WO2007061148A1 - Site-specific peg conjugates of glp-1 and methods for production thereof - Google Patents

Abstract

Disclosed are site-specific PEG conjugates of GLP-1 wherein a PEG unit is covalently bound to a side-chain amine of GLP-1 Lys26 or Lys34. Further, there is disclosed a preparation method thereof and a pharmaceutical composition for treating diabetes comprising the site-specific PEG conjugates of GLP-1.

Description

SITE-SPECIFIC PEG CONJUGATES OF GLP-I AND

METHODS FOR PRODUCTION THEREOF

TECHNICAL FIELD

The present invention relates to site-specific PEG conjugates of GLP-I

wherein PEG is covalently bound to a side-chain amine of GLP-I Lys²⁶ or Lys³⁴.

RELATED PRIOR ART

Glucagon-like-peptide-1 (referred to as 'GLP-I' hereinafter), a product of

proglucagon, is a peptide hormone secreted in intestinal mucosa in response to meal

ingestion. Bioactive GLP-I (7-36) amide and GLP-I (7-37) correspond to

proglucagon(78-107) amide and proglucagon(78-108), respectively. Both peptides

are present in plasma, but the amount of GLP-I (7-36) amide present in human body

is much higher than that of GLP-l(7-36) (C. Orskov et al., Diabetes 42:658-661, 1993).

GLP-I increases the insulin secretion in beta-cell in the pancreas in response to

glucose stimulation, inhibits the glucagons secretion in alpha-cell (C. Orskov,

Diabetologica 35:701-711, 1992, H. C Fehmann and J. F. Habener, TEM 3: 158-163,

1992), and promotes the insulin biosynthesis (H. C. Fehmann and J. F. Habener,

Endocrinology 130:159-166, 1992). GLP-I effectively inhibits the increase of the

blood glucose level by the effective insulin secretion even in an insulin-independent type-2 diabetes patient (M. A. Nauck et al., Exp. Clin. Endocrinol. Diabetes

105:187-195, 1997) and does not function when the blood glucose level is low, thus

being expected as a therapeutic agent for type-2 diabetes. However, GLP-I (7-36)

amide and GLP-I (7-37) cannot exhibit sufficient in vivo activity because they are

rapidly decomposed by dipeptidyl peptidase IV (referred to as 'DPP IV hereinafter;

EC.3.4.14.5), a specific aminopeptidase which is present in tissues and organs of

several kinds of mammal, and thus its biological half-life is only 3-5 minutes (T. J.

Kieffer et al., Endocrinology 136:3583-3596, 1995, C. Orskov et al., Diabetes

42:658-661, 1993).

To dissolve the drawbacks of GLP-I, various efforts have been made to

develop GLP-I derivatives with enhanced in vivo activity inhibiting the increase of

the blood glucose level. PCT/ US87/ 01005 discloses insulinotropin derivatives

comprising at least one amino acid sequence not occurring in nature.

PCT/ US89/ 01121 discloses insulinotropic polypeptide derivatives comprising

GLP-I (7-36), GLP-I (7-35) and GLP-I (7-34), which also includes polypeptides having

inconsequential amino acid substitutions, or additional amino acids not essential to

enhance coupling to carrier protein or to enhance the insulinotropic effect thereof.

Erhard G. Siegel et al. reported that, when the positions of 8, 8 and 14, 8 and 15, and

14 and 15 of GLP-l(7-36) are modified, the derivatives with modified positions of 14

or 15 lose their biological activity while the derivatives with modified position 8 show similar or more enhanced activity as compared to GLP-I (7-36) (Regulatory

Peptides 79:93-102, 1999). Remy Burcelin et al. reported that GLP-Gly8, which is a

GLP-I (7-36) with position 8 Ala substituted with GIy, shows superior activity in

inhibiting the blood glucose level increase as compared to GLP-I (7-36) in the glucose

tolerance test (Metabolism 48:252-258, 1999). P. M. Finbarr et al. reported that

His⁷-glucitol-GLP-l, which has glucitol attached to position 7, His, shows superior

activity in inhibition of blood glucose level increase and high stability in DPP-IV

metabolism (Biochimica et Biophysica Acta 1474:13-22, 2000). Further, GLP-I

derivates are disclosed in many prior arts, such as WO 1998/08871

(PCT/ DK1997/ 00340), WO 2000/34331 (PCT/ EP1999/ 009660), WO 2000/34332

(PCT/ US1999/ 28929), WO 2001/98331 (PCT/ US2001/ 16474) and WO 2003/018516

(PCT/ US2002/ 021325).

The technique of binding a peptide or a protein with polyethylene glycol

(referred to as 'PEG' hereinafter) is based on the research of Davis and Abuchowski

(Abuchowski A. et al., J. Biol. Chem., 252, 3571-3581, 1977; Abuchowski A. et al., J.

Biol. Chem., 252, 3582-3586, 1977). PEG is a hydrophilic biocompatible nontoxic

polymer having a structure of H(OCH2CH2)_nOH and is known to prolong the

bioactivity by sterically hindering enzymatic degradation, increasing molecular size

and thus decreasing the glomerular filtration like glyco chain of glycoprotein when

bound to a peptide or a protein. U.S. patent no. 4,179,337 discloses about the covalent bonds between a polypeptide and PEG that the modification of a protein or

an enzyme with PEG decreases immunity and antigenity and prolong half-life in

blood. As an example of increasing or prolonging the activity by conjugating a

peptide with PEG, there is WO 1999/27897 (PCT/EP1998/07748), which discloses

hGRF-PEG, human Growth hormone Releasing Factor-PEG conjugate.

However, there has been no report on the increase in the inhibitory activity of

GLP-I against the increase of blood glucose level by selectively binding PEG to a

specific site of GLP-I.

DETAILED DESCRIPTION

The present invention relates to site-specific GLP-I-PEG conjugates wherein

PEG is selectively bound to side-chain amine of Lys²⁶ or Lys³⁴ of GLP-I and a

preparation method thereof. The present invention also relates to a pharmaceutical

composition comprising the GLP-I-PEG conjugates for treating diabetes.

The present invention relates to site-specific GLP-I-PEG conjugates wherein a

PEG unit is covalently bound to side-chain amine of Lys²⁶ or Lys³⁴ of GLP-I.

As used herein, 'GLP-I' includes GLP-I, GLP-I fragment, insulinotropin or

derivatives thereof, and C- terminal ester or amide and salt of GLP-I.

PEG with various molecular weight of 2,000-40,000 may be bound to GLP-I,

upon necessity, and methoxylated PEG or branched PEG may also be used. The branched PEG may have a structure of R(-PEG-OH)_m/ wherein R is a central core

moiety such as pentaerythritol or glycerol and m is the number of branching arms

ranging from 3 to 100. A hydroxyl group may be also used in chemical

modification. Another form of branching PEG may be (CH3θ-PEG-)_pR-X_/ as

disclosed in WO 96/21469, wherein p is 2-3, R is a central core moiety such as lysine

or glycerol and X is an activating group such as a carboxyl group. A pendant PEG,

still another branching PEG, has an activating group such as a carboxyl group

attached to main chain instead of the end of PEG chain.

These PEGs are used in the form of 'an activated PEG' to be bound to free

amine. Examples of the activated PEG include alkylating agent such as PEG

aldehyde, PEG epoxide and PEG tresylate and acylating agent such as PEG ester.

Their representative examples are PEG-N-succinimidyl succinate,

PEG-N-succinimidyl carbonate, PEG-N-succinimidyl propionate.

The present invention also relates to a GLP-I wherein N^α amine and

side-chain amine of Lys³⁴ or Lys²⁶ are selectively protected, which is used to

selectively bind PEG to the side-chain of Lys²⁶ or Lys ³⁴ of the GCP-I. GLP-I

wherein N^α amine and other side-chain amine of lysine than the desired moiety are

selectively protected may be prepared according to Korean patent publication no.

2004-0066454. For example, to prepare N^α,Lys³⁴-diFmoc-GLP-l(7-36) amide, which

is used in manufacture of Lys²⁶-PEG-GLP-l(7-36) amide, C-terminal amino acid having N^α amine protected with 9-fluorenylmethoxycarbonyl ('Fmoc' hereinafter) is

coupled to resin, deprotected under basic condition, washed, and next amino acid

having N^α amine protected with Fmoc is introduced. The peptide may be prepared

by repeating theses reactions up to N-terminal amino acid. A lysine wherein N^α

amine and side-chain amine are protected with Fmoc and ivDde, respectively, is

used in the step of introducing Lys³⁴ which is not the target site of PEGylation. The

protecting group ivDde is not deprotected under the condition for Fmoc

deprotection for next step coupling, such as 1%

l_/δ-diazabicylofSAOJundec-Z-eneCDBU' hereinafter)-20% piperidine

/N,N-dimethylform amide (DMF). A lysine wherein N^α amine and side-chain

amine are protected with Fmoc and tert-butoxycarbonyl('Boc' hereinafter),

respectively, is used in the step of introducing Lys²⁶, the target site of PEGylation.

The protecting group Boc is not deprotected under the basic condition for

deprotecting Fmoc for next step coupling. After His⁷, N-terminal amino acid of

GLP(7-36), is introduced, the peptide-resin is treated under the strong basic

condition, for example under the condition of 2% hydrazine, thus deprotecting Fmoc

and ivDde of His⁷ and Lys³⁴, respectively, followed by the treatment of

9-fluorenylmethoxycarbonyl-succinamide('Fmoc-OSu' hereinafter) to protect the

deprotected 7-, 34- amines with Fmoc. This peptide-resin is treated with the

cleavage solution, for example, a mixture of triisopropyl silane(TIS) : water : trifluoroacetic acid(TFA) (2.5 : 2.5 : 95) to cleave the peptide from the resin and also

to deprotect Boc, the protecting group of side-chain amine of Lys²⁶, thus providing a

His⁷-, Lys³⁴-diFmoc-GLP-l(7-36) amide wherein every amine except one in the target

site is protected by Fmoc.

GLP-I, which is selectively protected by various amine-protecting groups,

may be prepared according to the following methods. For examples, Fmoc or

4-nitrophenylsulfonylethoxycarbonyl ('Nsc' hereinafter) may be used as the

amine-protecting group during the synthesis of protected peptide. For the

protection of target-site lysine amine, other amine-protecting groups may also be

used instead of Boc if the protecting group is not deprotected under the condition

for deprotecting Fmoc or Nsc. Further, other protecting groups may also be used to

protect the target-site lysine if the protecting group is not deprotected under the

condition for deprotecting Fmoc or Nsc, and cleaving peptide-resin, and

deprotecting a protecting group of amine in non-target-site lysine. For example,

the peptide-resin is prepared by protecting the target-site lysine amine with Boc and

protecting the non-target-site amine and N^α-amine of Hys⁷ with carbobenzoxy ('Cbz'

hereinafter), and the protected peptide may be cleaved to conjugate with PEG,

followed by deprotection of cbz. Further, during the protection of non-target-site

amine right before the peptide-resin cleavage, the protecting group may be replaced

with various other protecting groups which are appropriate for PEG conjugation condition and are not deprotected under the cleavage condition, and the stability of

peptide is maintained under the condition for deprotecting the protecting group.

Examples of amine protecting group of the site-protected GLP-I include

ivDde(l-(4_/4-dimethyl-2_/6-dioxocyclohexyllidene)-3-methylbutyl), which is disclsoed

⁵ in Examples herein, Fmoc(9-fluorenylmethoxycarbonyl),

Nsc(4-nitrophenylsulfonylethoxycarbonyl), Boc(tert-butoxycarbonyl),

Alloc(allyloxycarbonyl), Dde(l-(4,4-dimethyl-2,6-dioxocyclohex-l-yllidene)ethyl),

Adpoc(l-(l'-adamantyl)-l-methyl-ethoxycarbonyl),

2-Cl-Z(2-chlorobenzyloxycarbonyl), and Cbz(carbobenzoxy).

^ Another aspect of the present invention relates to a method of a method for

preparing a site-specific GLP-I-PEG conjugate, wherein a PEG unit is covalently

bound to a target-site amine, i.e., the side-chain amine of GLP-I Lys²⁶ or Lys³⁴, the

method comprising (a) reacting an activated PEG with GLP-I where non-target-site

amines are selectively protected, and (b) deprotecting the amine protecting group.

⁵ For example, for preparing Lys²⁶-PEG-GLP-l(7-36) amide, His7,

Lys³⁴-diFmoc-GLP-l(7-36) amide is dissolved in dimethylform amide containing

0.3% of triethylamine(triethylamine, TEA), added with 6 equivalents of

PEG-succinimidyl propionate, followed by reaction at room temperature for 60

minutes. After the reaction is terminated, a small amount of piperidine is added

⁰ for deprotection and 10% trifluoroacetic acid is also added for modification of pH, and then the solution is diluted ten times with 0.1% trifluoroacetic acid. This

solution is purified by using an ion-exchange chromatography to remove unreacted

GLP-I(Z-So), non-bound PEG and free protecting group. The eluate is adsorbed to

reversed-phase rein, for example C-18 Sep-Pak cartridge, washed with distilled

water, and deso

rbed with water-acetonitrile mixture, followed by evaporation of acetonitrile and

freeze drying, thus providing Lys²⁶-PEG-GLP-l(7-36) amide with high purity.

The method herein may further comprise a step of purifying the site-specific

PEG conjugates of GLP-I by using reversed-phase chromatography.

According to another aspect of the present invention, there is provided a

pharmaceutical composition for treatment of diabetes comprising the site-specific

PEG conjugates of GLP-I as an active ingredient.

The site-specific PEG conjugates of GLP-I may be prepared into a

pharmaceutical formulation comprising a therapeutically effective amount of the

site-specific PEG conjugates of GLP-I. The examples of the formulation include an

injection, a drop infusion, a depot, and an inhalant, which may contain a buffering

agent, a tonic agent, a stabilizer, a surfactant, a thickening agent, a preservative, a

coloring agent or an aromatic substance.

The pharmaceutical composition herein may be administered in various

formulations including the aforementioned ones. Appropriate dosage level of the pharmaceutical composition herein may be determined by considering various

kinds of information such as physical condition and weight of a patient, disease

severity, medicine formulation, administration route and dosing interval, which

may be easily determined by one with an average skill. In a preferred embodiment,

the daily dosage level for an adult is 0.0001-10 mg/ weight (kg). The medicine may

be administered once or several times a day or once per several days, and once per

week or six month or more in the case of the depot for injection. The compound

according to the present invention may be contained in the composition in the

amount of 0.01-99.9 wt% relative to total weight of the composition.

The present invention provides a method of treating diabetes comprising

administering to a mammal a therapeutically effective amount of the site-specific

PEG conjugates of GLP-I of claim 1.

The site-specific PEG conjugates of GLP-I herein may be used in treating

diabetes.

The site-specific PEG conjugates of GLP-I herein may be used in

manufacturing a pharmaceutical drug for treating diabetes.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1 is a HPLC chromatogram of Example 7 (Lys²⁶-PEG2K-GLP-l(7-36)

amide). Figure 2 is a Maldi-Tof mass spectrum of Example 7

(Lys²⁶-PEG2K-GLP-l(7-36) amide).

Figure 3 is an area under the blood glucose level-time curve (AUC) for 3 hours

after oral administration of glucose solution to a diabetic db/db mouse, which is

intraperitoneal^ administered with GLP-I (7-36) amide, Lys²⁶-PEG2K-GLP-l(7-36)

amide (Example 7) and Lys³⁴-PEG2K-GLP-l(7-36) amide (Example 8), followed by

the administration of glucose after 15 minutes.

EXAMPLES

The present invention is described more specifically by the following

Examples. Examples herein are only meant to illustrate the present invention, but

in no way to limit the scope of the claimed invention.

The abbreviations used herein have the meanings as set forth hereunder.

AC2O = acetic anhydride

Boc = tert-butoxycarbonyl

Bop = benzotriazol-l-yloxytris(dimethylamino)phosphonium

hexafluorophosphate

CLTR = 2-chlorotrityl resin

DCM = dichloromethane DIPEA = N,N-diisopropylethylamine

DMF = N,N-dimethylformamide

Fmoc = 9-fluorenylmethoxycarbonyl

HOBt = 1-hydroxybenzotriazole

ivDde = l-(4_/4-dimethyl-2_/6-dioxocyclohexyllidene)-3-methylbutyl

Nsc = 4-nitrophenylsulf onylethoxycarbonyl

Pbf = : l^SA^-pentamethyldihydrobenzofuran-δ-sulfonyl

PyBop = benzotriazol-l-yl-oxytris(pyrrolidino)phosphonium

hexafluorophosphate

tBu = tert-butyl

TFA = trifluoroacetic acid

Trt - trityl

Chloranil test

Chloranil test may be used to determine if Nsc or Fmoc related additives and

remaining piperidine are removed. The test solution is prepared by adding one

drop of saturated chloranil/ toluene solution in 1 mL of acetone. Dimethylform

amide washing may be tested by adding one drop of washing solution into chloranil

test solution. The solution turns to blue or violet if there is secondary amine. Kaiser test

To check the termination of reaction by using qualitative ninhydrin test, 2-10

mg of resin sample is washed with ethanol. Two drops of 80% phenol solution,

two drops of 0.02 niM potassium cyanide/ pyridine and two drops of 5%

ninhydrin/ ethanol are added into the sample. The sample is placed at 120 ⁰C heat

block for 4-6 minutes. The resin or solution turns into blue or violet if there exists

free amine.

HPLC Analysis condition

Device: Waters Alliance

Flow Rate: 1.0 ml/min

Gradient: A 0-100% B within 45 minutes

(A: 0.1% trifluoroacetic acid/ water, B: 0.1% trifluoroacetic acid/acetonitrile)

Column: Nova-Pak-Cis, 3.9 mm x 150 mm, 5 μm, IOOA

Mass Analysis Condition

Device: Voyager DE-STR Maldi-Tof mass spectrometer (Perseptive)

Operation Mode: Reflector

Extraction Mode: Delayed

Polarity: Positive Matrix: Alpha -cyano^-hydroxycinnamic acid

Example 1: Preparation GLP-I related Peptide Attached Resin-I ('GPAR-F

hereinafter)

Structure of GPAR-I

H-His(Trt)⁷-Ala-Glu(tBu)-Gly¹⁰-Thr(tBu)-Phe-Thr(tBu)-Ser(Trt)-Asp(tBu)¹⁵-Val-Ser(Trt)-

Ser(Trt)-Tyr(tBu)-Leu²⁰-Glu(tBu)-Gly-Gln(Trt)-Ala-Ala²⁵-Lys(Boc)-Glu(tBu)-Phe-lle-

Ala³⁰-Trp(Boc)-Lθu-Val-Lys(ivDde)-Gly³⁵-Arg(Pbf)-Rink AM-resin

Confirmed Material

(Lys³⁴(ivDde))-GLP-l(7-36) amide (human)

The usage and the order in use of the materials are provided in the following

Table 1.

1.5 g of Rink AM resin (Nova biochem company) is placed in a 50 mL

peptide reactor, which has a glass filter for filtrate resin support, and added with 20

mL of dichloromethane. Resin was allowed to be swelled sufficiently for 30

minutes and the solution was removed by filtration. Fmoc was deblocked by

performing a reaction with 3 x 20 mL of deprotecting solution 25% piperidine/DMF

for 5 minutes each, followed by washing with 5 x 20 mL DMF. Chloranil test was performed to confirm the removal of piperidine.

To introduce C-terminal amino acid (Arg) of GLP-I (7-36), Nsc-Arg(Pbf)-OH

(2.0 eq, 1.37g), 1-hydroxybenzotriazole (HOBt) (2.0 eq, 270 mg) and

N,N-diisopropylethylamine (DIPEA) (4.0 eq, 690 μL) were dissolved in 10 mL of

50% dichloromethane/dimethylformamide, and poured into the reactor containing

the aforementioned resin. Benzotriazol-l-yloxytris(dimethylamino)phosphonium

hexafluorophosphate (Bop) (2.0 eq, 885 mg, 10 mL of 5%

dichloromethane/dimethylformamide) was added and reaction was performed by

stirring at 35-40 ⁰C form 1 hour. After reaction was terminated, reaction solution

was removed by filtration, and the products were washed with 3 x 20 mL of

dimethylformamide, reacted with 3 x 20 mL of deprotecting solution (25%

piperidine/ dimethylformamide) for 5 minutes each, and sufficiently washed with 5

x 20 mL of dimethylformamide. Introduction of amino acids and removal of Nsc or

Fmoc were repeated as set forth above, and the sequential order of the introduced

amino acids after Arg³⁷ is provided in Table 2 from C-terminal of GLP-I .

Introduction of amino acids was determined according to the Kaiser test, and

reaction was further performed for from 30 minutes to 1 hour if the reaction was not

completed. If the reaction was still not completed despite the prolonged reaction

time, double coupling was performed with newly prepared reaction solution for

another 1 hour. If there is no progress in reaction after double coupling, acetylation was performed in 50 % aceticanhydride in DMF to avoid any possibile side reaction.

Peptide was prepare by performing condensation and deprotection in each

step as shown in Table 2. Thus prepared GPAR-I was washed with 5 x 50 mL of

dichloromethane and dried under nitrogen stream (dry weight: 6.8 g). 5-10 mg of

GPAR-I was taken to analyze the peptide synthesis, dried under nitrogen stream

and placed in 1.5 mL centrifuge tube. Materials were separated from the resin by

using 200 μL of cleavage solution (2.5% TIS - 2.5% water in trifluoroacetic acid), and

the protection groups of tBu, trt, Boc and Pbf were removed. Materials obtained by

ether precipitation were verified to be Lys³⁴(ivDde))-GLP-l(7-36) amide (human)

according to HPLC and Maldi-Tof mass spectrometry. The HPLC analysis showed

that main peak is 70-75% and the molecular weight of the target material is 3505

(M+l= 3505).

Example 2: Preparation of GLP-I related Peptide Attached Resin-II (GPAR-II):

Deprotecting ivDde of Lys³⁴ side-chain amine in GPAR-I Structure of GPAR-II

H-His(Trt)⁷-Ala-Glu(tBu)-Gly¹⁰-Thr(tBu)-Phe-Thr(tBu)-Ser(Trt)-Asp(tBu)¹⁵-Val-Ser(Trt)-

Sθr(Trt)-Tyr(tBu)-L^βu²⁰-Glu(tBu)-Gly-Gln0^"rt)-Ala-Ala²⁵-Lys(Boc)-Glu(tBu)-Phθ-llθ-

Ala³⁰-Trp(Boc)-Leu-Val-Lys(NH₂)-Gly³⁵-Arg(Pbf)~Rink AM-resin

Confirmed Material

GLP-I (7-36) amide (human)

The usage and the order in use of the materials are provided in the following

Table 3.

Table 3

Materials MW ΘQ mmols grams mi

Removal of ivDde

QPAR-I 1.0 6.8

DMF (Expansion and Washing) 60

2% hydrazuiaΦMF 120

OMF 200 OCM 200

GPAR-I prepared in Example 1 was washed with 3 x 2OmL of

dimethylformamide. 20 mL of 2% hydrazine/ dimethy If ormamide was added,

admixed with resin and stirred for 20 minutes. The 2% hydrazine solution was removed and admixed with stirring with 5 x 20 mL of 2% hydrazine /

dimethylformamide for 20 minutes, respectively. Progress of reaction was

determined by detection of

6,6-dimemyl-3-(2-methylpropyl)-4-oxo-4,5,6,7-tetrahydro-lH-indazole, which is

produced when ivDde is separated from a peptide, by using thin layer

chromatography (TLC). As a standard solution, 2% hydrazine /

dimethylformamide solution was used. Completion of reaction was determined by

dropping solution on TLC and observing at UV 254 nm. The solution was further

treated with 2% hydrazine / dimethylformamide solution when the reaction is not

completed.

After completion of the reaction, solution was removed and washed with 5 x

20 mL dimethylformamide, 5 x 20 mL dichloromethane, 5 x 20 mL

dimethylformamide, and 5 x 20 mL dichloromethane. Under nitrogen stream, resin

was dried to provide 5.8 g of GPAR-II. To verify the removal of ivDde, a part of

GPAR-II was taken, further dried under nitrogen stream and placed in 1.5 mL

centrifuge tube. Matrials were separated from resin and the protection groups of

tBu, Trt, Boc and Pbf were removed by using 200 μL of cleavage solution (2.5% TIS -

2.5% water in trifluoroacetic acid). The material obtained by ether precipitation

method was identified to be GLP-I (7-36) amide (human) according to HPLC and

Maldi-Tof mass spectrometry. The HPLC analysis showed that main peak is 50-60% and the molecular weight of the target material is 3298 (M+l= 3298).

Example 3: Preparation of GLP-I related Peptide Attached Resin-III (GPAR-III):

Protecting His⁷, Lys³⁴ side-chain amine in GPAR-II with Fmoc

Structure of GPAR-III

Fmoc-His(Trt)⁷-Ala-Glu(tBu)-Gly¹⁰-Thr(tBu)-Phθ-Thr(tBu)-Ser(Trt)-Asp(tBu)¹⁵-Val-

Ser(Trt)-Ser(Trt)-Tyr(tBu)-Leu²⁰-Glu(tBu)-Gly-Gln(Trt)-Ala-Ala²⁵-Lys(Boc)-Glu(tBu)-

Phθ-llθ-Ala³⁰-Trp(Boc)-Leu-Val-Lys(Fmoc)-Gly³⁵-Arg(Pbf)-Rink AM-resin

Confirmed Material

(Fmoc-His⁷, Lys(Fmoc)³⁴)-GLP-l(7-36) amide (human)

The usage and the order in use of the materials are provided in the following

Table 4.

Table 4

Materials MW eα mmols grams ml

Introduction of Ftwoc GPAR-Il 1.0 6,8

DCM (Expansion) - - 20

FstK>C-QSu 837,3 5.0 to 3,4 -

DMf (for reaction) - - - _ 20 DMF (for washing) - - - - 100 0CM (for wAshiog) 100

GPAR-II prepared in Example 2 was swelled with 20 mL of dichloromethane,

washed with 3 x 20 mL of dimethylformamide and admixed with stirring for 1 hour

with 9-fluorenylmethoxycarbonyl-succinamide (Fmoc-OSu) / dimethylformamide

(3.4 g Fmoc-OSu / 20 mL dimethylformamide). Reaction progress was determined

by Kaiser test, and solution was filtrated and removed when the reaction

termination was verified. Resin was washed with 5 x 20 mL of dimethylformamide

and 5 x 20 mL of dichloromethane and dried under nitrogen stream to provide 5.3 g

of GPAR-III. To confirm the introduction of Fmoc, a part of GPAR-III was taken,

further dried under nitrogen stream and placed in 1.5 mL centrifuge tube.

Materials were separated from resin by using 200 μL of cleavage solution (2.5% TIS -

2.5% water in trifluoroacetic acid) and the protecting groups of tBu, Trt, Boc and Pbf

were removed. The material obtained by ether precipitation was identified to be Fmoc-His⁷, Lys(Fmoc)³⁴)GLP-l(7-36) amide (human) according to HPLC and

Maldi-Tof mass spectrometry. HPLC analysis shows that the main peak is about

50% and molecular weight of the verified material is 3743 (M+l= 3743).

Example 4: Preparation of Fmoc-His⁷, Lys(Fmoc)³⁴)GLP-l(7-36) amide (human):

cleavage and purification of GPAR-III

Structure of (Fmoc-His⁷, Lys(Fmoc)³⁴)GLP-l(7-36') amide (human)

Fmoc-His^Ala-Glu-Gly^-Thr-Phθ-Thr-Ser-Asp^-Val-Ser-Ser-Tyr-Leu^-Glu-Gly- Gln-Ala-Ala²⁵-Lys-Glu-Phe-lle-Ala³⁰-Trp-Leu-Val-Lys(Fmoc)-Gly³⁵-Arg-NH₂

The usage and the order in use of the materials are provided in the following

Table 5.

Table 5

Mate-rials MW ©q inmofe grants ml

Eegin for cleavage

GPAfHIJ - 0.2 1.0 -

TFA (Trifluorøactiϋ add) for cleavage - ]Q TIS (Triisopropyl silane) - - - 0,5

H2O - 0.5

TfA for washing - - - *- 10

20 mL of cleavage solution was poured to 1.0 g of GPAR-III and stirred at

room temperature for 2 hours. Solution was separated from resin and collected

with the remaining solution after washing the resin with 10 mL of trifluoroacetic

acid. The collected trifluoroacetic acid solution was poured to 300 mL of cold ether

and precipitate was collected by centrifugation. White powders were washed with

2 x 100 mL of diethylether and dried under nitrogen condition. Thus obtained

white powders were further dried under vacuum for 10 hours to provide 295 mg of

mixture. HPLC analysis shows that the content of the target material is about 50%

as in Example 3 and characterized by Maldi-Tof mass spectrometry.

Purification of materials was performed by using HPLC. As a column,

Xtera (RPl 8, 7 μm, 19 x 300 mm) was used and a mixture of distilled water

containing 0.1% trifluoroacetic acid and acetonitrile as a mobile phase. 200 mg of

mixed materials were dissolved in 10 mL of water-acetonitrile-trifluoroacetic acid mixture and injected by 1.0 mL once. As a mobile phase, 30-60% acetonitrile

aqueous solution containing 0.1% trifluoroacetic acid was used at a flow rate of 10

mL/min. The fraction of target material peak was collected, evaporated to remove

acetonitrile and freeze dried. 200 mg of peptide mixture was first purified to

provide of 87.5 mg of target material (> 85%), and further purified to provide 28 mg

of target material (> 95%). Thus obtained materials were characterized by using

Maldi-Tof mass spectrometry (M+ 1=3298).

Example 5: Preparation of Fmoc-His⁷, Lys(Fmoc)²⁶)GLP-l(7-36) amide (human)

Structure of Fmoc-His⁷, Lys(Fmoc)²⁶)GLP-l(7-36) amide (human)

Fmoc-His^Ala-Glu-Gly^-Thr-Phθ-Thr-Ser-Asp^-Val-Ser-Ser-Tyr-Lθu^-Glu-Gly-

Gln-Ala-Ala²⁵~Lys(Fmoc)-Glu-Phe-lle-Ala³⁰-Trp-Leu-Val-Lys-Gly³⁵-Arg-NH₂

The construction of peptide backbone, the deprotection of ivDde, the Fmoc

protection, cleavage and purification were performed as described in Examples 1-4.

The construction of peptide backbone was performed following the order shown in

Table 6 except that Fmoc-Lys(ivDde) and Nsc-Lys(Boc) were introduced in positions

of 26 and 34, respectively.

Thus prepared peptide-resin, GPAR-IV, has the following structure. H-His(Trt)⁷-Ala-Glu(tBu)-Gly¹⁰-Thr(tBu)-Phe-Thr(tBu)-Ser(Trt)-Asp(tBu)¹⁵-Val-Ser(Trt)-

S^θr(Trt)-Tyr(tBu)-L^θU²⁰~Glu(tBu)-Gly-Gln(Trt)-Ala-Ala²⁵-Lys(ivDde)-Glu(tBu)-Phθ-llθ-

Ala³⁰-Trp(Boc)-Leu-VahLys(Boc)-Gly³⁵-Arg(Pbf)-Rink AM-resin

To confirm the reaction progress, a part of sample was cleaved and the

material was verified to be (Lys²⁶(ivDde))-GLP-l(7-36) amide (human). Thus

obtained material is 6.5 g of GPAR-IV and its HPLC purity was 75-80%. The

molecular weight of the target material was measured to be 3505 by using Maldi-Tof

mass spectrometry (M+l=3505).

GPAR-V, which was prepared by removing ivDde of GRAP-IV according to

Example 2 has the following structure.

H-His(Trt)⁷-Ala-Glu(tBu)-G!y¹⁰-Thr(tBu)-Ph_θ-Thr(tBu)-Ser(Trt)-Asρ(tBu)¹⁵-Val-Ser(Trt)-

Ser(Trt)-Tyr(tBu)-Leu²⁰-Glu(tBu)-Gly-Gln(Trt)-Ala-Ala²⁵-Lys(NH₂)-Glu(tBu)-Phe-lle-

Ala³⁰-Trp(Boc)-Lβu-Val-Lys(Boc)-Gly³⁵-Arg(Pbf)-Rink AM-resin

To confirm the reaction progress, a part of sample was cleaved and the

material was identified to be GLP-I (7-36) amide (human). Thus obtained material

is 6.5 g of GPAR-V and its HPLC purity was about 68%. The molecular weight of

the target material was measured to be 3298 by using Maldi-Tof mass spectrometry

(M+l=3298).

GPAR-VI, which was prepared by introducing Fmoc into GRAP-V according

to Example 3, has the following structure. Fmoc-His(Trt)⁷-Ala-Glu(tBu)-Gly¹⁰-Thr(tBu)-Phe-Thr(tBu)-Ser(Trt)-Asp(tBu)¹⁵-Val-

Ser(Trt)-S_θr(Trt)-Tyr(tBu)-Leu²⁰-Glu(tBu)-Gly-Gln(Trt)Αla-Ala²⁵-Lys(Fmoc)-Glu(tBu)-

Phe-ile-Ala³⁰-Trp(Boc)-Leu-Val-Lys(Boc)-Gly³⁵-Arg(Pbf)-Rink AM-resin

To confirm the reaction progress, a part of sample was cleaved and the

material was identified to be (Lys²⁶(ivDde))-GLP-l(7-36)amide (human). Thus

obtained material is 4.9 g of GPAR-VI and its HPLC purity was about 60%. The

molecular weight of the target material was measured to be 3743 by using Maldi-Tof

mass spectrometry (M+l=3743).

1 g of GPAR-VI was taken and cleaved according to Example 4, and 320 mg

of peptide mixture was thus obtained. Purification of materials was performed by

using HPLC. As a column, Xtera (RP18, 7 μm, 19 x 300 mm) was used and a

mixture of of distilled water containing 0.1 % trifluoroacetic acid and acetonitrile as a

mobile phase. 200 mg of mixed materials were dissolved in 10 mL of

water-acetonitrile-trifluoroacetic acid mixture and injected by 1.0 mL per once. As

a mobile phase, 30-60% acetonitrile aqueous solution containing 0.1% trifluoroacetic

acid was used at a flow rate of 10 mL/min. Materials around target peak area were

dried and concentrated to remove acetonitrile and freeze dried. 200 mg of peptide

mixture was first purified to provide of 110 mg of target material (> 90%), and

further purified to provide 58 mg of target material (> 95%). Thus obtained

materials were characterized by using Maldi-Tof mass spectrometry (M+l=3298).

Example 6: Preparation of Lys²⁶-PEG2k-GLP-l(7-36) amide

10 mg of (Fmoc-His⁷, Lys(Fmoc)³⁴)GLP-l(7-36) amide according to Example 4

and 32 mg of PEG2k(PEG 2000)succinimidyl propinonate were placed in a glass

container and dissolved with 5 mL of dimethylformamide containing 0.3%

triethylamine, followed by reaction at room temperature for 60 minutes. After reaction was completed, 0.25 niL of piperidine was added and Fmoc was

deprotected. 5 mL of 10% trifluoroacetic acid aqueous solution was added to

modify pH, diluted by adding 40 mL of 0.1 % trifluoroacetic acid aqueous solution

again, and purified by ion exchange chromatography.

Ion Chromatography Condition

Column: TSK SP-5PW (21.5 mm ID x 150 mm L)

Mobile Phase

A: 50 mM sodium acetate buffer solution (pH 4.0)

B: 50 mM sodium acetate buffer solution (pH 4.0) containing 0.5 M sodium

chloride

0 min: A, 100% / B₇ 0% → 40 min: A, 40% / B, 60%

Flow Rate: 6 ml/ min

Detection: UV 215 nm

The collected fraction solution was slowly passed through activated Sep-Pak

cartridge, thus allowing peptide to be adsorbed, and washed with 100 mL of

distilled water, followed by elution with 25 mL of 80% acetonitrile containing 0.1%

trifluoroacetic acid. This solution was evaporated under nitrogen stream to remove

acetonitrile and freeze dried to provide 7.6 mg of target materials. Thus obtained Lys²⁶-PEG2k-GLP-l(7-36) amide showed a purity of above

99% according to the reversed-phase chromatography (figure 1). The Maldi-Tof

mass spectrometry showed a mono-PEG conjugate peak of 5551 at a central

molecular weight (figure 2).

Reversed-phase Chromatography Condition

Column: Lichrosphere 100 RP-8 (4 mm ID x 250 mm L, 5 μm, Merck)

Mobile Phase

A: 0.1% trifluoroacetic acid/ distilled water

B: 0.1% trifluoroacetic acid/acetonitrile

0 min: A, 64% / B, 36% → 30 min: A, 56% / B, 44%

Flow Rate: 1 ml/ min

Detection: UV 215 nm

Example 7: Preparation of Lys³⁴-PEG2k-GLP-l(7-36) amide

8.9 mg of Lys³⁴-PEG2k-GLP-l(7-36) amide 8.9 mg was prepared as set forth

in Example 6 by using 10 mg of (Fmoc-His⁷, Lys(Fmoc)²⁶)GLP-l(7-36) amide

prepared in Example 5.

Example 8: Preparation of Lys26-PEG2k-GLP-l(7-36) amide 100 mg of (Fmoc-His⁷, Lys(Fmoc)³⁴)GLP-l(7-36) amide according to Example

4 and 160 mg of methoxypolyethyleneglycol2k(PEG 2000)succinimidyl propinonate

were placed in glass container and dissolved with 5 mL of dimethylformamide

containing 0.5% diisopropylethylamine, followed by reaction with stirring at 35 ⁰C

for 30 minutes. After reaction was terminated, 2 mL of piperidine was added and

stirred at 35 ⁰C for 30 minutes to deprotect Fmoc. Products were precipitated by

adding 120 mL of diethyl ether, centrifuged, dispersed in 120 mL of diethyl ether,

washed and centrifuged, thereby providing precipitates. Thus obtained

precipitates were dispersed in 120 mL of diethylether containing 0.1% of acetic acid,

washed, centrifuged and dried under vacuum at room temperature for 12 hours,

thus providing 149 mg of products. The products were dissolved in 25 mL of

distilled water and purified by injecting 5 mL of the products in pre-HPLC at a time

under the following conditions.

Prep-HPLC Condition

Column: Disogel SP Ci₈ 10 um (50 mm ID x 400 mm L)

Mobile Phase

A: 0.1% trifluoroacetic acid/ distilled water

B: 0.1% trifluoroacetic acid/acetonitrile

0 → 5 min: A 100% / B 0% 5 → 60 min: A 100% / B 0% → A 30% / B 70%

60 → 70 min: A 30% / B 70% → A 10% / B 90%

70 → 80 min: A 100% / B 0%

Flow Rate: 100 ml/ min

Detection: UV 216 ran

The collected eleuted solution was dried and concentrated. 132 mg of thus

obtained materials were dissolved in 10% of acetic acid and substituted into acetate

salt by using ion exchange chromatography, followed by freeze drying, thus

providing 122 mg of Lys²⁶-PEG2k-GLP-l(7-36) amide acetate.

Experimental Example 1: Inhibition of Blood Glucose Increase by

Lys²⁶-PEG2k-GLP-l(7-36) amide and Lys³⁴-PEG2k-GLP-l(7-36) amide

Diabetes-induced db/db mice were divided into 4 groups (n=5) and were

fasted for 16 hours. Saline solution, 100 μg/kg of GLP-I (7-36) amide, 100 μg eq/kg

of Lys²⁶-PEG2k-GLP-l(7-36) amide and 100 μg eq/kg of Lys³⁴-PEG2k-GLP-l(7-36)

amide were intraperitoneally injected and 1 g/kg of glucose solution was orally

administered after 10 minutes. After -10, 0, 10, 20, 30, 60, 90, 120 and 180 minutes,

blood samples were taken and blood glucose levels were measured. The effects of inhibiting blood glucose increase of GLP-I (7-36) amide,

Lys²⁶-PEG2k-GLP-l(7-36) amide and Lys³⁴-PEG2k-GLP-l(7-36) amide were

compared by calculating the area under the blood glucose level to time(0-180

minutes) curve (figure 3). AUC of GLP-l(7-36) amide group was 25165±4463

mg-min/dl, which is a decreased value by 27.8%, as compared to the saline solution

group (34864+4774 mg.min/dl). However, Lys²⁶-PEG2k-GLP-l(7-36) amide and

Lys³⁴-PEG2k-GLP-l(7-36) amide are 14470+5700 mg-min/dl and 17520+2484

mg-min/dl, respectively (i.e. 58.5% and 49.7% decreases each). Theses results show

that Lys²⁶-PEG2k-GLP-l(7-36) amide and Lys³⁴-PEG2k-GLP-l(7-36) amide have

much enhanced activity for inhibiting blood glucose level as compared to

GLP-I (7-36) amide.

INDUSTRIAL APPLICABILITY

Site-specific PEG conjugates of GLP-I, where PEG is selectively bound to

Lys²⁶ or Lys³⁴ side-chain amine of GLP-I, may be prepared according to the present

invention.

The site-specific PEG conjugates of GLP-I show superior activity for

inhibiting blood glucose level increase as compared to the GLP-I itself.

Claims

1. A site-specific PEG conjugates of GLP-I wherein a PEG unit is covalently bound

to a side-chain amine of GLP-I Lys²⁶ or Lys³⁴.

2. The site-specific PEG conjugates of GLP-I of claim 1, wherein C-terminal of the

GLP-I has a form selected from the group consisting of pharmaceutically

acceptable salts, amide and ester.

3. A site-protected GLP-I wherein N^α amine and side-chain amine of Lys³⁴ or Lys²⁶

are selectively protected with amine-protecting group.

4. The site-protected GLP-I of claim 3, wherein the amine protecting group is at

least one group selected from the group consisting of

l-(4,4-dimethyl-2_/6-dioxocyclohexylidene)-3-methylbutyl_/

9-f luorenylmethoxycarbonyl, 4-nitrophenylsulf onylethoxycarbonyl,

tert-butoxycarbonyl, allyloxycarbonyl,

l-(4,4-dimethyl-2_/6-dioxocyclohex-l-yllidene)ethyl_/

1-(1 '-adamantyty-l-methyl-ethoxycarbonyl, 2-chlorobenzyloxycarbonyl and

carbobenzoxy.

5. A method for preparing a site-specific PEG conjugates of GLP-I according to

claim 1, the method comprising:

(a) reacting the site-protected GLP-I according to claim 3 with an activated PEG,

and

(b) deprotecting the amine protecting group.

6. The method of claim 5, wherein the activated PEG is alkylating or acylating PEG.

7. The method of claim 5, further comprising a purification step using

chromatography.

8. A pharmaceutical composition for treating diabetes comprising the site-specific

PEG conjugates of GLP-I of claim 1 as an active ingredient.

9. A method of treating diabetes comprising administering to a mammal a

therapeutically effective amount of the site-specific PEG conjugates of GLP-I of

claim 1.

10. Use of the site-specific PEG conjugates of GLP-I of claim 1 for treating diabetes.

1. Use of the site-specific PEG conjugates of GLP-I of claim 1 in the manufacture of

a pharmaceutical drug for treating diabetes.