and / or physical instability of the protein in drying in general and especially during freeze-drying and subsequent storage. Exemplary lyoprotectants
. 19/97 res include sugars, such as sucrose or trehalose; amino acids such as arginine, glycine, glutamate or histidine; methylamines such as betaine; lyotropic salts such as magnesium sulfate; polyols such as trihydric or higher sugar alcohols, for example, glycerin, erythritol, glycerol, —arabitol, xylitol, sorbitol, and mannitol; ethylene glycol; propylene glycol; polyethylene glycol; pluronic; hydroxyalkyl starches, for example, hydroxyethyl starch (HES), and combinations thereof.
"Surfactant" refers to wetting agents that decrease the surface tension of a liquid.
"Isotonicity modifiers" refer to compounds that minimize the pain that can result from cell damage due to differences in. osmotic pressure in the injection tank. The term "stabilizers" refers to compounds used to: stabilize the polymer prodrug. Stabilization is achieved by reinforcing the stabilizing forces of protein, by destabilizing the denatured state, or by direct binding of excipients to the protein.
"Anti-absorption agents" refers to mainly ionic or non-ionic surfactants or other soluble proteins or polymers used to coat or competitively absorb the internal surface of the composition container. The chosen concentration and the type of excipient depend on the effect to be avoided, but typically a surfactant monolayer is formed at the interface just above the CMC value.
"Oxidation protection agents" refers to antioxidants such as ascorbic acid, ectoin, glutathione, methionine, monothioglycerol, —morine, polyethyleneimine (PEI), propyl gallate, vitamin E, chilling agents such as citric acid, EDTA, hexaphosphate, thioglycolic acid.
"Antimicrobial" refers to a chemical substance that kills or inhibits the growth of microorganisms, such as bacteria, fungi, yeasts, protozoa and / or destroys viruses.
"Sealing a container" means that the container is closed in such a way that it is airtight, allowing no gas exchange between the outside and the inside and keeping the contents sterile.
. 20/97 ril.
The term "reagent" or "precursor" refers to an intermediate or starting material used in the assembly process to induce a prodrug of the present invention.
The term "chemical functional group" refers to activated carboxylic acid and derivatives, amino, maleimide, thiol and derivatives, sulfonic acid and derivatives, carbonate and derivatives, carbamate and derivatives, hydroxyl, aldehyde, ketone, hydrazine, isocyanate, isothiocyanate, phosphoric acid and derivatives, 1 phosphonic acid and derivatives, haloacetyl, alkyl halides, acryloyl and other Michael unsaturated alpha-beta acceptors, arylating agents such as aryl fluorides, hydroxylamine, disulfides such as pyridyl disulfide, vinyl sulfone,. vinyl ketone, diazoalkanes, diazoacetyl compounds, oxirane, and aziridine. If a chemical functional group is coupled to another chemical functional group, the resulting chemical structure will be referred to as "bonding". For example, the reaction of an amine group with a carboxyl group results in an amide bond. "Reactive functional groups" are chemical functional groups of the skeleton portion, which are connected to the hyper-branched portion. "Functional group" is the collective term used for "reactive functional group", "degradable interconnected functional group", or "conjugated functional group". A "degradable interconnected functional group" is a bond comprising a biodegradable bond that is connected on one side to a spacer portion connected to a skeleton portion and on the other side is connected to the crosslinking portion. The terms "degradable interconnected functional group", "biodegradable interconnected functional group", "interconnected biodegradable functional group" and "interconnected functional group" are used synonymously. The terms "blocking group" or "buffering group" are "30 used synonymously and refer to portions that are irreversibly connected to reactive functional groups to render them unable to react with , for example, chemical functional groups.
| . '21/97 The terms "protecting group" or "protecting group" refer to a portion that is reversibly connected to reactive functional groups to render them unable to react with, for example, other chemical functional groups. The term "interconnectable functional group" refers to chemical functional groups, which participate in a radical polymerization reaction and are part of the crosslinking reagent or the skeleton reagent. The term "polymerizable functional group" refers to chemical functional groups, which participate in a polymerization reaction of the bonding type and are part of the crosslinking reagent and the skeleton reagent. A skeleton portion may comprise a spaced portion. dora that at one end is connected to the skeleton portion and at the other side to the cross-linking portion. Ê The term "derivatives" refers to chemical functional groups suitably substituted with protecting and / or activation groups or to the activated forms of the corresponding chemical functional group are known to the person skilled in the art. For example, activated forms of carboxyl groups include, but are not limited to, active esters, such as succinimidyl ester, benzotriazil ester, nitrophenyl ester, pentafluorophenyl ester, —azabenzotriazil ester, acyl halides, mixed or symmetric anhydrides, acyl imidazole.
The term "non-enzymatically cleavable linker" refers to linkers that are hydrolytically degradable under physiological conditions without enzymatic activity.
"Non-biologically active linker" means a linker that does not show the pharmacological effects of the drug (D-H) derived from the biologically active moiety.
The terms "spacer", "spacer group", "spacer molecule", and "spacer portion" are used interchangeably and if used to describe a portion present in the hydrogel vehicle of the invention, refer to any portion suitable for connecting two portions, such as C1-50 alkyl, C2.56 alkenyl or C2.55 alkynyl, whose fragment is optionally included
. 22/97 terminated by one or more groups selected from -NH-, -N (C14 alkyl) -, -O-, -S-, -C (O) -, -C (O) NH-, -C (O ) N (C14 alkyl) -, -0-C (O) -, -S (O) -, -S (O) 2-, phenyl, naphthyl or 4- to 7-membered heterocyclyl. The terms "terminal", "terminals" or "distal end" refer to the position of a functional group or bond with a molecule or moiety, whereby such a functional group can be a chemical functional group and the bond can be a degradable or permanent bond, characterized by being located adjacent to or a bond between two portions or at the end of an oligomeric or polymeric chain.
The phrases "in connected form" or "portion" refer to the substructures that are part of a larger molecule. The phrase "in linked form" is used to simplify reference to the portions by naming or listing the reagents, starting materials or hypothetical starting materials well known in the art, and by which "in linked form "means that, for example, one or more hydrogen radicals (-H), or one or more activation or protection groups present in the reagents or starting materials are not present in the portion.
It is understood that all reagents and moieties comprising polymeric moieties refer to known macromolecular entities exhibiting variability with respect to molecular weight, chain lengths or degree of polymerization, or the number of functional groups. The structures shown for the skeleton reagents, skeleton moieties, crosslinking reagents, and binding moieties are thus only representative examples.
A reagent or portion can be linear or branched. If the reagent or portion has two terminal groups, it will be referred to as a reagent or linear portion. If the reagent or portion has more than two terminal groups, it will be considered to be a branched or multi-functional reagent or portion.
The term "poly (ethylene glycol) based polymer chain" or "PEG based chain" refers to an oligo- or polymeric molecular chain.
: 23/97 Preferably, such a polymer chain based on poly (ethylene glycol) is connected to a branching core, it is a linear poly (ethylene glycol) chain, one end of which is connected to the branching core and the other to a hyper-branched dendritic portion. It is understood that a chain — based on PEG can be terminated or interrupted by alkyl or aryl groups optionally substituted with heteroatoms and chemical functional groups. If the term "polymer chain based on poly (ethylene glycol)" is | used in reference to a crosslinking reagent, it refers to a crosslinking chain or moiety comprising at least 20% by weight of ethylene glycol moieties. - In the following sections the invention is described in other details. The present invention relates to a prodrug or a pharmaceutically acceptable salt thereof, comprising an exendin ligand conjugate D-L, wherein D represents an exendin moiety; and, -L is a non-biologically active linker portion -L 'represented by formula (1), nº | Oo H * R
AS NOS H o q, where the dashed line indicates the attachment to one of the amino groups of the exendin moiety forming an amide bond; R 'is selected from C14 alkyl, preferably CH3; R , R º are independently selected from the group consisting of H and C ,, alkyl; where L 'is replaced with a L2-Z and optionally also - substituted, provided that the hydrogens marked with the asterisks in formula (1) are not replaced by a substituent and where [E is a simple chemical bond or a spacer; and
. 24/97 Z is a hydrogel. Preferably, in formula (1) R is replaced by Lº-Z. Preferably, in formula (1) Rº is CH> -L2-Z. Preferably, L 'is also not substituted. Preferably, an exendin moiety is linked to L 'via N-terminal nitrogen or via a lysine side chain nitrogen from the exendin moiety. More preferably, an exendin moiety is linked to L 'via the N-terminal nitrogen. Preferred drugs of the present invention comprise D-L linker conjugates, where L is represented by the formulas (la) or (Ib):. RE Ha Hº and pn SA H * O (la), 2 -Z Rr o | no. . R ANA Ro SAO x
À HH * Oo (1b), where D, R ', Rº, Rº, L , Z have the preferred meanings and meanings as indicated here and where L is optionally also substituted, provided that the hydrogens marked with asterisks in the formula (la) or (lb) are not replaced by a substituent, however preferably L is also not substituted (apart from the mandatory substituent Lº-Z already shown in (la) and (lb)). As shown, for example, in formulas (la) or (lb), a L 'hydrogen of formula (1) is replaced by the group L2-Z. In general, Lº can be linked to L 'in formula (1) in any position of the hydrogens marked with the asterisks. Preferably, one of the hydrogens supplied by R ', R , R º, directly or as hydrogen of the
: 25/97 C14 alkyl or other groups is replaced by Lº-Z.
In addition, L * of formula (1) can optionally also be substituted. In general, any substituent can be used as long as the cleavage is not affected. However it is preferred that L 'is also not replaced.
Preferably, one or more other optional substituents are independently selected from the group consisting of halogen; CN; COOR *; OR *; C (O) Rº; C (OIN (R'Rº); S (O) LN (R'R =); S (ON (RºRº); S (O) Rº; S (O) Rº; N (RS (O) N (RºR * ); SRº; N (R'Rº); NO; OC (OJRº; N (RIC (O) R *; N (R) S (O) 2R *; N (R) IS (O) R *; N ( RÚ) C (O0) OR *; N (RC (O) IN (R * R *); OC (OIN (RºR ); T; C1.59 alkyl; C2.59 alkenite; or Ca.50. Alkynyl, where T; C1.505 alkyl; C2.59 alkenyl; and C2.59 alkynyl are optionally substituted with one or more R "º, which are the same or different and À where Cr.55 alkyl; C2.59 alkenyl ; and C2.59 alkynyl are optionally interrupted by one or more groups selected from the group consisting of T, -C (0) O-; -O-; -C (O) -; -C (O) N ( R "'!) -; -S (O) 2N (R") -; -S (O) N (R "!) -; -S (O) 2-; - S (O0); -N (R ) S (OLN (R '! 9) -; -S-; -N (RU) -; -OC (OIR "; -N (R) C (O); - N (RS (O) a-; - N (R) S (O0) -; -N (RMC (OJO-; -N (RI) CIOIN (R-; e - OC (O) N (R "R!" A); Rº, Rº, R * * are independently selected from the group consisting of H; T; and C1.505 alkyl; C2.59 alkenyl; or C2.595 alkynyl, where T; C1-50 alkyl; C2.55 alkenyl; and C2.59 alkynyl are optionally replaced with one or more R'º, which are the same or different and where C; .59 alkyl; C2. are alkenyl; and C2.505 alkynyl are optionally interrupted by one or more groups selected from the group consisting of T, -C (0) O-; -O-; -C (O) -; “C (OIN (R) -; - -S (O) LN (R) -; —-S (OIN (R) -; —-S (0); -S (O); - N (R ') S (O) AN (R '* 9) -; -S-; -N (R "); -OC (O) R"'; -N (R) C (O) -; -N (R /) S (O) 2-; - N (R '!) S (O) -; -N (R) C (0) O-; -N (R /) C (O) N (R !! 3) - ; and -OC (O) IN (R! IR !! a); T is selected from the group consisting of phenyl; naphthyl; indenyl; indanyl; tetralinyl; C319 cycloalkyl; 4- to 7-membered heterocyclyl; or heterocyclic of 9 to 11 members, where T is optionally substituted with one or more R "º, which are the same or different;
and 26/97 R ** is halogen; CN; oxo (= O); COOR ; OR; COLOR" ; CIOIN (RARIP); S (O) LN (RRI); S (ON (RRIP); S (OLR ; S (O) R '; N (R) S (O) aN (R'22RI ); SR; N (R / 2R / 22); NO; OC (O) JR'2; N (RBC (O) R 2; N (RS (0) AR 2; N (R3S (O) R'2; N (RIC (O) OR 2; N (R) C ( O) N (R / 22R 2); OC (OIN (R RI ); Or C1.6 alkyl, where C1.6 alkyl is optionally substituted with one or more halogens, which are the same or different; R "', Rº, Rº, R / º, R / are independently selected from the group consisting of H; or C1 .; alkyl, where C; .6 alkyl is optionally substituted with one or more halogens, which are the same or different.
The term "interrupted" means that between two carbons a group is inserted or at the end of the carbon chain between carbon and - hydrogen. L it is a simple chemical bond or a spacer. In case Ê where L is a spacer, it is preferably defined as the one or more - optional substituents defined above, as long as L be replaced with Zz.
Consequently, when L it is different from a simple chemical bond, L -Z is COOR *; OR; C (O) Rº; C (O) N (RºR *); S (O) N (RºRº); S (OIN (RR *); S (OLR *; S (O) R; N (RS (O) LLN (RºR *); SR; N (RºRº); OC (O) R *; N (RIC (OIR) *; N (RIS (OLR * :; N (RIS (OIRº; N (RÓC (IO) JOR *: N (R) C (O) N (RºR *); OC (OIN (RºR); T; C1. 50 alkyl; Ca.59 alkenyl; or Cz.50 alkynyl, where T; C1.59 alkyl; C2.59 alkenyl; and C> .50 alkynyl are optionally substituted with one or more R'º, which are the same or different and in which C1.595 alkyl; C2.50 alkenyl; and Ca.59 alkynyl are optionally interrupted by one or more groups selected from the group consisting of -T-, -C (0) O-; -O -; -C (0) -; -C (O) N (R "!) -; -S (O) 2N (R"!) -; -S (O) N (R "!) -; -S (O) 2-; -S (0) -; -N (R) S (OLN (R! 9) -; -S-; -N (R) -; -OC (O) R '; -N ( RU) C (O) -; - N (RS (0) 2-: -N (R) S (O) -; -N (RIC (OJO-; -N (RICIOIN (R! 9) -; e - OC (O) N (R "R" * s); Rº, R * º, Rº * are independently selected from the group consisting of H; Z; T; and C1,509 alkyl; C2,505 alkenyl; or C2. 59 alkynyl, where T; C1.50 alkyl; C2.55 alenyl; and C2,505 alkynyl are optionally substituted
: 27/97 with one or more R "º, which are the same or different and where C.59 alkyl; C2.505 alkenyl; and C2.595 alkynyl are optionally interrupted by one or more groups selected from the group that consists of T, -C (0) O-; -O-; - C (O) -; -C (OIN (IR ') -; -S (O) LN (R ") -; -S (OIN ( R) -; -S (O)> -; -S (O) -; - N (RIIS (O) LLN (R! 9) -; -S-; -N (R) -; -OC (O) R "'; -N (R / C (O) -; -N (R /) S (O) 2-; - N (R *) S (O) -; -N (R) C (0) O -; -N (R! C (O) N (R! 1 ) -; and -OC (O) N (RUR! * ); T is selected from the group consisting of phenyl; naphthyl; indemnity; indanyl; tetralinyl; C3., 9 cycloalkyl; 4- to 7-membered heterocyclic; or 9 to 11-membered heterocyclic, where t is optionally substituted common or more R'º, which are the same or different; R "º is Z; halogen; CN; oxo (= 0); COOR ' ; OR'2; C (O) R'2;: CIOINIRERIE); S (O) LN (RPRIZ); S (OIN (RPRIP); S (OLR Z; S (OIR * ; N (RS (O) LN (RIPAR ! ); SR ' ; N (R / PR'29); NO; OC (O) JR'2; N (R) C ( O) R '=; - N (R3) S (O) .R 2; N (RBS (O) R'22; N (RBC (0) OR 22; N (RB) C (O) N (R / 22R128); OC (OIN (RRI 9); or C1.6 alkyl, where C1. 6 alkyl is optionally substituted with one or more halogens, which are the same or different; R '!, RUº, R2, R1º, R' they are independently selected from the group consisting of H; Z; or C1.6 alkyl, where C1.6 alkyl is optionally substituted with one or more halogens, which are the same or different; As long as only one among Rº, Rºº, Rº%, R1º, RU, R1º, R $ 2, R '*, R' be Z.
More preferably, Lº is a C1-20 alkyl chain, which is optionally interrupted by one or more independently selected groups - -O- names; and C (O) N (R **); optionally substituted with one or more groups independently selected from OH; and C (O) N (RºR **): and in which R *, Rº ** º are independently selected from the group consisting of H; and C ,, 4 alkyl.
Preferably, Lº has a molecular weight in the range of 14 g / mol to 750 g / mol.
Preferably, L is linked to Z via a selected terminal group of
'28/97 Is it the faith has such a terminal group, it is furthermore preferred that L has a molecular weight in the range of 14 g / mol to 500 g / mol calculated without such a terminal group.
Preferably, the covalent bond formed between the linker and Z] is a permanent bond.
Preferably, hydrogel Z is a water insoluble hydrogel based on biodegradable poly (ethylene glycol) (PEG).
The term "based on PEG" as understood here means that the mass ratio of PEG cha- rias in the hydrogen! is at least 10% by weight, preferably at least 25%, based on the total weight of the hydrogel.
The remainder can be prepared from other spacers and / or oliomers or polymers, such as oligo- or polylysines.
In addition, the term "water-insoluble" refers to an expandable three-dimensionally cross-linked molecular network forming the hydrogel.
The - hydrogel if suspended in a large excess of water or aqueous buffer of physiological osmolarity can absorb a substantial amount of water, for example, up to 10 times in a weight per weight basis, and is therefore dilatable, however after water removal physical stability still remains | of a gel and a shape.
Such a shape can be of any geometry and it is understood that such an object of hydrogen! individual should be considered as a simple molecule consisting of components in which each component is connected to another component through chemical bonds.
According to this invention, the hydrogel can be composed of skeletal portions interconnected by hydrolytically degradable bonds.
Preferably, the hydrogel is a PEG-based hydrogel comprised of backbone portions.
Preferably, Lº is connected to a skeleton portion.
Preferably, the skeleton portion has a molecular weight in the
. 29/97 range from 1 kDa to 20 kDa, more preferably from 1 kDa to 15 kDa and even more preferably from 1 kDa to 10 kDa. The backbone portions are also preferably based on PEG comprising one or more PEG strands.
In a hydrogel carrying exendin-linker conjugates according to the invention, a backbone portion is characterized by several functional groups, comprising interconnected biodegradable functional groups and hydrogel-connected drug binding conjugates, and optional. buffer groups. This means that a backbone portion is characterized by several hydrogel-connected drug-binding conjugates; functional groups, comprising interconnected functional groups of the biodegradables; and optionally buffer groups. Preferably, the sum of interconnected and conjugated biodegradable functional groups of drug binding and buffering groups is 16-128, preferable 20-100, more preferable 24-80 and more preferable 30-60.
Preferably, the sum of interconnected and conjugated functional groups of hydrogel-linked drug binders and buffer groups of a backbone is equally divided by the number of polymeric chains based on PEG extending from the branching core. For example, if there are 32 interconnected and conjugated functional groups of binder drug connected by hydrogel and buffering groups, eight groups can be provided for each of the four polymeric chains based on PEG extending from the core, preferably. through dendritic portions attached to the end of each chain — polymer based on PEG. Alternatively, four groups can be provided for each of the eight PEG-based polymer chains extending from the nucleus or two groups for each of the sixteen PEG-based polymer chains. If the number of PEG-based polymeric chains extending from the branching nucleus does not provide an equal distribution, it will be preferred that the deviation from the average number of the sum of interconnected and conjugated functional groups of hydrogel-bound drug groups and polymeric chain buffer based SN
: 30/97 in PEG is kept to a minimum.
In such vehicle-linked prodrugs according to the invention, it is desirable that almost the entire release (> 90%) occurred before a significant amount of release from the skeletal portions (<10%) occurred. This can be achieved by adjusting the half-life of the vehicle-bound prodrug versus the degradation of hydrogel kinetics according to the invention.
Preferably, a backbone portion is characterized by having a branching core, from which at least three PEG-based polymer chains extend. Consequently, in a preferred aspect of the present invention the backbone reagent comprises a branching core, from which at least three polymer chains based on PEG extend. Such branching nuclei can be comprised of poly- or oligo-alcohols in linked form, preferably pentaerythritol, tripentaerythritol, hexaglycerin, sucrose, sorbitol, fructose, mannitol, glucose, cellulose, starches, starches, hydroxyalkyl starches, polyvinyl alcohols, dextran, hyualuroananes, or branching nuclei can be comprised of poly- or oligo-amines such as ornithine, diaminobutyric acid, trilisine, tetralysin, penthalisin, hexalisin, heptalisin, octalisin, nonalisine, decalisin, undecalisine, d , tridecalisin, tetradecalisin, pentadecalisin or oligolysins, polyethyleneimines, polyvinylamines in bound form.
Preferably, the branching core extends from three to sixteen polymeric chains based on PEG, more preferably four to eight. Preferred branching nuclei can be comprised of penterythritol, ornithine, diaminobutyric acid, trilisine, tetralysin, pentalisin, hexalisin, heptalisine or oligolysin, low molecular weight PEI, hexaglycerine, tripentaerythrite! in connected form. Preferably, the branching core extends from three to sixteen polymer chains based on PEG, more preferably four to eight. Preferably, the polymer chain based on PEG is a linear poly (ethylene glycol) chain, one end of which is connected to the branching core and the other to a hyper-branched dendritic portion. It is understood that a polymeric chain based on PEG NM
. 31/97 can be terminated or interrupted by alkyl or aryl groups optionally substituted with heteroatoms and chemical functional groups. Preferably, the PEG-based polymer chain is a properly substituted poly (ethylene glycol) derivative (based on PEG). Preferred structures for corresponding PEG-based polymer chains extending from a branching nucleus contained in a skeleton portion are derived from multiple branching PEGs, for example, detailed in the JenKkem Technology, USA product list (accessed via download from www.jenkemusa.com on July 28, 2009), 4-branch PEG derivatives (pentaerythritol core), 8-branch PEG derivatives (hexaglycerin core) and 8-branch PEG derivatives (tripentaerythritol core) . Most preferred are 4-branch PEG amine (pentaerythritol core) and 4arm PEG Carboxyl (pentaerythritol nucleus), 8-branch PEG amine (hexaglycerine core), 8-branch PEG carboxyl (hexaglycerin core) , 8-branch PEG amine (tripentaerythritol core) and 8-branch PEG carboxyl (tripentaerythritol core). Preferred molecular weights for such multi-branched PEG derivatives in a skeletal portion are 1 kDaa20 kDa, more preferably 1 kDa to 15 kDa and even more preferably 1 kDa to 10 kDa.
The terminal amine groups of the aforementioned multi-branched molecules are understood to be linked in the schematic portion to provide other interconnected functional groups and functional recreational groups of a skeleton portion.
It is preferred that the sum of interconnected functional groups and reactive functional groups of a backbone portion is equally divided by the number of polymeric chains based on PEG extending from the branching nucleus. If the number of polymer chains based on PEG extending from the branching nucleus does not provide an equal distribution, it will be preferred that the deviation from the average number of the sum of reactive and interconnected functional groups per polymer chain based on PEG SN
. 32/97 is kept to a minimum.
Most preferably, the sum of reactive and interconnected functional groups of a backbone portion is equally divided by the number of polymeric chains based on PEG extending from the branching core. For example, if there are 32 interconnected functional groups and reactive functional groups, eight groups can be provided by each of the four PEG-based polymer chains extending from the nucleus, preferably through dendritic portions attached to the end of each polymer chain with based on PEG. Alternatively, four groups can be provided for each of the eight PEG-based polymer chains extending from the nucleus or two groups for each of the sixteen PEG-based polymer chains. Such additional functional groups can be provided by dendritic portions. Preferably, each dendritic portion has a molecular weight in the range of 0.4 kDa to 4 kDa, more preferably 0.4 kDa to 2 kDa. Preferably, each dendritic portion has at least 3 branches and at least 4 reactive functional groups, and at most 63 branches and 64 reactive functional groups, preferred at least 7 branches and at least 8 reactive functional groups and at most 31 branches and 32 reactive functional groups.
Examples for such dendritic portions are comprised of trilisine, tetralysin, pentalisin, hexalisin, heptalisin, octalisin, nonalisin, decalisin, undecalisin, dodecalisin, tridecalisin, tetradecalisin, pentadekalisine, hexadecalisin, hexadecalisine, hexadecalisine, hexadecalisine, hexadecalisine, hexadecalisine, hexadecalisine, hexadecalisine Examples for such preferred dendritic moieties are comprised of trilisine, tetralysin, pentalisin, hexalisin, heptalisin in bound form, more preferred trilisine, pentalisin or heptalisin, ornithine, diaminobutyric acid in bound form.
More preferably, the hydrogel vehicle of the present invention is characterized by the fact that the skeleton portion has a quarantine carbon of formula C (A-Hyp) a, where each A is independently the polymer chain based on poly (ethylene) l) terminally linked to carbon ND
. 33/97 by a permanent covalent bond and the distal end of the PEG-based polymer chain is covalently linked to a Hyp dendritic portion, each Hyp dendritic portion having at least four functional groups representing the interconnected functional groups and reactive functional groups.
Preferably, each A is independently selected from the formula - (CH2>)) :( OCH2CH2), X-, where n1 is 1 or 2; n is an integer in the range 5 to 50; and X is a chemical functional group covalently linking: Ae Hyp.
Preferably, A and Hyp are covalently linked by an amine bond. ju Preferably, the dendritic Hyp portion is a hyper-branched peptide. Preferably, the hyper-branched peptide comprises lysine in bound form. Preferably, each Hyp dendritic portion has a molecular weight in the range of 0.4 kDa to 4 kDa. It is understood that a skeletal C (A-Hyp) portion «can consist of the same or different dendritic portions Hyp and that each Hyp can be chosen independently. Each Hyp portion consists of between 5 and 32 lysines, preferably at least 7 lysines, i.e., each Hyp portion is comprised of between 5 and 32 lysines in linked form, preferably at least 7 lysines in linked form. More preferably, Hyp is comprised of heptalisinyl.
The reaction of polymerizable functional groups with a skeleton reagent, more specifically from Hyp with the polymerizable functional groups of crosslinking reagents based on poly (ethylene glycol!) Results in a permanent amine bond.
Preferably, C (A-Hyp), has a molecular weight in the range of 1 kDa to 20 kDa, more preferably 1 kDa to 15 kDa and even more preferably 1 kDa to 10 kDa.
A preferred skeleton portion is shown below, the dotted lines indicate biodegradable interconnection bonds to the connecting portions and n is an integer from 5 to 50: Di Dói TE>
. 34/97% | H o. NA 'ce deScca dances: SS o NH o NH, CoaA y S> ”9 ds o 7% l, É HN Vl, -. c- RA NX Nº. fot Ar H NH; a o —— Le rh N o F Saw NA
AS 4 o. );
It is NH, 4 The biodegradability of hydrogels according to the present invention is obtained by introducing hydrolytically degradable bonds. The terms "hydrolytically degradable", "biodegradable" or "hydrolytically cleavable", "self-cleavable", "self-cleavable", "self-cleavable", "transient" or "temporary" refer to the context of the present invention bonds and linkages that are hydrolytically degradable non-enzymatically or cleavable under physiological conditions (aqueous buffer at pH 7.4, 37ºC) with half-lives ranging from one hour to three months, including, but not limited to, are limited to aconityls, acetals, amides, —carboxylic anhydrides, esters, imines, hydrazones, maleamic acid amides, ortho esters, phosphamides, phosphoesters, phosphosyl esters, silyl esters, sulphonic esters, aromatic carbamates, combinations thereof, and similar. If present in a hydrogel according to the invention as a degradable interconnected functional group, preferred biodegradable bonds are carboxylic esters, carbonates, phosphoesters and esters of sulfonic acid and most preferred are carboxylic esters or carbonates.
. 35/97 Permanent bonds are hydrolytically degradable non-enzymatically under physiological conditions (aqueous buffer at pH 7.4, 37ºC) with half-lives of six months or more, such as, for example, amides.
To introduce the hydrolytically cleavable bonds into the hydrogel vehicle of the invention, the backbone portions can be directly linked to each other by means of biodegradable bonds.
In one embodiment, the skeleton portions of the biodegradable hydrogel vehicle can be connected together directly, that is, without crosslinking portions. The hyper-branched dendritic portions of two skeletal portions of such biodegradable hydrogel can be directly linked through an interconnected functional group that connects the two dendritic portions. hyper-branched structures. Alternatively, two hyper-ramified dendritic portions of two different skeletal portions can be interconnected through two spacer portions connected to a skeleton portion and on the other side connected to a crosslinking portion separated by interconnected functional groups.
Alternatively, skeleton portions can be connected together via crosslinking portions, each crosslinking portion is terminated by at least two of the hydrolytically degradable connections. In addition to the degradable terminating bonds, the bonding portions may contain other biodegradable bonds. In this way, each end of the crosslinking portion connecting to a skeleton portion comprises a hydrolytically degradable bond, and biodegradable bonds may optionally be present in the crosslinking portion.
Preferably, the biodegradable hydrogel vehicle is composed of skeletal portions interconnected by hydrolytically degradable bonds and the skeletal portions are connected together via crosslinking portions.
The hydrogen vehicle! biodegradable can contain one or more different types of cross-linking portions, preferably one. The cross-linking portion can be a linear or branched molecule and is preferably a linear molecule. In a preferred embodiment of the invention, the cross-linking portion is
. 36/97 connected to skeletal portions by at least two biodegradable bonds.
Preferably, the linker moieties have a molecular weight in the range of 60 Da to 5 kDa, more preferably 0.5 kDa to 5 kDa, even more preferably 1 kDa to 4 kDa, even more preferably 1 kDa to 3 —kDa In one embodiment, a crosslinker portion consists of a polymer.
In addition to the oligomeric or polymeric crosslinking portions, low molecular weight crosslinking portions can be used, especially when hydrophilic high molecular weight backbone portions are used to form a biodegradable hydrogel according to the invention.
Preferably, the linking portions based on poly (ethylene glycol) are hydrocarbon chains comprising ethylene glycol units, optionally comprising other chemical functional groups, wherein the connecting portions based on poly (ethylene glycol) comprise at least Ê each m ethylene glycol units, where m is an integer in the range 3 to 100, preferably 10 to 70. Preferably, the linking portions based on poly (ethylene glycol) have a molecular weight in the range of 0.5 kDa to 5 kDa.
If used in reference to a PEG-based crosslinker or polymeric chain connected to a branching core, the term "PEG-based" refers to a crosslinker or chain - polymer based on PEG comprising at least minus 20% by weight of ethylene glycol portions.
In one embodiment, monomers constituting the polymeric bonding portions are connected by biodegradable bonds. Such polymeric linker moieties may contain up to 100 or more biodegradable bonds, - depending on the molecular weight of the crosslinker moiety and the molecular weight of the monomer units. Examples for such linking portions are polymers based on poly (lactic acid) or glycolic polyphatide). It is understood that such poly (lactic acid) or glycolic polyfacid chains) can be terminated or interrupted by alkyl or aryl groups and that they can optionally - be replaced with heteroatoms and chemical functional groups.
Preferably, the linker moieties are based on PEG, preferably represented by a molecular chain based on PEG.
The E “: 9): = 2) 2> /2Ê2=.o$%“
. Preferably, the poly (ethylene glycol) based linker moieties are hydrocarbon chains comprising ethylene glycol units, optionally comprising other chemical functional groups, wherein the poly (ethylene glycol) based linker moieties comprise at least each m - ethylene glycol units, where m is an integer in the range of 3 to 100, preferably from 10 to 70. Preferably, the poly (ethylene glycol) based linking moieties have a molecular weight in the range of 0, 5 kDa to 5 kDa. In a preferred embodiment of the present invention the crosslinking portion consists of PEG, which is symmetrically connected via ester bonds to two alpha, omega-aliphatic spacers provided by the skeleton portions connected to the hyper-branched dendritic portion through. of permanent amide bonds.
The dicarboxylic acids of the spacer moieties connected to It is a skeleton moiety and on the other side connected to a crosslink moiety they consist of 3 to 12 carbon atoms, more preferably between 5 and 8 carbon atoms and can be substituted by one or more carbon atoms. Preferred substituents are alkyl groups, hydroxyl groups or substituted starch groups or amino groups. One or more of the methylene groups of aliphatic dicarboxylic acid can optionally be substituted by O orNHouN substituted by alkyl. Preferred alkyl is linear or branched alkyl having 1 to 6 carbon atoms.
Preferably, there is a permanent amide bond between the hyper-branched dendritic portion and the spacer portion connected to a backbone portion and on the other side is connected to a crosslinking portion.
A preferred cross-linking portion is shown below; dashed lines indicate biodegradable interconnection connections to the skeleton portions: KoDAQNXRTA where q is an integer from 5 to 50.
Preferably, the hydrogel vehicle is composed of backbone portions interconnected by hydrolytically degradable bonds.
J. 38/97 More preferably, the skeleton portions comprise a branching core of the following formula: Tot where the dashed line indicates the connection to the rest of the skeleton portion. More preferably, the skeleton portions comprise a structured following formula: DX “Cc Trot— NT 4 where n is an integer from 5 to 50 and the dashed line indicates the connection to the rest of the skeleton portion. Preferably, the skeletal portion comprises a Hyp hyperbranched portion. More preferably, the skeleton portion comprises a hyper-branched portion Hyp of the following formula:
NH NH Xk: NH /. The & /
H O x o PAN ND DENND
N NH E n | N to HH. H Hà N— = 2 AND.
. 39/97 in which the dashed lines indicate the connection to the rest of the molecule and carbon atoms marked with an asterist indicate the S configuration.
Preferably, the skeleton portions are attached to at least one spacer of the following formula: o,
wherein one of the dashed lines indicates the connection to the hybridized Hyp portion and the second dashed line indicates the connection to the rest of the molecule; and where m is an integer from 2 to 4. f Preferably, the backbone portions are linked together. 10 through linker portions having the following structure Xo AQ Azo * where q is an integer from 3 to 100, preferably from 5 to 50. In hydrogel prodrugs of the invention, the rate of hydrolysis of the biodegradable bonds between skeleton portions and portions linkers is influenced or determined by the number and type of connected atoms adjacent to the PEG ester carboxy group.
For example, by selecting succinic, adipic or glutaric acid to form PEG esters, it is possible to vary the half-life degradation of the biodegradable hydrogel vehicle according to the invention.
Preferably, L it is attached to Z via a thiosuccinimide group which in turn is attached to the backbone of the hydrogel via a spacer, such as an oligoethylene glycol chain.
Preferably, the attachment of this spacer chain to the backbone portion is a permanent bond, preferably an amide bond. "The biodegradability of hydrogels according to the present invention is achieved by introducing hydrolytically degradable bonds.
For interconnected functional groups, the term "hydrolytically
. 40/97 'te degradable' refers, in the context of the present invention, to bonds that are hydrolytically degradable non-enzymatically under physiological conditions (aqueous buffer at pH 7.4, 37ºC) with half-lives ranging from one hour to three months , include, but are not limited to, aconitilas,
acetals, carboxylic anhydrides, esters, imines, hydrazones, maleamic acid amides, ortho esters, phosphamides, phosphoesters, phosphosylyl esters, silyl esters, sulfonic esters, aromatic carbamates, combinations thereof, and the like.
Preferred biodegradable bonds are carboxylic esters, carbonates, phosphoesters and sulfonic acid esters and more preferred are carboxylic esters or carbonates.
It is understood that for in vitro studies accelerated conditions such as, for example, pH 9, 37ºC, aqueous buffer, po-. be used for practical purposes.
Permanent bonds are hydrolytically degradable non-enzymatically under physiological conditions (aqueous buffer at pH 7.4, 37 ° C) with half-lives of six months or more, such as, for example, amides.
The degradation of the biodegradable hydrogel vehicle according to the invention is a multi-step reaction where a large number of degradable bonds are cleaved resulting in degradation products that can be soluble in water or insoluble in water.
However, water-insoluble degradation products may also comprise degradable bonds so that they can be cleaved in which water-soluble degradation products are obtained.
These water-soluble gradation products may comprise one or more skeleton portions.
It is understood that the released skeletal moieties may, for example, be permanently attached to the spacer, blocking or ligand groups or affinity groups and / or prodrug ligand degradation products and that also soluble degradation products in water can comprise degradable connections.
The structures of the branching nucleus, polymer chains with PEG-based, hyper-branched dendritic portions and portions linked to the hyper-branched dendritic portions can be inferred from the corresponding descriptions provided in the sections covering the vehicle.
. 41/97 hydrogels of the present invention. It is understood that the structure of a degreaser depends on the type of hydrogel according to the invention undergoing degradation. The total amount of skeletal portions can be measured in the solution after complete degradation of the hydrogel according to the invention, and during the degradation, the fractions of soluble skeletal degradation products can be separated from the insoluble hydrogel according to the invention and can be quantified without interference from the other soluble degradation products released from the hydrogel according to the invention. A hydrogel object according to the invention can be separated from excess water of physiological osmolarity buffer by sedimentation or centrifuge. tion. Centrifugation can be carried out in such a way that the supernatant supplies at least 10% of the hydrogel volume! expanded according to: invention. Degradation products of soluble hydrogel remain in the aqueous supernatant after such a sedimentation or centrifugation step, and water-soluble degradation products comprising one or more skeletons are detectable by subjecting the aliquots of such supernatant to the methods of separation and / or appropriate analytics.
Preferably, water-soluble degradation products can be separated from water-insoluble degradation products by filtration through 0.45 µm filters, after which water-soluble degradation products can be found in full flow. Water-soluble degradation products can also be separated from water-insoluble degradation products by a combination of a centrifugation and a filtration step.
For example, skeleton portions can carry groups that exhibit UV absorption at wavelengths where other degradation products do not exhibit UV absorption. Such selectively UV-absorbing groups can be structural components of the backbone portion such as amide bonds or can be introduced into the backbone by binding to their reactive functional groups through aromatic ring systems
. 42/97 such as Indo-Indian groups.
In such hydrogel-bound exendin prodrugs according to the invention, it is desirable that almost all exendin release (> 90%) occurred before a significant amount of release of the skeletal degradation products (<10%) occurred . This can be achieved by adjusting the half-lives of the exendin prodrug attached by hydrogel versus the hydrogel degradation kinetics.
Preferably, an exendin D-L prodrug has a structure, where L is represented by the formula (11) z P On. Y DANA
H H f O (1) - 10 where the dashed line indicates the bond to a nitrogen, preferably the N-terminal nitrogen, of the exendin forming an amide bond and Z is a hydrogel; Preferably, the hydrogel in formula (II) is a hydrogel! insoluble in water based on biodegradable poly (ethylene glycol!) (PEG). Preferably, the hydrogel in formula (II) is composed of skeleton portions interconnected by hydrolytically degradable bonds. More preferably, the skeletal portions comprise a branching core of the following formula: Tot ”where the dashed line indicates the connection to the rest of the skeletal portion. More preferably, the skeleton portions comprise a structure of the following formula: + ADIA A] c O [N f, where n is an integer from 5 to 50 and the dashed line indicates
. 43/97 each binding to the rest of the molecule. Preferably, the backbone portion comprises a hyper-branched Hyp portion. More preferably, the skeleton portion comprises a hyper-branched portion Hyp of the following formula:] N NH
HT O NH Fá /] | í [) Y o. - / N "NO o YEAR H AX TN N NH
It is PEA T N Ha. HH Nº 0 $ | +. , where the dashed lines indicate the bond to the rest of the molecule and carbon atoms marked with an asterist indicate the S configuration. Preferably, the skeleton portions are attached to at least one spacer of the following formula: | o o where one of the dashed lines indicates the connection to the hyper-branched portion Hyp and the second dashed line indicates the connection to the rest of the molecule; and where m is an integer from 2 to 4.
: 44/97 Preferably, the skeleton portions are connected to at least one spacer of the following formula: Ad À> AÁDAIAD Ai “OPN ': where the dashed line marked with the asterisk indicates the connection between the hydrogel and the N of the thiosuccinimide group; where the other dashed line indicates the connection to the Hyp; and, where p is an integer from 0 to 10. Preferably, the backbone portions are joined together i via linker portions having the following structure Kg DA nor where q is an integer from 3 to 100.
The rate of hydrolysis of the biodegradable bonds between the backbone and linker moieties is determined by the number and type of atoms connected | adjacent to the PEG ester carboxy group. For example, by selecting succinic, adipic or glutaric acid to form PEG esters, it is possible to vary the half-life degradation of the crosslinker.
The hydrogel-bound exendin prodrug of the present invention can be prepared starting from the hydrogel of the present invention by convenient methods known in the art. It is evident to a clinic in the technique that several routines exist. For example, the prodrug binder mentioned above to which the biologically active moiety is covalently linked can be reacted with the reactive functional groups of the hydrogel of the present invention with or already transporting the active moiety in part or as a whole.
In a preferred method of preparation, the hydrogel is generated through chemical bonding reactions. The hydrogel can be formed from two macromolecular extracts with complementary functionality that undergo a reaction such as condensation or addition. One of these starting materials is a cross-linking reagent with at least two groups
. 45/97 identical functional and the other starting material is a homomuiltifunctional skeleton reagent.
Suitable functional groups present in the crosslinking reagent include amino terminal, carboxylic acid and derivatives, maleimide and other Michael unsaturated alpha, beta acceptors such as vinylsulfone, thiol, hydroxyl groups.
Suitable functional groups present in the skeletal reagent include, but are not limited to, amino, carboxylic acid and derivatives, maleimide and other alpha, beta unsaturated Michael acceptors such as vinylsulfone, thiol, hydroxyl groups.
If reactive cross-linking reagent functional groups are used sub-stoichiometrically with respect to reactive skeleton functional groups, the resulting hydrogel will be a reactive hydrogel with free reactive functional groups attached to the skeleton structure.
Optionally, the prodrug ligand can first be conjugated to exendin and the resulting prodrug ligand conjugate can then react with the reactive hydro-gel functional groups.
Alternatively, after activating one of the functional groups of the prodrug ligand, the ligand-hydrogel conjugate can be contacted with e-xendine in the second reaction step and the excess exendin can be removed by filtration after conjugation of the exendin to the ligand hydrogel bound prodrug.
A preferred process for the preparation of a prodrug according to the present invention is as follows: A preferred starting material for the synthetic reagent synthesis is a 4-branch PEG tetra amine or 8-branch PEG octa amine, with the PEG reagent having a molecular weight ranging from 2000 to 10,000 Dalton, more preferably from 2000 to 5000 Da.
For such multi-branched PEG derivatives, lysine residues are coupled sequentially to form the hyper-branched skeletal reagent.
It is understood that lysines can be partially or totally protected - by protection groups during the coupling steps and that the final skeletal reagent can also contain protection groups.
A block A preferred building block is bis-boc lysine.
Alternatively, instead of
- 46/97 sequential additions of residues and lysine, a dendritic polylysine moiety can be assembled first and subsequently coupled to the 4-branch PEG tetraamine or 8-branch PEG octamine. It is desirable to obtain skeletal reagent carrying 32 amino groups, therefore seven lysines can be linked to each branch of a 4-branch PEG, or five lysines can be linked to each branch of 8-branch PEG. In another embodiment, the multi-branched PEG derivative is a tetra- or octa carboxy PEG. In this case, the portions! dendritic can be generated from glutaric or aspartic acid, and the resulting skeletal reagent can carry 32 carboxy groups. It is understood that all or a fraction of the functional groups of the skeletal reagent can be »present in a free form, as salts or in conjunction with the protection groups. It is understood that due to practical reasons the number of lysine skeletal reactants per PEG branch will be between six and seven, more preferably approximately seven. A preferred skeletal reagent is shown below: ear O NH NH, 2 o and N md N 3 NH rat 2. x E o
N HN NH on — 28 f NH, 4 The synthesis of the crosslinking reagent starts from a linear PEG chain with a molecular weight ranging from 0.2 to 5 kDa, more preferably from 0.6 to 2 kDa, which is esterified with a semiester of a dicarboxylic acid ND
. 47/97 lico, such as adipic acid or glutaric acid. Preferred protection group for forming semiester is the benzyl group. The resulting bis dicarboxylic acid PEG semesters are converted to more reactive carboxy compounds such as acyl chloride or active esters, for example, pentafluorophenyl or N-hydroxysuccinimide esters, more preferred N-hydroxysuccinimide esters, of which the selected structure preferred is shown below. Oo o o o Qro 6 nº45 eo: Alternatively, PEG semi-esters of bis dicarboxylic acid. can be activated in the presence of a coupling agent such as DCCouHOBtouPyBOP.
In an alternative embodiment, the backbone reagent carries carboxy groups and the corresponding crosslinking reagent can be selected from PEG chains terminated by amino containing ester.
The skeletal reagent and cross-linking reagent can be polymerized to form the hydrogel according to the invention using reverse emulsion polymerization. After selecting the desired stoichiometry between the skeleton and crosslinker functional groups, the skeletons and crosslinkers are dissolved in DMSO and a suitable emulsifier with an appropriately selected HLB value, preferably Arlacel P135, is employed to form a reverse emulsion using a mechanical and controlled stirrer - turning the stirring speed. Polymerization is initiated by the addition of a suitable base, preferably with NNN'N-tetramethylethylene diamine. After stirring for an appropriate amount of time, the reaction is quenched by the addition of an acid, such as acetic acid and water. The beads are harvested, washed, and fractionated according to the particle size by mechanical sieving. Optionally, protection groups can be removed at this stage.
Furthermore, such a hydrogel according to the invention can be used NM
- 48/97 with a spacer carrying a different reactive functional group than that provided by the hydrogel. For example, reactive functional groups of maleimide can be introduced into the hydrogel by attaching a suitable heterobifunctional spacer such as Mal-PEG6-NHS to the hydrogel. Such a functionalized hydrogel can also be conjugated to the exendin-linker reagents, carrying a reactive thiol group in the linker to form hydrogel-linked exendin drugs according to the present invention. After loading the hydrogel-binding exendin conjugate containing the functionalized maleimide group, all remaining functional groups are buffered with a suitable blocking reagent, such as mer-. captoethanol, to prevent unwanted side reactions.
In another preferred embodiment of the invention, a conjugate of 'exendin binder carrying free thiol is connected to the binding portion, is reacted with a hydrogel functionalized by maleimide at temperatures between room temperature and 4 ° C, more preferred at room temperature, in an aqueous solution buffered pH 5.5 to 8, preferably pH 6.5 to 7.5. Subsequently, the resulting binder-hydrogel drug conjugate is treated with a low molecular weight compound comprising a thiol group, preferably with a compound containing 34-500 Da thiol, more preferably with mercaptoethanol at temperatures between room temperature and 4 ° C, more preferred at room temperature, in a buffered aqueous solution of pH 5.5 to 8, preferably pH 6.5 to 7.5. In another preferred embodiment of the invention, an exendin binder conjugate carrying a maleimide group is reacted with a thiol functionalized hydrogel according to the invention at temperatures between room temperature and 4 ° C, more preferred at room temperature, in a buffered aqueous solution of pH 5.5 to 8, preferably pH 6.5 to 7.5. Subsequently, the resulting corresponding linker-hydrogel drug conjugate is treated with a low molecular weight compound comprising a maleimide group, preferably a compound containing 100 to 300 Da macheimide, for example, N-ethyl-naleimide , at temperatures MN
& 49/97 between room temperature and 4 ° C, more preferred at room temperature, in a buffered aqueous solution of pH 5.5 to 8, preferably 6.5 to 7.5. Another aspect of the present invention is a process comprising & steps of (a) contacting at temperatures between room temperature and 4 ° C in a buffered aqueous solution of pH 5.5 to 8 an aqueous À suspension comprising maleimide-functionalized hydrogel microparticles with a solution comprising a exendin ligand reagent of the present invention, wherein the Lº * chemical functional group comprises a thiol group, resulting in an exendin ligand-hydrogel conjugate; . (b) optionally, treat the exendin binder-hydrogel conjugate from step (a) with a compound containing 34 Da to 500 Da thiol at temperatures between room temperature and 4 ° C in an aqueous buffered solution of pH5,5a8. Another aspect of the present invention is a process comprising the steps of (a) contacting at temperatures between room temperature and 4 ° C in an aqueous buffered solution from pH 5.5 to 8 an aqueous suspension comprising thiol functionalized hydrogel microparticles with a solution comprising a exendin ligand reagent of the present invention, wherein the chemical functional group of L * comprises a maleimide group, resulting in an exendin ligand-hydrogel conjugate; (b) optionally, treat the binder exendin-hydrogel conjugate of step (a) with a compound containing maleimide from 100 to 300 Da at temperatures between room temperature and 4ºC in a buffered aqueous solution of pH 5.5 to 8 A particularly preferred method for the preparation of a prodrug of the present invention comprises the steps of (a) reacting a compound of formula C (A'-X '), in which A ”-X' represents A before its binding to Hyp or a precursor to Hyp and X * is a suitable functional group with a compound of the formula Hyp'-X ND
: 50/97 Hyp'-X represents Hyp before its binding to A or a precursor to Hyp and X it is a functional group suitable for reacting with X '; (b) optionally reacting the compound resulting from step (a) in one or more other steps to produce a compound of formula C (A- - Hyp). having at least four functional groups; (c) reacting at least four functional groups of the compound resulting from step (b) with a crosslinking precursor based on poly (ethylene glycol), in which the active ester groups of the crosslinking precursor are used in a substoichiometric amount compared to the total number of C reactive functional groups (A-Hyp), to produce a hydrogel; (d) reacting the remaining unreacted functional groups (representing the reactive functional groups of the backbone comprised in the hydrogel) in the step hydrogel backbone (c) with a biologically active covalent conjugate and transient prodrug linker or first reactive functional groups not reacted with the transient prodrug ligand and subsequently with the biologically active portion; (e) optionally buffering the remaining unreacted functional groups to produce a prodrug of the present invention.
Specifically, the hydrogels for the exendin prodrugs of the present invention are synthesized as follows: For charge polymerization, the skeletal reagent and crosslinking reagent are mixed in an amine / active ester ratio from 2: 1 to 1.05: 1.
Both the skeletal reagent and the crosslinking reagent are dissolved in DMSO to provide a solution with a concentration of 5 to 50 g per 100 ml, preferably 7.5 to 20 g per 100 ml! and more preferably 10 to 20 g per 100 ml. To carry out the polymerization, 2 to 10% (volume) of N, N, N 'N-tertramethylethylene diamine (TMEDA) are added to the DMSO solution containing the crosslinking reagent and skeleton reagent and the mixture is stirred for 1 to 20 seconds and left to rest. The mixture solidifies in less than 1 minute. ND
Such a hydrogel according to the invention is preferably crushed by mechanical processes such as agitation, crushing, cutting press, or grinding, and optionally sieving.
For emulsion polymerization, the reaction mixture is comprised of the dispersed phase and the continuous phase.
For the dispersed phase, the skeleton reagent and the cross-linking reagent are mixed in an active amine / ester ratio from 2: 1 to 1.05: 1 and are dissolved in DMSO to provide a solution with a concentration of: 5 to 50 g per 100 ml, preferably 7.5 to 20 g per 100 ml and more preferably 10 to 20 g per 100 ml.
The continuous phase is any solvent, which is not miscible with. DMSO, non-basic, aprotic and shows a viscosity of less than 10 Pa * s.
Preferably, the solvent is not miscible with DMSO, non-basic, aprotic, shows a viscosity of less than 2 Pa * s and is non-toxic.
More preferably, the solvent is a straight or branched hydrocarbon saturated with 5 to 10 carbon atoms.
Most preferably, the solvent is n-heptane.
To form an emulsion of the dispersed phase in the continuous phase, an emulsifier is added to the continuous phase before adding the dispersed phase.
The amount of emulsifier is 2 to 50 mg per ml of dispersed phase, more preferably 5 to 20 mg per ml! dispersed phase, more preferably 10 mg per ml dispersed phase.
The emulsifier has an HLB value of 3 to 8. Preferably, the emulsifier is a sorbitol triester and a fatty acid or a hydroxyl fatty polylacid) -poly (ethylene glycol) conjugate. More preferably, the emulsifier is a conjugate of poly (hydroxy fatty acid) -polyethylene glycol, with a linear poly (ethylene glycol) of a molecular weight in the range of 0.5 kDa to 5 kDa and poly (acid) units hydroxy fat) of a molecular weight in the range of 0.5 kDa to 3 kDa at each end of the chain.
More preferably, the emulsifier is poly (ethylene glycol) dipolehydroxy ester, Cithrol DPHS (Cithrol DPHS, Arlacel P135 trainer, Croda International Plc). Droplets of the dispersed phase are generated by stirring with
| | . 52/97 axial flow sorter with a geometry similar to agitators such as Isojet, Intermig, Propeller (EKATO Rúhr- und Mischtechnik GmbH, Germany)), more preferably similar to Isojet with a diameter of 50 to 90% of the diameter of the reactor. Preferably, stirring is started before adding the dispersed phase. The agitator speed is set at 0.6 to 1.7 m / s. To the dispersed phase is added at room temperature, and the concentration of the dispersed phase is 2% to 70%, preferably 5 to 50%, more preferably 10 to 40%, and more preferably 20 to 35% of the total reaction volume. The mixture of the dispersed phase, emulsifier and continuous phase is stirred for 5 to 60 minutes before adding the base to carry out the polymerization.
5 to 10 equivalents (referred to each amide bond to be formed) of a base are added to the dispersed and continuous phase mixture. The base is aprotic, non-nucleophilic and soluble in the dispersed phase. Preferably, the base is aprotic, non-nucleophilic, well soluble in both the dispersed and DMSO phases. More preferably, the base is aprotic, non-nucleophilic, well soluble in both the dispersed phase and DMSO, an amine base and non-toxic. Most preferably, the base is NN diamine, N 'No. tertramethylethylene (TMEDA). Stirring in the presence of base is continued for 1 to 16 hours.
During stirring, droplets of dispersed phase are hardened to become crosslinked hydrogel beads according to the invention that can be collected and fractionated according to size is carried out in a continuous vibrating screening machine with a 75 um deck. and 32 µm to supply hydrogel microparticles according to the convention.
Another aspect of the present invention is an exendin ligand conjugate intermediate D-L ', where L' is of formula (Ill) [Ace in Hess AAA NI OSS 'o (1), where the dashed line indicates attachment to one of the amino groups NM
. 53/97 of the exendin portion forming an amide bond; Another aspect of the present invention is exendin linker reagents D-L *, where D represents an exendin moiety; and, L * is a non-biologically active ligand reagent represented by formula (IV), nº o H *, SR g and rs o Nm, where the dotted line indicates the bond to one of the amino groups h of the exendin forming if an amide bond; D R 'is selected from C1 + 4 alkyl, preferably CH3; R , Rº, are independently selected from the group consisting of H and C ;, 4 alkyl, where L * is substituted with a L * And optionally also substituted, provided that the hydrogens marked with the asterisks in formula (IV) are not replaced by a substituent and where L * is a spacer connected to L * and comprising a chemical functional group intended for conjugation to a hydrogel.
Preferably, R in formula (IV) is replaced by L2 *. Preferably, R * in formula (IV) is CHz-L *. Preferably, L * in formula (IV) is also not substituted.
Preferably, Lº * comprises a thiol group.
Preferably, Lº * comprises a maleimide group.
Preferably, L is L -H.
The hydrogel for the prodrug of the present invention can be obtained from methods of preparation in the form of a molded article, such as a mesh or a sfent or microparticles.
More preferably, the hydrogen! it is formed in microparticulate beads that can be administered as a subcutaneous or intramuscular injection using a standard syringe.
Such as soft beads can have a diameter of between 1 and 500 SS. ) ÕSIS.
. 54/97 micrometer.
Preferably, the microparticles have a diameter of between 10 € 100 micrometer if suspended in an isotonic aqueous formulation buffer, more preferably a diameter of between 20 and 100 micrometer, more preferably a diameter of between 25 and 80 micrometer.
Preferably, the microparticles can be administered by injection through a needle less than 0.6 mm in diameter, preferably through a needle less than 0.3 mm in diameter, more preferably through a needle less than 0.225 mm in internal diameter, even more preferably through a needle less than 0.175 mm in internal diameter, and more preferably through - a needle less than 0.16 mm in internal diameter. It is understood that the terms "can be administered by injection", "injectable" or "injectable" refer to a combination of factors such as a certain force applied to a syringe plunger containing the biodegradable hydrogel in accordance with the invention dilated in a liquid at a certain concentration (weight / volume) and at a certain temperature, a needle of a certain internal diameter connected to the outlet of such a ring, and the time required to extrude a certain volume of the hydrogel biodegradable according to the invention of the syringe through the needle In order to provide injectability, a volume of 1 ml of the exendin prodrugs according to the invention dilated in water to a concentration of at least 5% (weight / volume) and contained in a syringe holding a 4.7 mm diameter plunger can be extruded at room temperature within 10 seconds by applying a force of less than 50 Newtons, preferably injectability is obtained for an exendin prodrug from to according to the invention swelled in water to a concentration of ca. 10% (weight / volume). Another aspect of the present invention is a process for preparing a prodrug by injectable comprising the step of (a) preparing an exendin hydrogel prodrug of the present invention in the form of microparticles;
. 55/97 (b) sieve the microparticles (c) select a fraction with a prodrug bead diameter between 25 and 80 µm.
(d) suspend the step bead fraction (c) in an aqueous buffer solution suitable for injection.
Another aspect of the present invention is a needle injectable prodrug obtainable from the process described above, in which the needle injectable prodrug is injectable through a needle with an internal diameter less than 300 µm, preferably through a needle with an internal diameter less than 225 µm, and more preferably through a needle with an internal diameter less than 175 µm. bh Another aspect of the present invention is a pharmaceutical composition comprising a prodrug of the present invention or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable excipient. The pharmaceutical composition is also described in the following paragraphs.
The exendin hydrogel prodrug composition can be provided as a suspension composition or as a dry composition. Preferably, the exendin hydro-gel prodrug pharmaceutical composition is a dry composition. Suitable drying methods are, for example, spray drying and freeze drying (freeze drying). Preferably, the exendin hydrogel prodrug pharmaceutical composition is dried by lyophilization.
Preferably, the hydrogel prodrug exendin is sufficiently dosed into the composition to provide a therapeutically effective amount of exendin for at least three days in one application. More preferably, an application of the exendin hydrogel prodrug is sufficient for one week.
The pharmaceutical composition of a hydrogel prodrug of exence-dyad according to the present invention contains one or more excipients.
Excipients used in parenteral compositions can be categorized as buffering agents, isotonic modifiers.
. 56/97 de, preservatives, stabilizers, anti-absorption agents, oxidation protection agents, viscosifiers / viscosity enhancing agents, or other auxiliary agents.
In some cases, these ingredients can have dual or triple functions.
The exendin hydrogel prodrug compositions according to the present invention contain one or more than one excipient, selected from the groups consisting of:
(i) Buffering agents: physiologically tolerated buffers to maintain pH in a desired range, such as sodium phosphate, bicarbonate, succinate, histidine, citrate and acetate, sulfate, nitrate, chloride, pyrus
vato.
Antacids such as Mg (OH), or ZnCO; 3 can also be used.
The buffering capacity can be adjusted to match the conditions
- more sensitive to pH stability (ii) Isotonicity modifiers: to minimize the pain that can result from cell damage due to different osmotic pressures in the injection tank.
Effective concentrations can be determined by osmometry using an assumed osmolarity of 285 to 315 mOsmol / kg for serum
(il) Preservatives and / or antimicrobials: parenteral multi-dose preparations require the addition of preservatives in a concentration
sufficient to minimize the risk of patients becoming infected with the injection and corresponding regulatory requirements have been established.
Typical preservatives include m-cresol, phenol, methylparaben, ethylparaben, propylparaben, butylparaben, chlorobutanol, benzyl alcohol, phenylmercuric nitrate, thimerosol, sorbic acid, potassium sorbate, benzoic acid, chlorocresol, and
-benzalkonium chloride
(iv) Stabilizers: stabilization is achieved by reinforcing the stabilizing forces of protein, by destabilizing the denatured state, or by direct attachment of excipients to the protein.
Stabilizers can be amino acids such as alanine, arginine, aspartic acid, glycine, histidine,
lysine, proline, sugars such as glucose, sucrose, trehalose, polyols such as glycerol, mannitol, sorbitol, salts such as potassium phosphate, sodium sulfate, chelating agents such as EDTA, hexaphosphate, binders such as
- 57/97 divalent metal ions (zinc, calcium, etc.), other salts or organic molecules such as phenolic derivatives. In addition, oligomers or polymers such as cyclodextrins, dextran, dendrimers, PEG or PVP or protamine or HSA can be used (v) Anti-absorption agents: Mainly ionic or non-ionic surfactants or other soluble proteins or polymers are used to coat or absorb competitively to the inner surface of the composition or the composition container. For example, poloxamer (Pluronic F-68), PEG dodil ether (Brij 35), polysorbate 20 and 80, dextran, polyethylene glycol, - PEG polystidine, BSA and HSA and gelatines. The concentration and type of excipient chosen depends on the effect to be avoided, but typically a - surfactant monolayer is formed at the interface just above the value
CMC í (vi) Lyo- and / or cryoprotectants: During freeze or spray drying, excipients can counteract the destabilizing effects caused by rupture of the hydrogen bond and removal of water. For this purpose, sugars and polyols can be used, but corresponding positive effects have also been observed for surfactants, amino acids, non-aqueous solvents, and other peptides. Trealose is particularly effective in reducing moisture-induced aggregation and also improves the thermal stability potentially caused by exposure of hydrophobic protein groups to water. Mannitol and sucrose can also be used, as exclusive lio / cryoprotectant or in combination with each other where higher ratios of mannitol: sucrose are known to enhance the physical stability of a lyophilized mass. Mannitol can also be combined with trehalose. Trealose can also be combined with sorbitol or sorbitol used as the sole protector. Starch or starch derivatives can also be used (vii) Oxidation protection agents: antioxidants such as | ascorbic acid, ectoin, methionine, glutathione, monothioglycerol, morine, polyethyleneimine (PEI), propyl gallate, vitamin E, agents such as citric acid, EDTA, hexaphosphate, thioglycolic acid
E 58/97
(viii) Viscosifiers or viscosity enhancers: delay the sedimentation of particles in the vial and syringe and are used to facilitate mixing and resuspension of the particles and make the suspension easier to inject (ie low force under the syringe plunger ). Suitable viscosifiers or viscosity enhancers are, for example, carbomer viscosifiers such as Carbopol 940, Carbopol Ultrez 10, cellulose cellulose derivatives such as hydroxypropylmethylcellulose (hypromellose, HPMC) or diethylaminoethyl cellulose (DEAE or DEAE-C), silicate colloidal magnesium (Vee gum) or sodium silicate, hydroxyapatite gel, tricalcium phosphate gel, xanthans, carrageenans such as Satia UTC 30 gum, aliphatic poly (hydroxy acids), such as poly (D, L- or L-lactic acid) (PLA) and poly (glycolic acid) (PGA) - and their copolymers (PLGA), D, L-lactide, glycolide and capro-lactone terpolymers, poloxamers, hydrophilic poly (oxyethylene) blocks and blocks of hydrophobic poly-lifoxypropylene) to prepare a poly (oxyethylene) -poly (oxypropylene) -poly (oxyethylene) triplet (eg Pluronicº), polyether copolymer copolymer, such as a polyethylene glycol terephthalate copolymer / polybutylene terephthalate, acetate isobutyrate sucrose (SAIB), dextran or derivatives thereof, combinations of dextrans and PEG, polydimethylsiloxane, collagen, chitosan, polyvinyl alcohol (PVA) and derivatives, polyalkylimides, polyacrylamide-co-diallyldimethyl ammonium (DADMA), polyvinylpyrrolidine- dona (PVP), glycosaminoglycans (GAGs) such as dermatan sulphate, chondroitin sulphate, keratin sulphate, heparin, heparan sulphate, hyaluronan, ABA triblock copolymers or AB block composed of A t blocks hydrophobic, such as polylactide (PLA) or poly (lactide-co-glycolide) (PLGA), and hydrophilic B blocks, such as polyethylene glycol (PEG) or polyvinyl pyrrolidone.
Such block copolymers as well as the aforementioned poloxamers may exhibit reverse thermal gelation behavior (fluid state at room temperature to facilitate administration and gel state above the transition temperature of solution-gel in temperature).
after injection). (ix) Diffusion or dispersion agent: changes the permeability of connective tissue through the hydrolysis of extracellular matrix components
- 59/97 home in the intrastic space such as, but not limited to, hyalaluronic acid, a polysaccharide found in the intercellular space of connective tissue. A dispersing agent such as, but not limited to, hyaluronidase temporarily decreases the viscosity of the extracellular matrix and promotes the diffusion of injected drugs.
(x) Other auxiliary agents: such as wetting agents, viscosity modifiers, antibiotics, hyaluronidase. Acids and bases such as hydrochloric acid and sodium hydroxide are necessary auxiliary agents for pH adjustment during manufacture.
Preferably, the exendin hydrogel prodrug composition contains one or more of the viscosifier and / or modifying agent -,. viscosity.
The term "excipient" preferably refers to a diluent, adjuvant, or vehicle with which the therapeutic is administered. Such pharmaceutical excipient may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, including, but not limited to, peanut oil, soybean oil, mineral oil, gerbera limuca oil and the like. Water is a preferred excipient when the pharmaceutical composition is administered orally. Saline and aqueous dextrose are preferred excipients when the pharmaceutical composition is administered intravenously. Saline and aqueous dextrose solutions and glycerol solutions are preferably employed as liquid excipients for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, prolonged release formulations and the like.
The composition can be formulated as a suppository, with traditional binders and excipients such as triglycerides. The oral formulation may include
- 60/97 standard excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical excipients are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions will contain a therapeutically effective amount of the therapeutic, preferably in purified form, together with an adequate amount of excipient in order to provide the form for appropriate administration to the patient. The formulation must adjust the mode of administration.
In a general embodiment, a pharmaceutical composition of the present invention either in dry form or as a suspension or in another form can be provided as a single or multiple dose composition. if. In one embodiment of the present invention, the dry exendin hydrogel prodrug composition is provided as a single dose, meaning that the container in which it is provided contains a pharmaceutical dose.
Thus, in another aspect of the present invention the composition is provided as a single dose composition.
Preferably, the suspension composition is a multiple dose composition, meaning that it contains more than one therapeutic dose. Preferably, a multiple dose composition contains at least 2 doses. Such a multi-dose composition of exendin-hydrogel can be used for different patients in need of it or is intended for use in one patient, in which the remaining doses are stored after the first dose is applied until necessary.
In another aspect of the present invention the composition is comprised in a container. Preferably the container is a dual chamber syringe. Especially the dry composition according to the present invention is provided in a first chamber of the dual chamber syringe and - reconstitution solution is provided in a second chamber of the dual chamber syringe.
Before applying the dry hyacinth prodrug composition ND
. 61/97 exendin drogel to a patient in need of it, the dry composition is reconstituted. Reconstitution can take place in the container in which the dry exendin hydrogel prodrug composition is provided, such as in a vial, syringe, dual chamber syringe, ampoule, and cartridge. Reconstitution is done by adding a predefined amount of reconstitution solution to the dry composition. Reconstitution solutions are sterile liquids, such as water or buffer, which may contain other additives, such as preservatives and / or antimicrobials. If the exendin hydrogel prodrug composition is provided as a single dose, the reconstitution solution may contain one or more preservatives and / or antimicrobials. Preferably, the reconstitution solution is sterile water. If the | exendin hydrogel prodrug composition is a multi-dose composition, it will be preferred that the reconstitution solution contains one: or more preservatives and / or antimicrobials, such as, for example, benzylalcoholecresol.
A further aspect of the present invention is the administration of a reconstituted exendin hydrogel prodrug composition. An exendin hydrogel prodrug composition can be administered by injection or infusion methods, including intradermal, subcutaneous, intramuscular, intravenous, intraosseous, and intraperitoneal.
Another aspect is a method of preparing a reconstituted composition comprising a therapeutically effective amount of an exendin hydrogel prodrug, and optionally one or more pharmaceutically acceptable excipients, in which the exendin is transient- linked to a hydrogel, the method comprising the step of * contacting the composition of the present invention with a reconstitution solution.
Another aspect is a reconstituted composition comprising a therapeutically effective amount of an exendin hydrogel prodrug, and optionally one or more pharmaceutically acceptable excipients, wherein the exendin is transiently linked to a hydrogel obtainable by the above method. NM
62/97 Another aspect of the present invention is the method of making a dry exendin hydrogel prodrug composition. In one embodiment, such a suspension composition is made by (i) mixing the exendin hydrogel prodrug with one or more excipients, (ii) transfer quantities equivalent to single or multiple doses in a suitable container, (ii) dry the composition in said container, and (iv) seal the container. Suitable containers are vials, syringes, dual chamber syringes, ampoules, and cartridges - Another aspect is a kit of parts. When the delivery device is simply a hypodermic syringe then the kit can comprise the syringe, a needle and a container comprising the drug composition. dry exendin hydrogel for use with the syringe and a second container comprising the reconstitution solution.In more preferred embodiments, the injection device is different from a simple hypodermic syringe and this So the separate container with reconstituted exendin hydrogel product is adapted to compromise with the injection device so that the use of the liquid composition in the container is in fluid connection with the outlet of the injection device. dog. Examples of delivery devices include, but are not limited to, hypodermic syringes and chain injector devices. Particularly preferred injection devices are pen injectors in — the container is a cartridge, preferably a disposable cartridge.
A preferred kit of parts comprises a needle and a container containing the composition according to the present invention and optionally also containing a reconstitution solution, the container - being adapted for use with the needle. Preferably, the container is a dual chamber syringe.
In another aspect, the invention provides a cartridge containing an exendin hydrogel prodrug composition as described hereinbefore for use with a pen injector device. The cartridge can contain a single dose or multiple doses of exendin.
In one embodiment of the present invention the exendin hydrogel prodrug suspension composition not only comprises an exendin hydrogel prodrug and one or more of the excipients, but also other biologically active agents, in its free form or as a prodrug. Preferably, such one or more additional biologically active agents are a prodrug, more preferably a hydrogel prodrug. Such biologically active agents include, but are not limited to, compounds of the following classes: - (i) Sulphonylureas, such as, for example, chlorpropamide, tollazamide, tolbutamide, glyburide, glipizide, glimepiride, glibenclamide, gliclazide f and the like; (ii) Meglitinides, such as, for example, repaglinide, nateglinide or mitiglinide; (iii) Glucagon-like peptide 1 (GLP-1) and its mimetics, insulinotropic glucose peptide (GIP) and its mimetics, exendin and its mimetics, and dipeptyl protease inhibitors (DPPIV); (iv) Biguanides, such as, for example, metformin; (v) Thiazolidinediones, such as, for example, rosiglitazone, pyoglobitazone, troglitazone, isaglitazone (known as MCC-555), 2-2 - [(2R) 4-hexyl-3,4-di acetic acid -hydro-3-0x0-2H-1,4-benzoxazin-2-ylJetoxy] - benzene, ciglitazone, rosiglitazone or the compounds described in WO 97/41097 by Dr. Reddy'is Research Foundation, especially 5 - [[4- [(3,4-dihydro-3-methyl-4-0x0-2-quinazolinylmethoxyphenyl] methyl] -2,4-thiazolidinedione and the like; (vi) GW2570, and the like; (vii) Retinoid-receptor modulators X (RXR), such as, for example, targretin, 9-cis-retinoic acid, and the like; (viii) Other insulin sensitizing agents, such as, for example, INS-1, PTP-1B inhibitors, inhibitors of GSK3, inhibitors of
- 64/97 glycogen phosphorylase a, fructose-1,6-bisphosphatase inhibitors, and the like; (ix) Insulins, including regular, short-acting, intermediate-acting, and long-acting insulins, inhaled insulin, insulin derivatives and insulin-like logos, such as insulin molecules with minor differences in the natural amino acid sequence; (x) Small molecule insulin mimetics, including, but not limited to, L-783281, TE-17411, and the like; (xi) sodium-dependent glucose transporter 1 / o0u 2 inhibitors (SGLT1, SGLT2), for example KGA-2727, T-1095, T-1095A, SGL- - 0010, AVE 2268, SAR 7226, SGL-5083, SGL-5085, SGL-5094, ISIS-388626, sergliflozin, dapagliflozin or remogliflozin etabonate, canagliflozin,. florizine, and the like; (xii) Amylin agonists that include, but are not limited to, pranlintide, and the like; (xiii) Glucagon antagonists such as AY-279955, and the like. (xiv) Intestinal modulators and hormones of gut hormone activity, such as Somatostatin, Oxintomodulin, Cholecystokinin, Incretins, Ghrelin, PYY3.36, and the like. Insulins as described above can be independently selected from the group consisting of bovine insulin, porcine insulin, and human insulin. Most preferably, insulin is independently selected from human insulins. An insulin can be selected from unmodified insulin, more particularly from bovine insulin, porcine insulin, and human insulin.
Insulin derivatives are derived from naturally occurring and / or insulin analog insulin, which are obtained by chemical modification. The chemical modification can consist, for example, of the addition of one or more chemical groups defined in one or more amino acids.
Insulin analogs that are described in EP 0214826, EPO0375437, EP0678522, EP O 885 961, EP 0 419 504, WO 92/00321, German Patent Applications 10 2008 003 568.8 and 10 2008 003 566.1, and EP- 0 368 187 can be part of the combinations of the invention. The documents MM
-. 65/97 EP 0 214 826, EP 0 375 437, EP 0 678 522, EP 0 419 504, WO 92/00321, and EP-A O 368 187 are included here by reference. A preferred insulin analogue can be selected from the group consisting of human insulin GIy (A21) -Arg (B31) -Arg (B32) (insulin glargine, Lantus); human insulin amide Arg (AO) -His (A8) -Glu (A15) - F ASP (A18) -GI (A21) -Arg (B31) -Arg (B32), human insulin Lys (B3) -Glu (B29 ); human insulin Lysº ºPro * (insulin lispro), human insulin B28 Asp (insulin aspart), human insulin in which the proline in position B28 has been replaced by Asp, Lys, Leu, Val or Ala and where Lys in position B29 can be replaced by Pro; human insulin AlaB26; des human insulin (B28-B30); human insulihta des (B27) or human insulin B29Lys (s- - tetradecanoil), des (B30) (insulin detemir). A preferred insulin derivative can be selected from the group consisting of human insulin B29-N-myristoil-des (B30), human insulin B29-N-palmitoyl-des (B30), human insulin B29-N- myristoyl, human insulin B29-N-palmitoyl, human insulins B28-N-myristoil LysººSPro , human insulin B28-N-palmitoil-Lysº "ºSPro , human insulin B30-N-myristoil-ThrP ºLys *, human insulin B30-N-palmitoil- Thrº29A ysEº, human insulin B29-N- (N-palmitoyl-Y-glutamyl) -des (B30), human insulin B29-N- (N-litocoli-Y-glutamil ) -des (B30), human insulin B29-N- (w-carboxyptadecanoyl) -des (B30), and human insulin B29-N- (w-carboxyptadecanoyl).
A most highly preferred insulin derivative is selected from the group consisting of human insulin GIy (A21) -Arg (B31) -Arg (B32), human insulin Lys " ºPro (insulin lispro), human insulin B28 Asp (as - part of insulin), human insulin B29Lys (e-tetradecanoil), desB30 (insulin detemir).
Preferably, such one or more additional biologically active agents is an insulin hydrogel drug as described in WOZ2011 / 012718 and WO2011 / 012719.
In addition to antidiabetic agents, bioactive compounds can be anti-obesity agents such as orlistat, a pancreatic lipase inhibitor.
- 66/97 tica, which prevents the breakdown and absorption of fat; or sibutramine, an appetite suppressant and serotonin, norepinephrine and dopamine uptake inhibitor in the brain, growth factors increasing fat mobilization (eg growth hormone, IGF-1, release factor - growth hormone ), modulators of oxintomodulin and ghrelin. Other potential bioactive anti-obesity agents include, but are not limited to, appetite suppressants that act through adrenergic mechanisms such as benzfetamine, femetrazine, phentermine, diethylpropion, mazlin, sibutramine, phenylpropanolamine or, ephedrine; appetite suppressing agents that act through serotonergic mechanisms such as quipazine, fluoxetine, sertraline, fenfluramine, or dexfenfluramine; surprised agents. appetite sensors that act through dopamine mechanisms, for example, apomorphine; appetite suppressant agents that act through histaminergic mechanisms (eg, histamine mimetics, H3 receptor modulators); energy consumption enhancers such as beta-3 adrenergic agonists and non-coupling protein function stimulators; leptin and leptin mimetics (for example, metreleptin); neuropeptide Y antagonists; melanocortin-1, 3 and 4 receptor modulators; cholecystokinin antagonists; analogs and mimetics of glucagon-like peptide 1 (GLP-1) (eg, Exendin); androgens (for example, dehydroepiandrosterone and derivatives such as ethiocolandione), testosterone, anabolic steroids (for example, oxandrolone), and steroidal hormones; galanin receptor antagonists; cytokine agents such as ciliary neurotrophic factor; amylase inhibitors; enterostatin agonists / Mimimetics; orexin / hypocretin antagonists; urocortin antagonists; bombesin agonists; protein kinase A modulators; corticotropin releasing factor mimetics; cocaine- and amphetamine-regulated transcription mimetics; peptide mimetics related to the calcitonin gene; and fatty acid synthase inhibitors.
In an alternative embodiment, the exendin hydrogel prodrug composition according to the present invention is combined with a second biologically active compound such that the exendin hydrogel bp 67/97 prodrug is administered to a patient in need of the same first, followed by administration of the second compound. Alternatively, the exendin hydrogel composition is administered to a patient in need of it after another compound has been administered to the same patient.
Yet another aspect of the present invention is a prodrug of the present invention or a pharmaceutical composition of the present invention for use with a drug.
Yet another aspect of the present invention is a prodrug of the present invention or a pharmaceutical composition of the present invention for use in a method of treating or preventing diseases or disorders that can be treated by exendin. Said compositions are for use in a method of treatment or prevention of diseases or disorders known to exendin and exendin agonists, for example, for the treatment and prevention of hyperglycemia and for the treatment and prevention of diabetes mellitus of any type, for example, insulin-dependent diabetes mellitus, non-insulin-dependent diabetes mellitus, pre-diabetes or gestational diabetes mellitus, for the prevention and treatment of metabolic syndrome and / or obesity and / or eating disorders, insulin resistance syndrome , decreased plasma lipid level, reduced cardiac risk, reduced appetite, reduced body weight, etc. Patients in need of treatment with long-acting exendin compositions described in the present invention are at high risk of developing comorbidities. Consequently, a combination of the present long-acting exendin with appropriate bioactive compounds can be used, for example, for the prevention, delay of progression or treatment of diseases and disorders selected from the group consisting of hypertension (including, but not limited to systolic hypertension and familial dyslipidemic hypertension), congestive heart failure, left ventricular hypertrophy, peripheral arterial disease, diabetic retinopathy, macular degeneration, cataracts, diabetic nephropathy, glomerulosclerosis, chronic renal failure, diabetic neuropathy, syndrome X, pre-
- 68/97 menstrual, coronary heart disease, angina pectoris, thrombosis, atherosclerosis, myocardial infarction, transient ischemic attacks, stroke, vascular restenosis, hyperglycemia, hyperinsulinemia, hyperlipidemia, hypertriglyceridemia, insulin resistance, impaired glucose metabolism - do, impaired glucose tolerance conditions, impaired fasting plasma glucose conditions, obesity, erectile dysfunction, skin and connective tissue disorders, foot ulcers and ulcerative colitis, endothelial dysfunction and impaired vascular compliance.
Prevention, progression delay or treatment of diseases and disorders selected from the group above can be achieved by combining the long-acting exendin composition of the present invention with at least one bioactive compound selected from the drug classes used for the treatment of said conditions, including AT1 receptor antagonists; angiotensin-converting enzyme (ACE) inhibitors; renin inhibitors; beta-adrenergic receptor blockers; alpha-adrenergic receptor blockers; calcium channel blockers; aldosterone synthase inhibitors; aldosterone receptor antagonists; neutral endopeptidase (NEP) inhibitors; dual angiotensin converting enzyme / neutral endopetidase inhibitors (ACE / NEP); an enothelin receptor antagonist; diuretics; statins; nitrates; anticoagulant agents; natriuretic peptides; digital compounds; PPAR modulators.
In the case of biologically active agents; prodrugs, especially hydrogel prodrugs, contain one or more acidic or basic groups, the invention also comprises their corresponding pharmaceutically or toxicologically acceptable salts, in particular their pharmaceutical salts
maceutically usable.
Thus, prodrugs that contain groups
acidic powders can be used according to the invention, for example,
as alkali metal salts, alkaline earth metal salts or as ammonium salts.
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.
Prodrugs that contain one or more basic groups, ie
. 69/97 is, groups that can be protonated, can be present and can be used according to the invention in the form of their addition salts with inorganic or organic acids. Examples for suitable acids include hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acids, oxalic acid, acetic acid, tartaric acid, lactic acid, acid salicyclic, benzoic acid, formic acid, propionic acid, pivalic acid, diethylacetic acid, malonic acid, succinic acid, pyelic acid, fumaric acid, maleic acid, malic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic, citric acid, adipic acid, and other acids known to the person skilled in the art. If - the prodrugs contain both acidic and basic groups in the molecule, the invention will also include, in addition to the salt forms mentioned, It is internal salts or betaines (zwitterions). The respective salts can be obtained by customary methods that are also known to the person skilled in the art, for example by contacting them with an organic or inorganic acid or base in a solvent or dispersant, or by anion exchange or exchange of cation with other salts. The present invention also includes all salts of the prodrugs which, due to low physiological compatibility, are not directly suitable for use in pharmaceutical products, but which can be used, for example, as intermediates for chemical reactions or for the preparation pharmaceutically acceptable salts.
The term "pharmaceutically acceptable" means approved by a regulatory agency such as EMEA (Europe) and / or the FDA (United States) and / or any other national regulatory agency for use in animals, preferably in humans. Yet another aspect of the present invention is a method of treatment, control, delay or prevention in a mammalian patient, preferably in a human, in need of treating one or more conditions comprising administering to said patient a therapeutically effective amount of a prodrug of the present invention or a
. 70/97 pharmaceutical composition of the present invention or a pharmaceutically acceptable salt thereof.
Fig. 1 shows the release of kinetics of compounds 8, 10, 12, 14 and 16 at pH 7.4, 37ºC.
Examples Materials and Methods Exendina4 [Sec ID No.: 1) in the resin (approximately 0.1 mmol / g load) synthesized by Fmoc strategy was obtained from CASLO Labora- | Tory Aps, Lyngby, Denmark.
Lixisenatide [Seg ID No. 21] and GLP-1 [Seg ID No. 13] naresin (approximately 0.1 mmol / g load) synthesized by Fmoc strategy was obtained from Peptide Specialty Laboratories, Heidelberg, Germany.
The peptides were fully side-protected and had a free N-terminus. í 5kDa 4-branch amino PEG was obtained from JenKkem Te- —chnology, Beijing, P. R.
China.
NH- ester of N- (3-maleimidopropyl) - 21-amino-4,7,10,13,16,19-hexaoxa-heneicosanoic acid (Mal-PEG6-NHS) was obtained from Celares GmbH, Berlin, Germany .
6- (S-tritillmercapto) hexanoic acid was purchased from Polipeptide, Strasbourg, France.
The amino acids used were of L configuration, if not established otherwise.
All chemicals were from Sigma-ALDRICH Chemie GmbH, Taufkirchen, Germany.
Deprotection of Fmoc: To remove the Fmoc protection group, the resin was stirred with 2/2/96 (volume / volume / volume) of piperidine / DBU / DMF (twice, 10 minutes each) and washed with DMF (ten times). Purification of RP-HPLC: RP-HPLC was performed on a 100x20 mm or 100x40 mm C18 ReproSil-Pur 300 ODS-3 column (Dr.
Maisch, Ammerbuch, Germany) connected to a Waters 600 HPLC System and Waters 2487 Absorbance detector, unless otherwise stated.
Linear gradients of solution A (0.1% TFA in H7O) and solution B (0.1% TFA in acetonitrile) were used.
HPLC fractions containing the product were pooled and lyophilized.
. 71/97 Flash Chromatography Purifications by flash chromatography were performed on an Isolera One system from Biotage AB, Sweden, using Biotage KP-Sil silica and n-heptane cartridges and ethyl acetate as eluents. The products were detected at 254 nm. For hydrogel beads, syringes equipped with polyethylene chips were used as reaction vessels or for washing steps. Ultra-performance analytical LC (UPLC) was performed in a Waters Acquity system equipped with a Waters column BEH300 C18 (21x50mm, particle size 1.7 µm) coupled to an LTQ Orbitrap Discovery mass spectrometer from Thermo Scientific. - MS of PEG products showed a series of portions of (CH2CH20O), due to the polydispersity of PEG starting materials. For easier interpretation, only a single representative m / z sign is provided - examples are required. Hydrogel peptide content: Peptide content is expressed as% weight of peptide in relation to the gross weight of hydrogel (sum of hydrogel weight functionalized by maleimide and exendin-binding thiol peptide-binding). The weight of exendin-binding thiol peptide-binding in hydrogel (and thus, the weight of peptide alone) was determined by consumption of exendin-binding thiol-binding peptide during the conjugation reaction with maleimide-functionalized hydrogel. The consumption of exendin-binding peptide-binding thiol was determined by the Ellman test (El-Iman, G. L. and another, Biochem. Pharmacol., 1961, 7, 88-95). Example 1 Synthesis of skeletal reagent 1g
NM k 72/97 Pe O NH NH, 2 o o ne ne N NH 8 AS OS Fr N NH, 8 ANS To cg Ho 1g and n — 28 NH, 4. The skeletal reagent 1g was synthesized from PEG5000 of 4 amino branches 1a according to the following scheme: Boc-Lys (Boc) -OH EDC, HOBL, DMSO, Collidine HCI Dioxane / MeOH [PeS1aso — n], Ra [ Pestaso — tyst8o0)], —— [PeGtaso sans], 1a 1b te Boc-Lys (Boc) -OH HCI Dioxane / MeOH BocLys (Boo) -OH —— - | Pesiaso — tsisções, | E | PETI Lyetss, 0oi), | Ea1aDo9 o 1 1e HCI Dioxane / MeOH [PEG1I250 —— ysLysLys, (Boc), l ————— [PEGI2So theta us.00, |, 1 1st For the synthesis of compound 1b, PEG5000 of 4 amino branches 1a ( MW of approximately 5200 g / mol, 5.20 g, 1.00 mmol, HCl salt) was dissolved in 20 ml of DMSO (anhydrous). Boc-Lys (Boc) -OH (2.17 9, 6.25 mmol) in 5 ml DMSO (anhydrous), EDC HCI (1.15 9 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 for 30 minutes at room temperature.
The reaction mixture was diluted with 1200 ml of DCM and washed
. 73/97 with 600 ml! H2SO, 0.1 N (2 x), brine (1 x), 0.1 M NAOH (2x), and 1/1 (volume / volume) brine / water (4 x). The aqueous layers were re-extracted with 500 ml! of DCM. The organic phases were dried over Na> SO ;, filtered and evaporated to provide 6.3 g of crude product 1b as colorless oil. Compound 1b was purified by RP-HPLC.
Yield 3.85 g (59%) of colorless glassy product 1b.
MS: m / z 1294.4 = [M + 5H] ”* (MW calculated for [M + 5H] ** = 1294.6).
Compound 1c was obtained by stirring 3.40 g of compound 1b (0.521 mmol) in 5 ml of methanol and 9 ml of HCla4 Nor dioxane 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] ”* (MW calculated for [M + 5H] ** = f 1152.0).
For the synthesis of compound 1d, 3.26 g of compound 1c (0.54 mmol) were dissolved in 15 ml of DMSO (anhydrous). 2.99 g Boc-Lys (Boc) -OH (8.64 mmol) in 15 ml DMSO (anhydrous), 1.55 g EDC HCI (8.1 mmol), 1.24 g HOBt: H2O (8.1 mmol), and 5.62 ml of collidine (43 mmol) were added. The reaction mixture was stirred for 30 minutes at room temperature. The reaction mixture was diluted with 800 ml of DCM and washed with 400 ml of 0.1 N H2SO4 (2 x), brine (1 x), 0.1 M NaOH (2x), and 1/1 (volume / volume) of brine / water (4 x). The aqueous layers were re-extracted with 800 ml of DCM. The organic phases were dried with Na> SO ;, filtered and evaporated to provide a glassy crude product.
The product was dissolved in DCM and precipitated with cooled diethyl ether (- 18 ºC). This procedure was repeated twice and the precipitate was dried in vacuo.
Yield: 4.01 g (89%) colorless glassy product 1d, which was used in the next step without further purification.
MS: m / z 14054 = [M + 6H] ** (MW calculated for [M + 6H] "* = 14054).
. 74/97 Compound 1e was obtained by stirring a solution of compound 1d (3.96 g, 0.47 mmol) in 7 ml of methanol and 20 ml of 4 N HCl in dioxane 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] * (MW calculated for [M + 7H] "* = 969.7).
For the synthesis of compound 1f, compound 1e (3.55 9, 0.48 mmol) was dissolved in 20 ml! of DMSO (anhydrous). Boc-Lys (Boc) -OH (5,329,15.4 mmol) in 18.8 ml DMSO (anhydrous), EDC HCI (2.76 9, 14.4 mmol), HOBt: H2zO (2.0 g , 14.4 mmol), and 10.0 ml of collidine (76.8 mmol!) - were added. The reaction mixture was stirred for 60 minutes at room temperature. "The reaction mixture was diluted with 800 ml of DCM and washed with 400 ml of H SO, ab0.1N (2x), brine (1 x), 0.1 M NAOH (2x), and 1/1 (volume / volume) brine / water (4 x) The aqueous layers were re-extracted with 800 ml of DCM The organic phases were dried over Na2SO2, filtered and evaporated to provide crude product 1f as colorless oil.
The product is dissolved in DCM and precipitated with cooled diethyl ether (-18ºC). 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] ** (MW calculated for [M + 8H] ** = 15054).
Skeletal reagent 1g was obtained by stirring a solution of compound 1f (MW approximately 12035 g / mol, 4.72 9, 0.39 mmol) in 20 ml of methanol and 40 ml of 4 N HCl in dioxane at room temperature - environment for 30 minutes. The volatiles were removed in vacuo.
Yield: 3.91 g (100%), vitreous skeleton reagent
19. MS: m / z 977.2 = [M + 9H] ** (MW calculated for [M + 9H] * =
NM p 75/97 977.4). Alternative synthetic routine for 19 For the synthesis of compound 1b, a suspension of 4-branch PEG5000 tetraamine (1a) (50.0 g, 10.0 mmol) in 250 ml of i-ProH (anhydrous), boc-Lys (boc) -OSu (26.6 g, 60.0 mmol) and DIEA (20.9 mL, 120 mmol) were added at 45 “ºC and the mixture was stirred for 30 minutes. Subsequently, n-propylamine (2.48 mL, 30.0 mmol) was added. After 5 minutes the solution was diluted with 1000 ml of MTBE and stored overnight at -20 ºC without stirring. Approximately 500 ml! of the supernatant were decanted and discarded. 300 ml! of cold MTBE were added and after 1 minute of shaking the product were 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 with a lumpy solid MS: m / z 937.4 = [M + 7H] ”* (MW calculated for [M + 7H]" * = 937.6). 1c was obtained by stirring compound 1b from the previous step (48.89, 7.44 mmol) in 156 ml of 2-propanol at 40 ° C. A mixture of 196 ml of 2-propanol and 78.3 ml of acetylchloride was added under stirring within 1 to 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, the suspension was stirred for 1 minute and cooled for 1 hour at -30 º C. The product was collected by filtration through a glass filter and washed with 200 ml of cold MTBE The product was vacuum dried for 16 hours Yield: 38.9 g (86% ) 1c as a white powder MS: m / z 960.1 = [M + 6H] "* (MW calculated for [M + 6H] * =
960.2). For the synthesis of compound 1d, boc-Lys (boc) -OSu (16.79, 37.7 mmol) and DIPEA (13.1 mL, 75.4 mmol) were added to a 1c suspension from the previous step (19.0 g, 3.14 mmol) in 80 ml of 2-
. y 76/97 propanol at 45 ° C and the mixture was stirred for 30 minutes at 45 ° C.
Subsequently, n-propylamine (1.56 ml, 18.9 mmol) was added.
After 5 minutes the solution was precipitated with 600 m! of cold and centrifuged MTBE (3000 min ”, 1 minute) 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 ºC for 16 hours without stirring.
The suspension was filtered through a glass filter and washed with 300 ml of cold MTBE.
The product was dried in vacuo for 16 hours. | Yield: 21.0 g (80%) of 1d as a white solid MS: m / z 14054 = [M + 6H] ** (MW calculated for [M + 6H] * "= 1405.4) - The compound 1e was obtained by dissolving compound 1d from the previous step (15.6 g, 1.86 mmol) -9 in 3 N HCl in methanol (81 mL, i 243 mmol) and stirring for 90 minutes at 40 ° C. 200 ml of MeOH and 700 ml of iPrOH were added and the mixture was stored for 2 hours at -30 ° C. For crystallization integrity, 100 ml of MTBE was added and the suspension was stored at -30 ° C overnight. ml of cold MTBE was added, the suspension was stirred for 1 minute and filtered through a glass filter and washed with 100 ml of 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] '"** (MW calculated for [M + 10H]' ** = 679.1) For the synthesis of compound 1f, boc-Lys (boc) -OSu (11.99, 26.8mmol) and DIPEA (9.34 mL, 53.6 mmol) were added to a suspension of 1e from the previous step , (8.22g, 1.12 mmol) in 165 ml of 2-propanol at 45 ° C and the mixture was stirred for 30 minutes.
Subsequently, n-propylamine (1.47 mL, 17.9 mmol) was added.
After 5 minutes the solution was cooled to -18 ºC for 2 hours, then 165 ml! de - “Cold MTBE were added, the suspension was stirred for 1 minute and filtered through a glass filter.
Subsequently, the filter mass was washed with 4x 200 ml of cold MTBE / IPrOH 4: 1 and 1x 200 ml! of cold MTBE. THE
The product was dried in vacuo for 16 hours.
Yield: 12.8 g, MW (90%) of 1f as a lumpy yellow solid MS: m / z 1505.3 = [M + 8H] ** (MW calculated for [M + 8H] ** = 15054) . The 1g skeletal reagent was obtained by dissolving 4ArmPEGSKDa (-LysLys2Lysa (boc) s), a (1f) (15.5 g, 1.29 mmol) in 30 ml of MeOH and cooling to 0 ° C.
4 N HCl in dioxane (120 mL, 480 mmol, | cooled to 0 ° C) was added over 3 minutes and the ice bath was removed.
After 20 minutes, 3 N HCl in methanol (200 mL, 600 mmol, cooled to 0 ºC) was added for 15 minutes and the solution was stirred. for 10 minutes at room temperature.
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! of MeOH, precipitated with 240 ml of cold MTBE and the suspension was centrifuged at 3000 rpm for 1 minute.
The 1g product was dried in vacuo Yield: 11.5 g (89%) as pale yellow flakes.
MS: m / z 1104.9 = [M + 8H] ** (MW calculated for [M + 8H] ** = 1104.9). Example 2 Synthesis of crosslinking agent 2d Crosslinking agent 2d was prepared from adipic acid mono benzylester (English, Arthur R. and others, Joumal of Medicinal Chemistry, 1990, 33 (1), 344-347) and PEG2000 accordingly with the following scheme:
. 78/97
O À 2 AA oH "HO FO Iron o 2a n — 45 | DCC, DMAP, DCM O j OA all 2D o | H, 2, Pd / C, EtOHACOEt oo Po Ag O to o 2 oo | DCC, NHS, DCM oo Cro o o o o O 2d O o A solution of PEG 2000 (2a) (11.0 g, 5.5 mmol) and benzyladipate semiester (4.8 9, 20.6 mmol) in DCM (90, 0 ml) was cooled to 0. C. Di-cyclohexylcarbodiimide (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 + 4ºC for 5 hours The solid was filtered and the solvent completely removed by vacuum distillation The residue was dissolved in 1000 mL 1/1 (volume / volume) diethyl ether / ethyl acetate and stored at room temperature for 2 hours at the same time that a small amount of a scaly solid is formed. The solid was removed by filtration through a pad of Celite®. The solution was stored in a vial. tightly closed at -30ºC in the freeze for 12 hours until crystallization is complete. The crystalline product was filtered through a glass frit and washed with cooled diethyl ether (-30ºC). The filter paste was dried SN
79/97 to the vacuum. Yield: 11.6 g (86%) 2b as a colorless solid. The product was used without further purification in the next step. MS: m / z 813.1 = [M + 3H] ** (MW calculated for [M + 3H] "* = 8133) In a 500 ml glass autoclave, bis adipic bis benzyl ester of PEG2000 2b ( 13.3 g, 5.5 mmol) was dissolved in ethyl acetate (180 mL) and 10% palladium on charcoal (0.4 g) was added to the solution, hydrogenated at 600 kPa (6 bar), 40ºC until hydrogen hydrogen consumption (5 to 12 hours) Catalyst was removed by filtration through a pad of Celiteº and the solvent was evaporated in vacuo - Production: 12.3 g (quantitative) 2c as yellowish oil. The product was used without further purification in the next step. 'MS: m / z 753.1 = [M + 3H] ** (MW calculated for [M + 3H] "* = 753.2) A semi-acid solution of acid PEG2000-bis-adipic 2c (9.43 g, 4.18 mmol), N-hydroxysuccinimide (1.92 g, 16.7 mmol) and di-cyclohexylcarbodiimide (3.44 g, 16.7 mmol) in 75 ml of DCM (anhydrous) was stirred overnight at room temperature. The reaction mixture was cooled to 0 ° C and the precipitate was filtered. DCM was evaporated and the residue was recrystallized from THF. Yield: 8.73 g (85%) of 2d crosslinking agent as color solid. MS: m / z 817.8 = [M + 3H] ** (MW calculated for [M + 3H] ** = 817.9 gimol). Example 3 Preparation of hydrogel beads (3) containing free amino groups A solution of 1200 mg of 1g and 3840 mg of 2d in 28.6 ml of —DMSO was added to a solution of 425 mg of Arlacel P135 (Croda In- ternational Plc) in 100 mL of heptane. The mixture was stirred at 650 rpm with a propeller shaker for 10 minutes at 25 ° C to form a suspension NM
| '80/97 pension in a 250 ml reactor equipped with deflectors. 4.3 mL of TMEDA was added to the effect polymerization. After 2 hours, the agitator speed was reduced to 400 rpm and the mixture was stirred for an additional 16 hours. 6.6 ml of acetic acid were added and then, after 10 minutes, 50 ml of water and 50 ml of saturated aqueous sodium chloride solution were added. After 5 minutes, the stirrer was stopped and the aqueous phase was drained. For fractionation by bead size, the water-hydrogel suspension was sieved wet through 75, 50, 40.32e20um steel mesh sieves. The bead fractions that were retained in the 32, 40, and 50 µm sieves were mixed and washed 3 times with water, 10 times with -. ethanol! and dried for 16 hours at 10 Pa (0.1 mbar) to provide 3 as a white powder. 'The content of the hydrogel amino group was determined by coupling a fmoc-amino acid to the free amino groups of the hydrogel and the subsequent fmoc determination as described by Gude, M., J. Ryf, and another, (2002 ) Letters in Peptide Science 9 (4): 203-206. For the different batches the amino group content of 3 was determined to be between 0.11 and 0.16 mmol / g. Example 4 Preparation of functionalized hydrogel by maleimide beads (4) and determination of maleimide substitution
O / O Chorus EIN Õ NS, Mal-PEG6-NHS Hydrogel beads 3 were pre-washed with 99/1 (volume / volume) DMSO / DIPEA, washed with DMSO and incubated for 45 minutes with a Mal-PEG6-NHS solution (2.0 equivalents relative to the theoretical amount of amino groups in hydrogel) in DMSO. The accounts 4 NM
. 81/97 were washed five times with DMSO and five times with pH 3.0 succinate (20 MM, 1 MM EDTA, 0.01% Tween-20). The sample was washed three times with pH 6.0 sodium phosphate (50 mM, 50 mM ethanolamine, 0.01% Tween-20) and incubated in the same buffer for 1 hour at room temperature. After the beads were washed five times with succinate and sodium of pH 3.0 (20 mM, 1 mM EDTA, 0.01% Tween-20). For the determination of maleimide content, an aliquot of con- | Hydrogel 4 was washed three times with water and ethanol each. The sample was lyophilized and weighed. Another aliquot of hydrogel beads 4 was reacted with excess mercaptoethanol (in 50 mM sodium phosphate buffer, 30 minutes at room temperature), and consumption of mercaptoethanol was detected by Ellman test (Ellman, GL and other , Biochem, Pharmacol., 1961, 7, 88-95). The maleimide content was determined to be between 0.10 and 0.13 mmol / g of hydrogelsic. Example 5 Synthesis of binding reagent 5c The binding reagent 5c was synthesized according to the following scheme:
1. MIC! no ANH: Lupo o Detio Hooe Neta TIO SAS o s. | 1.BH, -THF
2.boc, O, DIPEA
3.HCh, DS LI "stormania to pray Tas ARARAS No - ns NH, if * sb Synthesis of intermediate reagent ligand 5a: m-Methoxytritylchloride (3 g, 9.71 mmol) was dissolved in DCM (20 mL) and added dropwise drop to a solution of ethylenediamine (6.5 ml, 97.1 mmol) in DCM (20 ml). After two hours the solution was poured into diethyl ether (300 ml) and washed three times with 30/1 (volume / volume) ) solution NM
| brine / 0.1 M NaOH (50 ml each) and once with brine (50 ml). The organic phase was dried over Na-SO, and the volatiles were removed under reduced pressure. Mmt protected intermediate (3.18 g, 9.568 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). 6- (S-tritylmercapto) hexanoic acid (4.48 9, 11.47 mmol), PyBOP (5.67 g, 11.47 mmol) and DIPEA (5.0 mL, 28.68 mmol) were added and the The mixture was stirred for 30 minutes at room temperature. The solution was diluted with diethyl ether (250 mL) and washed three times with 30/1 (volume / volume) brine / 0.1 M NaOH solution (50 mL each) and once with brine (50 mL ). The organic phase was dried - over Na> SO, and the volatiles were removed under reduced pressure. Sa was purified by flash chromatography. r Yield: 5.69 g (8.09 mmol). MS: m / z 705.4 = [M + H] * (calculated MW = 705.0). Synthesis of binding reagent intermediate 5b: To a solution of Sa (3.19 g, 4.53 mmol) in anhydrous THF (50 mL) was added BH3-THF (1 mM solution, 8.5 mL, 8.5 mmol) and the solution was stirred for 16 hours at room temperature. Another BH3-THF (1 M solution, 14 mL, 14 mmol) was added and stirred for 16 hours at room temperature. The reaction was quenched by the addition of methane! (8.5 mL). N, N-dimethyl-ethylenediamine (3 ml, 27.2 mmol) was added and the solution was heated to reflux and stirred for three hours. The reaction mixture was allowed to cool to RT and was then diluted with ethyl acetate (300 ml), washed with Na; CO; aqueous, saturated (2 x 100 mL) and solution and NaHCO; aqueous, saturated (2 x 100 mL). The organic phase was dried over Na7SO, and the volatiles were removed under reduced pressure to obtain crude amine intermediate (3.22 g). The amine intermediate (3.22 g) was dissolved in DCM (5 ml). —Boc7rO (2.97 g, 13.69 mmol) dissolved in DCM (5 mL) and DIPEA (3.95 mL, 22.65 mmol) were added and the mixture was stirred at room temperature for 30 minutes. The mixture was purified by flash chromatography RN
- 83/97 to obtain the intermediate protected by Mmt and raw Boc and intermediate protected by Mmt (3.00 g). MS: m / z 791.4 = [M + H] ", 519.3 = [M-Mmt + H] * (calculated MW = 791.1).
0.4 M aqueous HCI (48 mL) was added to a solution of the intermediate protected by Mmt and Boc in acetonitrile (45 mL). The mixture was diluted with acetonitrile (10 ml) and stirred for 1 hour at room temperature. Subsequently, the pH value of the mixture reacts! it was adjusted to 5.5 by adding 5M NaOH solution. Acetonitrile was removed under reduced pressure and the aqueous solution extracted with DCM (4 x 100 mL). The combined organic phases were dried over Na> SO, and the vo-. latexes were removed under reduced pressure. Raw 5b was used in the next step without further purification. T Yield: 2.52 g (3.19 mmol). MS: m / z 519.3 = [M + H] * (calculated MW = 519.8 g / mol). Synthesis of binding reagent 5c: Intermediate 5b (985 mg, 1.9 mmol) and p-nitrophenylchloroformate (330 mg, 2.5 mmol) were dissolved in anhydrous THF (10 mL). DIPEA (0.653 ml, 3.7 mmol) was added and the mixture was stirred for 2 hours at room temperature. The solution was acidified by adding acetic acid (1 ml). Sc was purified by RP-HPLC. Yield: 776 mg, (1.43 mmol). MS m / z 706.3 = [M + Na] * (calculated MW = 706.3). Example 6 Exendin 6d binding reagent synthesis The exendin 6d binding reagent was synthesized according to the following scheme:
"84/97: 1. Fmoc-D-Ala-OH, PyBOP, DIPEA, DMF: H, N— Exedina-4 -O LDALoretane NE. Ao Exedina-4 OD 9 6a po oz oz Sc, DIPEA, DMF and ARNO , À, No Exedina-4 A ”)
HH 6b No, because NPySCI, DCM CER ro Exsdina-s ON o 6c Í 1. TFARtivanisol / o-cresol / b 2. TerP, WecNágua 8 xi É Exedina4 o nst>) "The 6d Synthesis of Binding Reagent Intermediate exendin 6a: Exendin-4 protected by a fully lateral chain with free N terminal on the resin (2.00 g, 0.2 mmol, approximately 0.1 mmol / g charge) was transferred in a 20 mL syringe equipped with a frit 8 ml of anhydrous DMF were withdrawn in the syringe and the syringe was agitated (600 rpm) for 15 minutes to pre-dilate the resin, the solvent was discarded, and a solution of Fmoc-D-alanine-OH (187 mg, 0.6 mol), PyBOP (312 mg, 0.6 mmol), and DIPEA (174 µL, 1.0 mmol) in anhydrous DMF (4 mL) was withdrawn in the syringe, the syringe was stirred at room temperature and 600 rpm for 60 minutes The solution was discarded, and the resin was washed ten times with DMF. Fmoc deprotection was performed according to "Materials and Methods". Exendin Binding Reagent Intermediate Synthesis 6b: One solution of 5c (137 mg, 0.4 mmol) in anhydrous DMF (3 mL) was added to resin 6a (0.2 mmol), followed by a solution of DI-PEA (80 µL, 0.46 mmol) in DMF anhydrous (4.5 mL), and the reaction mixture was SS SõS $ A
. 85/97 stirred (600 rpm) at 22ºC for 15 hours. The resin was washed ten times with DMF and ten times with DCM and dried in vacuo. Synthesis of Exendin Binding Reagent Intermediate 6c: 3-nitro-2-pyridine-sulfenyl chloride (48 mg, 0.25 mmol) was supplied in a syringe containing 6b (0.05 mmol, 0.5 g) . Anhydrous DCM (4 ml) was withdrawn in the syringe and the mixture was stirred (600 rpm) 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 exendin 6d binding reagent Intermediate: In a round-bottom vial o-cresol (1.5 ml), ticanisol (1.5 ml), DTT (1.1259 g), TES (1.125 ml), and water (1.5 (1.5 ml) were dissolved in TFA (37.5 ml). 6c (0.15 mmol, 1.5 g) was added to the stirred solution (250 to 350 rpm) at room temperature in order to to obtain a homogeneous suspension. Stirring was continued for 45 minutes. The solution was separated from the resin beads by filtration, the beads were washed with TFA twice (2 ml each) and the solutions and wash were combined with the filtrate. TFA was removed from the combined solutions in a nitrogen cream 6 crude was precipitated from the concentrated solution (approximately 10 ml) by adding diethyl ether (30 ml) and vigorous stirring After centrifugation (2 min, 5000 rpm) the supernatant was discarded and the precipitate was washed with diethyl ether twice (20 ml each) The dried precipitate was dissolved in a solution of TCEP (114 mg, 0.39 mmol) in 30 ml 1/19 ( volume / volume) of acetonitrile / water containing 0.01% TFA (volume / volume). The mixture was incubated for 15 hours at room temperature. 6d was purified by RP-HPLC as described in Materials and Methods using a 150 x 30 mm Waters XBridge "" BEH300 C18 10 pm column and a flow rate of 40 ml / minute. Up to 12 ml of the mixture was loaded onto the column. Elution was performed using a linear gradient of 5% to 30% solvent B (5 minutes) followed by a linear gradient of 30% to 35% solvent B (40 minutes). Fractions containing 6d product were mixed and lyophilized. Purity: MD
| - 86/97 86% (215 nm) | Yield: 85.2 mg (19.2 u mol, starting from 2.00 g of resin). MS m / z 1486.7 = [M + 3H] **, (calculated MW = 4460.0 g / mol). Example 7 Synthesis of exendin-binding reagent 7 ARRNERA. à A Eae nst SON ON HH õ 7 - Exendin reagent binder 7 was synthesized as described for exendin reagent binder 6a-6d starting from exendin-4 protected by a fully lateral chain in the resin with free N terminal (336 mg, 34 umol) ), except the use of Fmoc-L-alanine-OH instead of Fmoc-D-alanine-OH. The rea- “10 people were consequently represented in scale in order to obtain the same relations as those used in 6a-6d. Yield: 13.4 mg MS: m / z 1487.4 = [M + 3HP * (calculated MW: 4460.0) Example 8 Synthesis of exendin hydrogel binder 8
The hydrogel —NS Hs i No Exedinas PA dA, Oo O 8 242.5 mg of hydrogel functionalized by maleimide 4 (25.0 umol of maleimido groups), as a suspension in succinate buffer pH 3.0 (20 MM, 1 mM EDTA, 0.01% Tween-20) was loaded into a syringe equipped with a filter frit. The hydrogel was washed ten times with 1/1 (volume / volume) of acetonitrile / water containing 0.1% TFA (volume / volume). A solution of exendin 6d binding reagent (122.7 mg, 27.5 pumol) in 1/1 (volume / volume) acetonitrile / water plus 0.1% TFA (3.7 mL) was pulled and stirred for 2 minutes at room temperature
D 87/97 to obtain a balanced suspension. 334 µl of phosphate buffer (pH 7.4, 0.5 M) was added and the syringe was stirred (600 rpm) at room temperature for 15 minutes. Exendin-binding thiol consumption was monitored by Ellman's test. The hydrogel was washed 10 times with 1/1 (volume / volume) of acetonitrile / water containing 0.1% TFA (volume / volume). Mercaptoethane! (47 uL) was dissolved in 1/1 (volume / volume) acetonitrile / water plus 0.1% TFA (3 mL) and phosphate buffer (0.5 mL, pH 7.4, 0.5 M) . The solution was drawn into the syringe and the sample was stirred (600 rpm) for 1 hour at room temperature. The solution was discarded and the hydrogel was washed ten times with 1/1 (volume / volume) of acetonitrile / water plus 0.1% TFA. After the hydrogel was washed ten times with buffer - succinate (10 mM succinate, 46 g / L mannitol, 0.05% Tween-20, adjusted with Tris to pH 5.0) and stored at 4 ºC. Exendin containing exendin hydrogel binder was determined according to Materials and Methods. An exendin content of 30% (weight) was obtained. Example 9 In vitro release kinetics An aliquot of exendin 8 hydrogel binder (0.5 mg exendin) was transferred into a syringe equipped with a glass frit and washed 5 times with pH 7.4 phosphate buffer (60 mM , 3 mM EDTA, 0.01% Tween-20). The hydrogel was suspended in the same buffer and incubated at 37 ºC. At defined time points (after 1 to 7 days of incubation time each) the supernatant was exchanged and released and released exendin was quantified - by RP-HPLC at 215 nm. The UV signals that correlate with the released exentin were integrated and plotted against the incubation time. Curve adjustment software was applied to estimate the corresponding release interval. First-order release kinetics with an interval of 45 days has been obtained (see Fig. 1). Example 10 Synthesis of exendin hydrogel binder 10 NM
. 88/97 o
H X H hydrogel —N N N— Exedin-4 TEN
H H: O o 10 The exendin hydrogel binder 10 has been synthesized as described for the exendin hydrogel binder 8 except for the use of exendin binder thiol 7 instead of exendin binder thiol 6d. Exendin containing exendin hydrogel binder was determined according to Materials and Methods. An exendin content of 30.5% (weight) was obtained. : Example 11 Synthesis of lixisenatide binding reagent 11d o = - H: H N> as> Lixisenatide o 11d Synthesis scheme:
1. Fmoc-D-Ala-OH PyBOP, DIPEA, DMF. -
2. DBU / piperidine / DMF: Ho. : NLixisenatide: o N — Lixisenatide- Hy ixisenati O NO O o 11a E Ro; If. DIPEA OMF "AM A A reserataa- (O) Ex 11b o NO, oc o Z H NPySCL DEM Cê AR NIN ÇA, ge tisenatida- (O)
H H AN 1c º
1. TFA / IDTTITES / water / thioaniso / NBU.Br o "
2. TCEP, acetonitrilafagua À z [and N. N — Lixisenatide DO AOS,
TRA 11d
, '89/97 Synthesis of lixisenatide binding reagent intermediate 11a: Lixisenatide protected by fully lateral chain in the free N-terminated resin (300 mg, approximately 0.1 mmol / g charge) was transferred in a 5 mL syringe equipped with a glass frit. 4 ml! anhydrous DMF were removed from the syringe and the syringe was stirred (600 rpm) for 15 minutes to pre-gel the resin.
The solvent was discarded, and a solution of Fmoc-D-alanine-OH (28 mg, 90 umol), PyBOP (47 mg, 90 upmol), and DIPEA (26 ul, 150 umol) in anhydrous DMF (2 ml) withdrawal in | syringe.
The syringe was stirred at room temperature and 600 rpm for 60 minutes.
The solution was discarded, and the resin was washed ten times with DMF.
Deprotection of Fmoc was carried out according to "Materials and - Methods". Synthesis of lixisenatide binder reagent intermediate 11b: fi A solution of 5c (41 mg, 60 umol) in anhydrous DMF (1.5 ml) was added to resin 11a (30 umol), followed by addition of DIPEA (13 µL, 75 umol), and homogenized reaction mixture was stirred (600 rpm) at 22 ° C for 22 hours.
The resin was washed ten times with DMF and ten times with DCM and dried in vacuo.
Synthesis of lixisenatide binding reagent intermediate 11c: 3-nitro-2-pyridine-sulfenyl chloride (38 mg, 0.20 mmol) was supplied in a syringe equipped with a glass frit, containing 11b.
Anhydrous DCM (2 mL) was withdrawn in the syringe and the mixture was stirred (600 rpm) at room temperature.
After 3.5 h the solution was discarded and the resin was washed 15 times with DCM and dried in vacuo.
Synthesis of lixisenatide binding reagent 11d: In a 50 ml Falcon tube! of NBu, 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). 11c (30 umol) was added to the stirred solution (200 rpm) at room temperature in order to obtain a homogeneous suspension.
Stirring was continued for 1 hour.
The beads were filtered and washed with TFA twice (1 ml each). The washing solutions were combined and 90/97 with the filtrate.
h Crude 11d was precipitated from the filtrate (approximately 10 ml) by adding cold diethyl ether (-18 ° C, 40 ml) and vigorous stirring. The suspension was cooled to -18 ºC for a further 15 minutes and centrifuged (2 minutes, 5000 rpm). The supernatant was discarded and the precipitate was washed with diethyl ether twice (20 ml each) and dried under reduced pressure. The precipitate was dissolved in a solution of TCEP (27 mg, 0.94 umol) in 2.5 ml 1/1 (volume / volume) of acetonitrile / water containing 0.01% TFA (volume / volume) ). The mixture was incubated for 15 hours at room temperature. 20 mL of water was added and 11d was purified by RP-HPLC in two cycles using a linear gradient of 5% to 30% solvent B (5 - minutes) followed by a linear gradient of 30% to 35% solvent B (40 minutes). A 150 x 30 mm Waters XBridge "" BEH300 C18 10 µm column and a flow rate of 40 ml / min was used. Fractions containing product 11d were mixed and lyophilized. Yield 6.1 mg MS: m / z 1284.3 = [M + 4H] ** (calculated MW = 5131.9). Example 12 Hydrogen systems! of lixisenatide12 ligand H 1 hydrogel —N and NX À, et Tanta o W Rs o 12 The lixisenatide ligand 12 hydrogel was synthesized as described for the exendin 8 hydrogel ligand except for the use of ligand thiol lixisenatide 11d instead of exendin binder thiol 6d. The lixisenatide content of lixisenatide binder hydrogel was determined according to Materials and Methods. A lixisenatide content of 32.4% was obtained. Example 13 Synthesis of lixisenatide binding reagent 13
- 91/97 | H 7 H usT E Ad "Ns Lixisenatide 13 The lixisenatide binding reagent 13 was synthesized as described for lixisenatide binding reagent 11a-11d starting from lixisenatide protected by a fully lateral chain in the resin with free N terminal (335 mg , 34 umol), except the use of Fmoc-L-alanine-OH instead of Fmoc-D-alanine-OH.
The reagents were scaled accordingly in order to obtain ratios equal to those used in 11a-11d.
Yield 7.3 mg 'MS: m / z 1283.9 = [M + 4H] "** (calculated MW = 5131.9). Example 14 Hydrogen synthesis of 14 o lixisenatide binder; H 9 H hydrogel - N y ANA NX A IA riserataa o Ha [o 14 lixisenatide ligand hydrogel 14 has been synthesized as described for exendin ligand hydrogel 8 except for the use of lixisenatide thiol ligand 13 instead of exendin thiol ligand 6d.
The lixisenatide content of lixisenatide binder hydrogel was determined according to Materials and Methods.
A lixisenatide content of 34.5% (weight) was obtained.
Synthesis of binding reagent: GLP-115 H z ns Th AAA Nice o | 15 The GLP-1 binding reagent 15 was synthesized as described for lixisenatide binding reagent 11a-11d except starting from fully lateral chain protected GPL-1 in the resin with free N-terminus (258 mg, 30 umol) instead exendin in the resin. The reagents were plotted accordingly to obtain ratios equal to those used in 11a-11d. Yield 5.0 mg MS: m / z 1191.4 = [M + 3H] * (calculated MW = 3571.1).
Example 16 Synthesis of GLP-1 hydrogel binder 16 o i H TS hydrogel —N. ALA O "À a tr, Oo O 16 Í The GLP-1 hydrogel ligand 16 was synthesized as described for the exendin hydrogen ligand! 8 except for the use of the GLP-1 thiol ligand 15 instead of the thiol ligand 15 exendin 6d.
The content of GLP-1 GLP-1 hydrogel binder was determined according to Materials and Methods. A GLP-1 content of 26.3% (weight) was obtained.
Example 17 In vitro release kinetics The release half-life time at pH 7.4, 37 ° C of hydrogel exendin 10, hydrogel lixisenatide 12 and 14, and hydroelectric GLP-1 16 was determined as described in example 9. Release kinetics “of compounds 8, 10, 12, 14 and 16 are shown in Fig. 1. ligand structure o fee gas | 2 [esenstga Tags to [mint fi orgas - and aero sagas le Jessie from Esdas
Example 18 Synthesis of binding reagent 18e The binding reagent 18e was synthesized according to the following scheme: o 1 AXN | Ts A DNADNAH inSTTTESEASEESSES Ts ONA NA 182 ú 18 à Fx ENNetn | 7 Da a ho Tas Deferred NH PT nsOOSON A 'Nenth 18c E 18d | E E E P-nitrophenyl chloroformate
LL mea 18e The synthesis of ligand reagent intermediate 18b was performed under a nitrogen atmosphere. A solution of amine 18a (1.69 g, 4.5 mmol,] for preparation, see WO-A 2009/133137) in 30 ml! of THF (dry, molecular sieve) was cooled to 0 ºC. Butyl chloroformate (630 ul, 4.95 mmol) in 3 m! of THF (dry, molecular sieve) and DIPEA (980 µl, 5.683 mmol) were added. The mixture was stirred for 10 minutes at 0 ° C, cooling was removed and the mixture stirred for another 20 minutes at room temperature. LiAIH, 1 M in THF (9 mL, 9 mmol) was added and the mixture was refluxed for 1.5 hours. The reaction was quenched by slowly adding methanol (11 ml) and 100 ml! of saturated Na / K tartrate solution. The mixture was extracted with ethyl acetate, the organic layer was dried over NasSO, and the solvent was evaporated under reduced pressure. The crude product 18b (1.97 g) was used in the next step without further purification. MS: m / z 390.2 = [M + H] "(calculated MW = 389.6). 'A solution of crude product 18b (1.97 g), N- (bromoethyl) -phthalimide (1.43 g, 5.63 mmol) and KCO; s (1.24 g, 9.0 mmol) in 120 ml of acetonitrile was refluxed for 6 hours. 60 ml of a saturated NaHCO solution;
. 94/97 were added and the mixture was extracted 3x with ethyl acetate.
The combined organics were dried (Na2SO4) and the solvent was removed under reduced pressure.
Phthalimide 18c was purified on silica using heptane (containing 0.02% NEts) and an ascending amount of ethyl acetate (containing 0.02% NEt;) as eluents.
Yield: 0.82 g (1.46 mmol) MS: m / z 563.3 = [M + H] * (calculated MW = 562.8). Phthalimide 18c (819 mg 1.46 mmol) was dissolved in 35 ml! of e-! tanol and hydrazine hydrate (176 µl, 3.64 mmol) was added.
The mixture was refluxed for 3 hours.
The precipitate was filtered.
The solvent was removed under reduced pressure and the residue was treated with 15 ml of dichloromethane.
The precipitate was filtered and dichloromethane was removed under reduced pressure.
The residue was purified by RP HPLC.
The pooled HPLC fractions were adjusted to pH 7 by adding NaHCO; and extracted several times with dichloromethane.
The combined organics were dried (NasSO.) And the solvent was removed under reduced pressure to produce amine 18d.
Yield: 579 mg (1.34 mmol) MS: m / z 433.3 = [M + H] * (calculated MW = 432.7). Para-nitrophenyl chloroformate (483 mg, 2.40 mmol) was dissolved in 10 ml of dichloromethane (dry, molecular sieve). A solution of 18d amine (1.00 g, 2.31 mmol) in 5 ml of dichloromethane (dry, molecular sieve) and 1.8 ml of sym-collidine were added and the mixture was stirred at room temperature for 40 minutes.
Dichloromethane has been removed | under reduced pressure, the residue was acidified with acetic acid and purified by RP-HPLC to produce 18e para-nitrophenyl carbamate.
Yield: 339 mg (0.57 mmol) MS: m / z 598.3 = [M + H] * (calculated MW = 597.8). Synthesis of The GLP-1 Binding Reagent 19 | dA 1 ns Ds NO Sep o 19
- 95/97 The GLP-1 19 binding reagent was synthesized as described for the GLP-1 15 binding reagent except for the use of 18e binding reagent instead of 5c binding reagent, starting from fully protected chain GPL-1 side in the resin with free N terminal (150 mg, 16.5 umol). The reagents were scaled accordingly in order to obtain ratios equal to those used in 11a-11d.
Yield 1.33 mg DM: m / z 1196.0 = [M + 3H] ** (calculated MW = 3585.1). | Example 20 u of GLP-1 hydrogel linker 20 'hydrogel “x qt XxX x à N — etP Ss s N OA R 3 HH õ 20 GLP-1 linker hydrogel 20 was synthesized as described for the exendin linker hydrogel 8 except for the use of GLP-1 thiol 19 ligand instead of exendin 6d ligand thiol.
Abbreviations: AcOH Acetic acid AcCOEt ethyl acetate Bn benzyl Boc t-butyloxycarbonyl DBU 1,3-diazabicyclo [5.4.0Jundecene DCC N, Ndi-cyclohexylcarbodi-imide DCM dichloromethane DIPEA —di-isopropylethyl DMAP - dimethylamine-pyrethine-dimethylamine dimethylsulifoxide DTT DL EDC dithiothreitol 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide
96/97 EDTA ethylenediaminetetraacetic acid and stoichiometric equivalent EtoH ethanol! Fmoc 9-fluorenylmethoxycarbonyl HPLC high performance liquid chromatography HOBt N-hydroxybenzotriazole IPrOH 2-propanol LCMS liquid chromatography coupled to mass spectrometry - 3-maleimido mal propyl Mal-PEG6-NHS N- (3-maleimidopropyl) NHS ester -21-amino- 4,7,10,13,16,19-hexaoxa-henoicosanoic - Me methyl MeOH methanol: Mmt 4-methoxytrile MS mass spectrum / mass spectrometry MTBE terc.butil methyl ether MW molecular weight NHS N -hydroxy succinimide PEG poly (ethylene glycol) PyBOP benzotriazol-1-yl-oxy-tris-pyrrolidine-phosphonium Phth phthalimido RP-HPLC reverse phase high performance liquid chromatography rpm cycles per minute RT ambient temperature SEC exclusion chromatography size TCEP tris (2-carboxyethyl) hydrochloride TES triethylsilane TFA trifluoroacetic acid THF tetrahydrofuran: ”TMEDA diaminadeNN, N'N-tetramethylethylene Tris tris (hydroxymethyl) aminomethane Trt triphenylmethyl, trityl
- 97/97 UPLC ultra high performance liquid chromatography Vv volume

权利要求:
Claims (22)
[1]
1. Prodrug or a pharmaceutically acceptable salt thereof comprising an exendin ligand conjugate D-L, wherein D represents an exendin moiety; and, -L is a non-biologically active linker -L 'represented by formula (1), in OR AS | and the one where the dashed line indicates the bond to one of the amino groups. exendin forming an amide bond; R 'is selected from C,., Alkyl; “A10 R , Rº are independently selected from the group consisting of H and C1, alkyl, where L 'is substituted with an L2-2 and optionally also substituted, provided that the hydrogens marked with the asterisks in formula (1) are not substituted by a substituent and where LP is a single chemical bond or a spacer; and, Z is a hydrogel.
[2]
Prodrug according to claim 1, wherein L 'is also not substituted.
[3]
Prodrug according to claim 1 or 2, wherein R 'is -CH3
[4]
Prodrug according to any one of claims 1 to 3, wherein L it is a C1.20 alkyl chain, which is optionally interrupted by one or more groups independently selected from -O-; and C (O) N (R3aa); optionally substituted with one or more groups independently selected from OH; and C (O) N (R3aaR3aaa); and wherein R3aa, R3aaa are independently selected from the group consisting of H; and C14 alkyl.
[5]
Prodrug according to any one of claims 1
. 27 to 4, in which L is linked to Z through a terminal group selected from O.. H . AH Li PO O oe O '
[6]
Prodrug according to any one of claims 1 to 5, wherein L is represented by the formula (la) Ro O 1 |
The ATE pot SE H * Oo (la), is where the dashed line indicates the nitrogen bond of the exen "5 - forming an amide bond; and where L2 is a simple chemical bond or a spacer, Z it is a hydrogel.
[7]
Prodrug according to any one of claims 1 to 6, wherein L is represented by the formula (1!) O z. H õ Ho Ho N RAR AÔRAANIX, A a Ss N N
H H f (1), where the dashed line indicates the nitrogen bond of the exendin forming an amide bond and, Z is a hydrogel.
[8]
Prodrug according to any one of claims 1 to 7, wherein the hydrogel is a PEG-based hydrogel comprised of backbone portions.
[9]
Prodrug according to claim 8, wherein the skeletal portions comprise a branching nucleus of the following formula: 1 to A
"37 where the dashed line indicates the connection to the rest of the skeleton portion.
[10]
Prodrug according to claim 8, wherein the backbone portions comprise a structure of the following formula: OR. * efron f H where n is an integer from 5 to 50 and the dashed line indicates the connection to the rest of the molecule.
[11]
Prodrug according to any one of claims 8 to 10, wherein the skeletal portion comprises a hyper-branched portion Hyp of the following formula:. N NH HE / o FL. NH H "f .. | o YZ o E) X CO O
N NO 4N Hd H AX
PR N NH
AEE N a Ho
HH NX o $ +. where the dashed lines indicate the connection to the rest of the molecule; and carbon atoms marked with an asterist indicate the S configuration.
+ 417
[12]
Prodrug according to any one of claims 8 to 11, wherein the skeletal portions comprise at least one spacer of the following formula: O [9) in which one of the dashed lines indicates the connection to the hybridized portion Hyp and the second dashed line indicates the link to the rest of | molecule; and where m is an integer from 2 to 4.
[13]
13. Prodrug according to any one of claims 8 to 12, wherein the skeleton portions comprise at least one spacer of the following formula: Oo o ADADA> Ani 'Oo PN is' in which the dashed line is marked with an asterisk indicates the link between the hydrogel and the N of the thiosuccinamide group according to claim 5; where the other dashed line indicates the connection to the Hyp; and in that foot an integer from 0 to 10.:
[14]
Prodrug according to any one of claims 8 to 13, wherein the backbone portions are connected together via linker portions comprising the following structure x PAP Ar o “, where q is an integer from 3 to 100.
[15]
A pharmaceutical composition comprising a prodrug as defined in any one of claims 1 to 14, or a pharmaceutical salt thereof together with at least one pharmaceutically acceptable excipient.
"s7!
[16]
Prodrug according to any one of claims 1 to 14, or the pharmaceutical composition as defined in claim 15, for use with a drug.
[17]
Prodrug according to any one of claims 1a14.0u pharmaceutical composition as defined in na15, for use in a method of treating or preventing diseases or disorders that can be treated by exendin.
[18]
18. Exendin ligand D-L * reagent, where | D represents an exendin portion; and, -L * is a non-biologically active binding reagent represented by formula (IV), ã in AY R: ROD] NOS IO (N), where the dashed line indicates the bond to one of the exendin amino groups forming an amide bond; R 'is selected from C ,, 4 alkyl; R , Rºº, are independently selected from the group consisting of H and C ;, alkyl, | where L * is substituted with a L ** and optionally also substituted, provided that the hydrogens marked with the asterisks in formula (IV) are not replaced by a substituent and where L * is a spacer connected to L * and comprising a chemical functional group intended for conjugation to a hydrogel.
[19]
19. Exendin intermediate linker D-L 'conjugate, where L' is of formula (Ill)
OH TO RS to HpesOODADANAODA SP 7 o (11),
: Ã 67 'where the dotted line indicates the bond to one of the amino groups of the exendin portion forming an amide bond; It D represents an exendin portion.
[20]
20. Process for the preparation of a prodrug as defined in any one of claims 1 to 14, comprising the steps of (a) contacting at temperatures between room temperature and 4 ° C in a buffered aqueous solution of pH 5.5 to 8 um aqueous suspension - comprising maleimide-functionalized hydrogel microparticles with a solution comprising the exendin binding reagent according to claim 17, wherein the L * chemical functional group comprises a thiol group, resulting in an exendin binding conjugate - hydrogel; f (b) optionally, treat the binder exendin-hydrogel conjugate of step (a) with a compound containing 34 Da to 500 Da thiol at temperatures between room temperature and 4ºC in a buffered aqueous solution of pH 5.5a8.
[21]
21. Process for the preparation of a prodrug as defined in any one of claims 1 to 14, comprising the steps of (a) contacting at temperatures between room temperature and 4 ° C in a buffered aqueous solution of pH 5.5 to 8 um aqueous suspension comprising thiol-functionalized hydrogel microparticles with a solution comprising the exendin binding reagent according to "claim 18, wherein the L * chemical functional group comprises a maleimide group resulting in an exendin-hydrogel conjugate; (b) optionally, treat the exendin binder-hydrogel conjugate of step (a) with a compound containing maleimide from 100 to 300 Da at temperatures between room temperature and 4ºC in a solution: - quosatamponada of pH5,5a8.
[22]
22. Process for preparing a prodrug by injectable comprising the step of e TM (a) preparing a prodrug according to any one of claims 1 to 14, in the form of microparticles; (b) sieving the microparticles (c) selecting a fraction with a pro-bead diameter —macodentre25 and 80 um.
(d) suspend the step bead fraction (c) in an aqueous buffer solution suitable for injection.
a E Pá: ê 40 E ca AA - 14) Lxseatda (| Ala: PD AO * 10) beings (Ala) 8 - 12) Lixisenatida D-Ala & | 20 O h À E Ve. 7 8 | girl [(D-Wing) Na + 16 GLP- Dates o o 10 20 30 40 td] Fig. 1

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法律状态:
2020-08-11| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |

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2020-08-18| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2020-12-08| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2020-12-29| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-04-13| B11B| Dismissal acc. art. 36, par 1 of ipl - no reply within 90 days to fullfil the necessary requirements|

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2021-11-03| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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EP10177327.3|2010-09-17|

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EP10177327A|EP2438930A1|2010-09-17|2010-09-17|Prodrugs comprising an exendin linker conjugate|

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PCT/EP2011/066097|WO2012035139A1|2010-09-17|2011-09-16|Prodrugs comprising an exendin linker conjugate|

---