Poly(ethylene glycol) - American Chemical Society

Chapter 16

Site-Specific Poly(ethylene glycol)ylation of Peptides Arthur M. Felix

Downloaded by FORDHAM UNIV on August 15, 2014 | http://pubs.acs.org Publication Date: August 5, 1997 | doi: 10.1021/bk-1997-0680.ch016

Department of Chemistry and Physics, William Paterson College, Wayne, NJ 07470

In contrast to proteins, peptides are ideal targets for PEGylation since they may be PEGylated using modern orthogonal protection strategies. Synthetic conditions have been developed for the site-specific solid phase PEGylation (Fmoc/tBu strategy) at the NH2-terminal, internal (at the side-chain of Lys/Orn or Glu/Asp) and at the COOH-terminal positions. Conditions for site-specific solution phase PEGylation have also been developed in which a Cys residue is introduced at any position into the peptide and subsequently PEGylated using an activated dithiopyridyl-PEG reagent. Using these procedures, a number of biologically active mono-PEGylated peptides were prepared in which the PEG moieties were varied in molecular weight (i.e. degree of PEGylation; 750, 2000, 5000 and 10,000). Site-specific PEGylation of peptides enables the evaluation of the structure-activity relationships of biologically important peptides (i.e. the effect of site of PEGylation and degree of PEGylation on biological activity). These studies provide an important groundwork for the design of PEGylated peptides which retain the desirable properties imposed by the incorporation of P E G (enhanced solubility, increased resistance to proteolytic degradation, decreased antigenicity and immunogenicity) and have improved in vivo profiles. Bioactive peptides are known to play key pharmacological roles and have found widespread application in a variety of fields. Active biomedical research using peptides includes their application for autoimmune, inflammatory, cardiovascular, hormonal and neurological disorders and for wound healing, infectious diseases and cancer therapy. Peptides have found utility in the diagnosis and treatment of such diverse fields as diabetes, HIV, Alzheimer's Disease, asthma and growth deficiencies. Although many peptides have found application for a variety of clinical indications, their use has been limited due to their rapid metabolism from the action of extracellular proteases. Chemists have therefore developed ingenious methods for the modification of peptides (e.g. peptidomimetic design, cyclization etc.) with the goal of maintaining or improving their receptor selectivity while stabilizing them to enzymatic degradation. Although these efforts have yielded important progress, the problems of poor absorption of peptides, rapid proteolytic degradation and potential 218

© 1997 American Chemical Society

In Poly(ethylene glycol); Harris, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

16.

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Site-Specific PEGylation of Peptides

219

immunogenicity have severely hampered the application of peptides as potential therapeutics.


In the last decade considerable progress has been made on the application of poly(ethylene glycol) (PEG) as a covalent linking agent to polypeptides and proteins. The resultant PEG-protein conjugates have been compared to the parent protein and have been shown to have enhanced resistance to proteolytic degradation (increased plasma half life). In addition, PEG-proteins have been reported to have improved water solubility, increased resistance to proteolytic degradation, reduced antigenicity and decreased immunogenicity {1-6). Unfortunately, these important advantages of PEG-proteins have been offset by their decrease in biological activity in vitro. These observations may be rationalized as a result of the mdiscrirninate incorporation of PEG into the protein by the standard methods of protein PEGylation (attachment to the ε-ΝΗ of lysine). Unfortunately, the specific position of PEGylation in proteins usually cannot be controlled using the conditions of protein PEGylation thereby resulting in the incorporation of a multitude of PEG units per mol of protein. 2

In contrast to proteins, peptides are ideal targets for PEGylation since they may be specifically PEGylated using modern orthogonal protection strategies. The enormous progress that has been made in peptide synthetic methodology has provided an important basis for the site-specific PEGylation of peptides. This manuscript describes the chemical procedures that our laboratories have developed for sitespecific PEGylation of peptides at the NH2-terminal, internal (side-chain of Lys/Orn or Glu/Asp) and COOH-terminal positions (Figure 1). In addition, we have prepared several biologically important PEGylated peptides using this methodology, evaluated structure-activity relationships, and compared their biological and physical properties to those of the parent peptides. Solid Phase PEGylation

Synthesis

of

PEGylated

Peptides:

NH2-Terminal

The derivatization of the terminal hydroxyl function of monomethoxypoly(ethyleneglycol), via a urethane linkage covalently linked to norleucine (7,8) enables the analytical determination of the number of PEG units/mol of protein by amino acid analysis. We recently modified this procedure in our laboratory using derivatives derived from monomethoxypoly(ethyleneglycol), 1 (Figure 2). Synthetic methods were developed for the preparation of PEG -CH2-CO-Nle-OH, 2, in 3 steps from monomethoxypoly-(ethyleneglycol) (9). We observed that PEG -CH2-CO-Nle-OH could be used as an acylating reagent for NH2-terminal PEGylation in solid phase synthesis by standard procedures using BOP-activation (10). PEGylation typically proceeds in (1:9 CH2CI2-DMF or 1:9 CH2CI2-NMP) using 2 equivalents of BOP followed by 4 equivalents of DIEA for 24 hours. Completion of PEGylation is monitored in the usual way using the Kaiser ninhydrin test (77). The PEGylation is repeated if the Kaiser test is positive. Initially, we compared the purity of the PEGylated peptide using TFA cleavage to that from the low-high H F cleavage. In a model study, we observed that TFA cleavage was preferred since it resulted in the preparation of PEGylated peptides that revealed a single peak by analytical H P L C . In contrast, using the H F procedure we observed impurities by H P L C which corresponded to small amounts of low molecular weight fragments resulting from degradation of poly(ethyleneglycol) on exposure to HF. From these studies it was concluded that the Fmoc/tBu solid phase strategy for the synthesis of PEGylated peptides was preferred since this is compatible with TFA cleavage in the final stage. N

N


220

POLY(ETHYLENE GLYCOL)

IIII-W^^Y "

h

η vC0NH2

^ ύ

1

NH -Terminal Pegylated Peptide 2

0

0

H N ^γΝΗ O'VJH Ύ*Η vxCONH,

H N ^ -ΝΗγ^NH>^CONH

2

0

2

|Γ

10

Ε

^ j J ^


2

I

ο

ο

î ° NH 1

Side-Chain Pegylated (linked vw ε-NHj)

Side-Chain Pegylated (linked via Cû-COOH)

H N J^VNH x^^NH vCO-NH 2

0

0 COOH-Terminal Pegylated Peptide

Figure 1. Peptide pegylation sites.

CH -0-CH2-CH -0-(CH2-CH2-0) -CH -CH2-OH 3

2

n

2

PEG 1CH -0-CH -CH2-0-(CH2-CH2-0) -CH2-CH -0-CH2-CO-NH-Nle-COOH 3

n

2

n

2

Fmoc-Lys-OH

Fmoc-Orn-OH

2a

PEGn-CHi-CO-Nle-^ PEG -CH -CO n

2

n

2

a

N -PEG -CH -CO-Nle-OH

2b

2

CH -0-CH2-CH2-0-(CH2-CH2-0) -CH2-CH2-NHCO-Nle-NH 3

n

H-Nle-NH-CH -CH -PEG 2

2

2

3

n

Fmoc-Asp-OH

r 3a

Γ

ϋτ

S

^-Nle-NH-CH -CH -PEG 2

C H

2

Ο

2

n

>Sle-NH-CH CH -(OCH CH ) -OCH 2

2

2

Pyr-S-S-CH -CH -CO-Nle-NH-CH -CH -PEG 2

2

2

2

2

n

n

4

Figure 2. Monomethoxypoly(ethylene glycol) derivatives. In Poly(ethylene glycol); Harris, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

3

16.

221


FELIX

Accordingly, resins that are cleaved with T F A [e.g. Wang-resin (72), Rink-resin (73), PAL-resin (14) and Rink amide MBHA-resin (75)] are compatible with the solid phase synthesis of PEGylated peptides. The procedure for site-specific solid phase NH2-terminal PEGylation is outlined in Figure 3 starting with Rink amide MBHA-resin.


Solid Phase Synthesis of PEGylated Peptides: Side-Chain PEGylation The preparation of side-chain-PEGylated amino acids has been reported and used for the liquid-phase synthesis of hydrophobic peptides of medium size (16). We have extended these observations and found that side-chain solid phase PEGylation of Lys or Asp can be readily achieved by one of two pathways as outlined in Figure 4; direct side-chain PEGylation or post side-chain PEGylation (77). Starting with Rink amide MBHA-resin, we assemble the C-terminal portion of the peptide (Peptidea) by Fmoc/tBu solid phase peptide synthesis. This intermediate is subjected to direct sidechain PEGylation utilizing an N -Fmoc-protected derivative of Lys or Asp in which poly(ethyleneglycol) is already linked to the side-chain of Lys or Asp. This derivative was prepared by BOP-catalyzed solid phase coupling with Fmoc-Lys(PEG -CH2CO-Nle)-OH, 2a [prepared by the condensation of Fmoc-Lys(Nle)-OH with P E G CH2-COOSU (76)]. Direct side-chain PEGylation of Asp was accomplished in a similar manner by BOP-catalyzed coupling with Fmoc-Asp(Nle-NH-CH2-CH2P E G ) - O H , 3a [prepared by the condensation of Fmoc-Asp-OtBu with H-Nle-NHC H 2 - C H 2 - P E G , 3], followed by TFA deprotection. Alternatively, the intermediate may be subjected to post side-chain PEGylation using an N -Fmoc-protected derivative of Lys or Asp with an allylic side-chain protecting group. Following the complete assemblage of the peptide-resin, the allylic group was selectively removed by palladium-catalyzed deprotection using PdCl2[P(C6H5)3]2. Solid phase PEGylation of the side-chain of Lys or Asp was then carried out in the final stage by BOP-catalyzed acylation with PEG -CH2-CO-Nle-OH, 2, or H-Nle-NH-CH -CH2P E G , 3, respectively. Deprotection of the tBu-protected side-chains and resin cleavage was accomplished by standard procedures using TFA. The two pathways for side-chain PEGylation are outlined in Figure 4 for the site-specific Lys-side-chain PEGylation starting with Rink amide MBHA-resin -resin. a

n

n

n

n

a

n

2

n

Solid Phase PEGylation

Synthesis

of

PEGylated

Peptides:

COOH-Terminal

Fmoc-Orn(PEG -CH2-CO)-OH, 2b [prepared by the condensation of Fmoc-Orn-OH with PEG -CH2-COOSu], was attached directly to Rink amide MBHA-resin. The use of ornithine as the COOH-terrninal residue, with PEG attached at the side-chain position, also enables the analytical determination of the number of PEG units/mol of peptide by amino acid analysis. Following this attachment stage, solid-phase assemblage of the PEGylated peptide-resin was carried out using the standard Fmoc/tBu strategy (77). The presence of the PEG moiety at the COOH-terminus did not alter the course of solid phase synthesis. Deprotection of the tBu-protected sidechains and resin cleavage was accomplished by standard procedures using TFA. The procedure for site-specific solid phase COOH-terrninal PEGylation is outlined in Figure 5 starting with Rink amide MBHA-resin -resin. n

n

Multiply Site Specific PEGylated Peptides Using the solid phase procedures outlined in Figures 3, 4 and 5 we have prepared NH2-terminal, side-chain and COOH-terrninal PEGylated peptides using



222

POLY ( E T H Y L E N E G L Y C O L )

Fmoc-]

Fmoc/tBu SPPS

/tBu

\

Η-PEPTIDE -NH- [RINK-BHA] - \ \

a

(a)

N -PEG-CH -CO-Nle-OH, 2

(b)

TFA

2

a

N -PEG-CH -CO-Nle -PEPTIDE-NH 2

2

Figure 3. Site-specific solid phase NH2-terminal pegylation.


16.

FELIX

223


Fmoc-NH CH 0 Downloaded by FORDHAM UNIV on August 15, 2014 | http://pubs.acs.org Publication Date: August 5, 1997 | doi: 10.1021/bk-1997-0680.ch016

3

Fmoc/tBu SPPS

\

tBu H-PEPTIDE -NH-[RINKBHA]-

^

A

DIRECT SIDE-CHAIN P E G Y L A T I O N

p

o

s

x

SIDE-CHAIN P E G Y L A T I O N (a) Fmoc-Lys(Alloc)-OH

(a)

Fmoc-Lys-OrT

PEG-CH -CO-Nle-^

(b) Fmoc/tBu SPPS

2 a

2

(c) P d C l [ P ( C H ) ] 2

(b) Fmoc/tBu SPPS x

(d)

6

2

Ν

H-PEPTIDEb-Lys-PEPTIDE -NH-[RINK-BHA]—1\ a

PEG-CH -CO-Nle

TFA

2

H-PEPTIDE -Lys-PEPTIDE -NH b

3

a

2

PEG-CH -CO-Nle-OH, 2

,tBu

tBu

5

2

J PEG-CH -CO-Nle — ^ 2

Figure 4. Site-specific solid phase Lys-side-chain pegylation.


224



Fmoc-NH

Fmoc-Orn-OH

JL

2B

PEG-CH ô 2

Fzmoc-Orn-N PEG-CH ô

OCH

2

CH 0

3

3

Fmoc/tBu SPPS

,tBu H-PEPTIDE-Orn -NH-[RINK-BHA] — ^ PEG-CH ô 2

TFA

H-PEPTIDE-Orn -NH

2

PEG-CH ô 2

Figure 5. Site-specific solid phase COOH-terrninal pegylation.


16. F E L I X

225


poly(ethyleneglycol) of varying molecular weight ( P E G 7 5 0 , P E G 2 0 0 0 » P E G 5 0 0 0 P E G 1 0 0 0 0 ) . Attempts to extend these solid phase site-specific PEGylation methods to diPEGylated peptides were also successful. However, the second stage of PEGylation proceeded with some difficulty when PEGylation was attempted with high molecular weight poly(ethyleneglycol) derivatives (i.e. P E G 5 0 0 0 and PEG 1 0 0 0 0 ) · We observed that multiple solid phase PEGylation proceeds more efficiently with poly(ethyleneglycol) derivatives of lower molecular weight (i.e. P E G 7 5 0 and PECJ2000)- In addition, the second stage of PEGylation at the N H 2 terminus is less efficient when the NH2-terminal residue is sterically hindered (e.g. Ile) (18). a


Solution Synthesis PEGylation

of

PEGylated

Peptides:

n

d

COOH-Terminal

The methodology for site-specific COOH-terrninal PEGylation was extended to include a novel solution phase PEGylation methodology. This procedure features the incorporation of a cysteine residue at the COOH-terminus. Earlier biotinylation studies had shown that it is preferred to incorporate the cysteine residue at a site distant from the bioactive core of the peptide and a spacer arm consisting of -GlyG l y - C y s - N H 2 was incorporated into the peptide (79). We have extended these earlier observations and found that the - G l y - G l y - C y s - N H 2 extended peptides are ideally suited for solution phase PEGylation. The conversion of H - N l e - N H - C H 2 - C H 2 - P E G , 3, to the dithiopyrridyl-activated P E G , 4, was carried out by reaction with N succinimidyl 3-(2-pyridyldithio)propionate (SPDP) (20). Figure 6 outlines the general scheme for site-specific solution phase COOH-terrninal PEGylation through disulfide formation at COOH-terrninal cysteine. This process is not confined to COOH-terrninal PEGylation. Using this procedure, a residue of Cys may be introduced at any position into the peptide and subsequently site-specifically PEGylated using dithiopyrridyl-activated PEG, 4. Purification of PEGylated Peptides The PEGylated peptides are best purified by a 2-stage process using dialysis and preparative HPLC. Dialysis was carried out for 48 hours using dialysis membrane tubing with a nominal cutoff of 1000, 3500 or 7000 (used respectively for P E G 7 5 0 peptides, P E G 2 0 0 0 " P P d e s PEGsooo-peptides). This process removes all salts and low molecular impurities including any non-PEGylated peptides. The final purification step, reversed-phase preparative HPLC was carried out in the same manner as for the free peptide on Vydac C-18 columns using a gradient of H 2 O C H 3 C N (containing 0.1% TFA). The PEGylated peptides usually elute from the column after the free peptide and can therefore be separated from trace amounts of free peptide that were not removed in the dialysis step. e

u

o r

Characterization of PEGylated Peptides Since the elution properties of PEGylated peptides are similar to those of the corresponding free peptides, it is essential to thoroughly characterize the final product and to determine whether the PEGylated peptide is contaminated with free peptide. Surprisingly, analytical HPLC of PEGylated peptides results in homogeneous, sharp peaks. Moreover, PEG-peptides elute -1-5 minutes after the corresponding free peptides. Application of "spiking" studies using analytical HPLC enables the detection of small amounts of free peptide in the presence of PEG-peptide. Although poly(ethyleneglycol) does not absorb U V light, HPLC of the PEG-peptides are tracked at 214 nm where the peptide chromophore enables their detection. Figure 7


In Poly(ethylene glycol); Harris, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997. 2

2

+

2

2

2

4

2

2

n

3

S ^CH Nle-NH-CH CH -(OCH CH2) -OCH +

2

2

2

n

Ο

3

,SH

CH Nle-NH-CH CH2-(OCH CH ) -OCH

Figure 6. General scheme for site-specific solution phase COOH-terminal pegylation through disulfide formation at cysteine.

H-PEPTIDE-Gly-Gly-Cys-NH

/CH

pH4

2

H-PEPTIDE-Gly-GIy-Cys-NH


S)

r *n or

w ζ w O

r

*!

r

ON

FELIX


227


16.

TIME (minutes) 1

2

8

15

27

Figure 7. Analytical HPLC of a mixture of (1 [His ,Val ,Gln ,Ala ,Leu ]GRF(l-32)- Gly-Cys-NH (5μ£) with (2) [His ,Val ,Gln ,Ala ,Leu ]GRF(l-32)-Gly-Cys(S-CH2-CH2-CO-Nle-NH-PEG5()()0)-NH2(10μg).zzzz Column: Lichrosorb RP-8 (5μ); Eluant: (A) 0.1M NaC10 (pH 2.5)-(B) C H C N ; Gradient: 40-60% (B) in 20 min; Detection: 214 nm; Flow rate: 1 ml/min. 1

2

8

15

27

2

4


3

228


demonstrates that baseline resolution can be achieved for a mixture of peptide and PEGylated peptide. In this admixture chromatogram, the peptide, [His ,Val ,Gln ,Ala ,Leu ]-GRF(l-32)-Gly-Cys-NH , elutes at 11.5 min and the PEGylated analog, [His ,Val ,Gln ,Ala ,Leu ]-GRF(l-32)-Gly-Cys(S-CH CH2-CO-Nle-NH-PEG5ooo)-NH2 elutes at 13 min. Similar resolution have been observed for a number of peptides and their corresponding PEGylated peptides. 1

2

8

15

27

2

1

2

8

15

27

2


>

As mentioned earlier, amino acid analysis is used to determine the extent of PEGylation (i.e. the number of PEG moieties in the PEGylated peptide). Since norleucine and ornithine elute at different retention times than any of the natural amino acids, when these residues are covalently linked to PEG their detection by amino acid analysis can be used to determine the number of PEG moieties in the final PEGpeptide product. Therefore, tetemiination of Nle (from PEG-CH2-CO-Nle-OH, 2) was used to confirm the presence of P E G in NH2-terminal PEGylated peptides. Similarly, determination of Nle (from either Fmoc-Lys(PEG-CH2-CO-Nle)-OH, 2a, or Fmoc-Asp(Nle-NH-CH2-CH2-PEG )-OH, 3a) was used to confirm the presence of PEG in side-chain PEGylated peptides. Finally, the determination of Om (from Fmoc-Orn(PEG-CH2-CO)-OH, 2b) was used for confirmation of PEG in COOHterrninal PEGylated peptides. n

Since internal and COOH-terrninal PEGylated peptides undergo Edman degradation, sequence analysis of these site-specific PEGylated peptides can be used to provide indirect evidence of the position of PEGylation in the PEGylated peptide. The cycle of Edman degradation corresponding to the release of the PEGylated amino acid is undetected since the resultant thiohydantoin is PEGylated and does not co-elute with the known thiohydantoins. This absence of a thiohydantoin derivative may be used to confirm the position of the PEGylated residue in the sequence. *H-NMR spectroscopy is also extremely useful for characterization of the sitespecific PEGylated peptides. !H-NMR spectra of PEGylated peptides reveals a major off-scale peak at δ 3.5-3.8 from -(CH CH20) - which integrates for the expected number of protons (e.g. 452 Η for P E G 5 0 0 0 ) . In addition, the îH-NMR spectra of PEGylated peptides also reveal a singlet at δ 3.39 which is used as a standard and integrates for the expected number of protons (3H). 2

n

Laser desorption mass spectrometry of PEGylated peptides results in a bell-shaped spectrum centered at the expected position for the PEGylated peptide with a family of adjacent peaks at 44±1 Da corresponding to the varying molecular weights of poly(ethyleneglycol). The laser desorption mass spectrum of poly(ethyleneglycol), P E G 5 0 0 0 » shown in Figure 8 reveals the bell-shaped curve with a maxima at 5027.4 Da and a family of peaks at 44±1 Da starting with adjacent peaks at 4983.5 and 5072.4. Similar spectra have been observed for PEGylated amino acids and PEGylated peptides of poly(ethyleneglycol) of varying molecular weights. Site-Specific PEGylation of Growth Hormone-Releasing Factor Human growth hormone-releasing factor, hGRF(l-44)-NH2, is an amidated peptide containing 44-amino acids. It is produced and released by the hypothalamus and transported via a portal network system to the pituitary gland where it triggers the release of human growth hormone. The physiological profile of endogenous human growth hormone-releasing factor has been recently reviewed (27). Studies have demonstrated the potential use of human growth hormone-releasing factor for the



Figure 8. Laser desorption mass spectrum of poly(ethyleneglycol), P E G 5 0 0 0 . The spectrum was obtained on a Bruker ReflexTime of Flight Mass Spectrometer in the reflector mode at 30 kV.


230


treatment of dwarfism resulting from failure of the hypothalamus to produce sufficient quantities of the releasing factor. Structure-activity studies on human growth hormone-releasing factor have shown that an analog, [Ala ]-hGRF(l-29)N H 2 , is 3-4 times more potent in vitro than the parent peptide, h G R F ( l - 4 4 ) - N H 2 (22). With the goal of producing potent, long acting analogs of human growth hormone releasing-factor, a series of monoPEGylated derivatives of [Ala ]h G R F ( l - 2 9 ) - N H 2 were prepared. Site-specific PEGylation was carried out by the procedures outlined earlier at the NH2-terminus (position 1), side-chain positions (Asp , L y s , L y s and Asp ) and at the COOH-terminus (position 30). Each PEGylated peptide was purified by dialysis and preparative HPLC and characterized by laser desorption mass spectrometry, analytical HPLC, amino acid analysis and ïH-NMR spectrometry. 15

15


8

1 2

2 1

In vitro Biological Factor

25

Activity of PEGylated Growth Hormone-Releasing

Each [ A l a ] - h G R F ( l - 2 9 ) - N H 2 analog was studied in vitro to determine the effect of (a) site of PEGylation and (b) degree of PEGylation (i.e. molecular weight of PEG moiety) on the growth hormone releasing potency (23). The results of these studies are summarized in Table I and demonstrate that both the site of PEGylation and, in some cases, the degree of PEGylation are critical to biological activity. In each case the biological activity of the PEGylated peptide was compared to that of the corresponding acetylated or amidated peptide. This ensures an objective evaluation of the effect of PEGylation since the acetylated or amidated derivatives have different potencies than the parent peptide, [ A l a ] - h G R F ( l - 2 9 ) - N H 2 . 15

15

Acetylation at the NH2-terminus (compound 7) resulted in reduced potency compared to [ A l a ] - h G R F ( l - 2 9 ) - N H 2 , 6. Compound 6 showed no further change when the NH2-terminus was PEGylated, regardless of the degree of PEGylation (compounds 7a, 7b). Amidation at position 8 (compound 8) did not significantly alter the potency of 6. However, PEGylation at this site resulted in a drastic decrease of potency, which decreased further with increased molecular weight of PEG moiety (compounds 8a, 8b). Acetylation at position 12 (compound 9) resulted in a decrease of potency compared to 6, which decreased further with increasing molecular weight of PEG moiety (compounds 9a, 9b). On the other hand, acetylation at position 21 (compound 10) or amidation at position 25 (compound 11) had no significant effect on biological activity vs 6. In addition, an increase in the molecular weight of the PEG moiety at position 21 (compounds 10a, 10b) or at position 25 (compounds 11a, lib) also did not result in any change in relative potency. Finally, acetylation at the COOH-terminus (compound 12) provided the most potent analog, which retained its high potency regardless of the degree of PEGylation (compounds 12a, 12b). 15

These observations demonstrate the importance of both the site and degree of PEGylation on the in vitro biological activity in the growth hormone releasing factor peptide. It is anticipated that similar results will be observed for other biologically important peptides. These studies reveal that it is not possible to predict the effect of PEGylation on the biological activity of the peptide. Indiscriminate PEGylation of a peptide should be avoided since biological activity may be drastically reduced. Each peptide should be thoroughly evaluated to determine the optimal site of PEGylation.


In Poly(ethylene glycol); Harris, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997. 15

2

2

2

15

15

15

15

1

15

1

10b

11

11a

lib

12

21

2

12

15

2

21

1

12b

2

30

2

5

2

2

lAla 5,Orn(PEG ooo-CH -CO)30j-hGRF(l-30)-NH

2

2

30

30

4.83

4.63

4.36

2

—

15

25

[Ala ,Orn(PEG ooo-CH -CO) ]-hGRF(l-30)-NH

12a

5

[Ala 5,Orn(Ac)30]-hGRF(l-30)-NH

2

3.27

2

3.09

3.20 25

2

2.86

—

—

0.03

12

[Ala ,Asp(Nle-NH-CH CH -PEG ooo) ]-hGRF(l-29)-NH2

20

25

0.34

12

3.21 3.24

0.77

—

21 21

1.59 0.17

8

4.68

—

8

1.28

1 1

1.04

1.02

—

1.00 3.81

25

2

2

2

2

[Ala Âsp(Nle-NH-CH CH -PEG oo) ]-hGRF(l-29)-NH 2

[Ala ,Asp(NHEt) 5]-hGRF(l-29)-NH

2

2

5

[Ala ,Lys(PEG ooo-CH -CO-Nle) l-hGRF(l-29)-NH

21 2

2

2

15

[Ala ,Lys(PEG oo-CH -CO-Nle) ]-hGRF(l-29)-NH

20

[Ala ,Lys(Ac) ]-hGRF(l-29)-NH

10a

2

2

5

20

[Lys(PEG ooo-CH -CO-Nle) -Ala ]-hGRF(l-29)-NH

12

15

2

15

2

[Lys(PEG oo-CH -CO-Nle) -Ala ]-hGRF(l-29)-NH

12

2

[Lys( Ac) Ala ]-hGRF( 1 -29)-NH

10

9b

9a

9

8b

1

Relative Potency*

...

Pegylation Site

Analogs

Potency relative to hGRF(l-44)-NH as determined by ability to stimulate growth hormone release by rat pituitarycells in vitro (concentrations tested: 3.1-400 pM) (reproduced with permission from reference 23)

a

2

[Asp(Nle-NH-CH2CH -PEG ooo)-Ala ]-hGRF(l-29)-NH [Asp(Nle-NH-CH CH -PEG5000)-Ala ]-hGRF(l-29)-NH

1

[Asp(NHEt)8Ala 5]-hGRF(l-29)-NH

8a

15

8

5

2

2

15

a

2

N -PEG ooo-CH -CO-Nle-[Ala ]-hGRF(l-29)-NH

2

7b

20

2

7

a

2

Ac-[Alal5]-hGRF(l-29)-NH N -PEG oo-CH -CO-Nle-[Ala ]-hGRF(l-29)-NH

2

7a

15

[Ala ]-hGRF(l-29)-NH

hGRF(l-44)-NH

hGRF Analog

6

5

Compound

Table I. Biological Activity (in vitro) of Pegylated [Ala ]-GRF(l-29)-NH2

15


232


In vivo Biological Factor

Activity of PEGylated Growth Hormone-Releasing 15

The potent analog of hGRF, [Ala ]-hGRF(l-29)-NH2, undergoes rapid NH2terminal proteolytic degradation (24) initiated by dipeptidylpeptidase-IV (25). Other more potent analogs have been developed in which the NH2-terminus is protected against dipeptidylpeptidase-IV degradation. These include [desNH2Tyr ,DAla ,Ala ]-hGRF(l-29)-NH , 13, and [ H i s , V a l , G l n , A l a , L e u ] - h G R F ( l 32)-OH, 14, which are potent in vivo since they have enhanced plasma stability (26, 27). Homologs of these potent hGRF analogs were prepared with -Gly-Gly-CysN H 2 at the COOH terminus and shown to have similar in vivo biological activity. These peptides were then subjected to site-specific solution phase PEGylation through disulfide formation at the COOH-terrninal cysteine using dithiopyrridylactivated PEG, 4, as outlined in Figure 6. This procedure enabled us to prepare a series of PEGylated hGRF analogs with varying degree of PEGylation. The PEG5000 analogs (compounds 13a and 14a) were evaluated in vivo as summarized in Table II (20). 1

2

15

1

2

8

15

27


2

1

2

15

These in vivo studies (Table Π) revealed that [ d e s N ^ T y r ,D-Ala ,Ala ]-hGRF( 1 29)-NH2, 13, was -10 times more potent than hGRF(l-44)-NH2 , 5, in both mice and pigs in vivo. CCK3H-Terminal PEGylation of this peptide (compound 13a) resulted in a further increase of in vivo potency by factors of 3.4 and 5.2 times in the mouse and pig in vivo models, respectively. Similar observations were made for [His ,Val ,Gln ,Ala ,Leu ]-hGRF(l-32)-OH, 14, which was -7-10 times more potent than 5. When this analog was PEGylated at the COOH-terminus (compound 14a) the in vivo potency increased by factors of 1.7 and 5.2 times, respectively, in the mouse and pig in vivo models. Therefore COOH-terrninal PEGylation of 13 and 14 resulted in an increase of in vivo potency by 50-fold (pigs) and 20-fold (mice) when compared to the parent peptide, hGRF(l-44)-NH2 , 5. Since in vitro plasma stability is not altered by COOH-teiminal PEGylation, the increased potency of these PEGylated peptides may be attributed to longer half lives resulting from decreased clearance from the blood and tissues (20). 1

2

8

15

27

Conformational studies of PEGylated peptides Conformational studies were undertaken to better understand the observation that the PEGylation of [desNH Tyr ,D-Ala ,Ala ]-hGRF(l-29)-NH2, 13, to [desNH . Tyr^D-AlaÂla^l-hGRFa^) N H 2 , 13a, dramatically increases the in vivo potency. Figure 9 shows the circular dichroism of 13 and 13a and reveals that the spectra are superimposable in the helixsupporting solvent, 75% methanol-H20. The CD spectra were also shown to be superimposable in the helix-breaking solvent, H2O. Furthermore, the degree of PEGylation had no effect on the C D spectra. PEGylation of 13 with P E G 7 5 0 , PEG2000» or PEG10000, resulted in the corresponding PEGylated peptides which gave C D spectra that were superimposable with the PEGsooo-peptide, 13a in both 75% methanol-H 0 and in pure H2O. Similar observations were made for the C D spectra of [His ,Val ,Gln ,Ala ,Leu ]-hGRF(l-32)-OH, 14, and that of [ms ,Val ,Gln ,Ala ,Iû ]-hGRF(l-32)GlyCys(S-CH2-CH -CO-NleNHPEGn)NH2,14a, which were also shown to be superimposable in both 75% methanol-H20 and in pure H2O. The CD spectra were also superimposable regardless of the degree of PEGylation; i.e. PEG750, PEG2000, or P E G 5 0 0 0 and PEG10000 gave identical CD spectra. From these observations we conclude that COOH-terrninal PEGylation of hGRF peptides does not alter the conformation of the parent peptide. Therefore the 1

2

15

2

2

2

1

1

2

8

2

15

8

15

27

27

2



12.0

[His ,Val ,Gln ,Ala ,Iû ]-hGRF(l-32)-Gly-Cys(NH^

2

2

1

8

2

1

15

15

27

2

Injected into C57BL/6 mice (retro-orbital, 0-30 μg/kg i.v.). Mice were bled at 0, 5, 10, 15,30, 60 min post-injection (n=5)

1

1

2

2

47.1

9.48 6.97

[His ,Val ,Gln8,Ala 5,Leu 7]-hGRF(l-32)-OH

5

54.9

2

33.6

2

[desra Tyr ,D-Ala ,Ala ]-hGRF(l-29)-Gly-Gly-Cys(NH )-S-Nle-PEG ^

15

10.5

2

9.81

2

[desNH Tyr ,D- A l a , Ala ]-hGRF( 1 -29)-NH

1

hGRF(l-44)-NH

1.00

1

Relative Potency Mice Pigs* 1.00

hGRF Analog

b Injected into pigs (0-30 μg/kg s.c). 1st hr post-dosing, blood was collected at 10 min intervals. 2nd hour post-dosing blood was collected at 15 min intervals. 3rd hr post-dosing blood was collected at 30 min intervals. Total collection time: 6.5 hr.

a

14a

14

13a

13

5

Compound

Table II. Biological Activity (in vivo) of COOH-Terminal Pegylated hGRF Analogs


234

GLYCOL)


POLY(ETHYLENE

280

220

WAVE LENGTH, ι Figure 9. Circular dichroism of non-pegylated and pegylated peptides: ο [desNH Tyr ,D-Ala ,Ala ]-hGRF(l-29)-NH and · [desNH Tyr\ D-AlaÂla 5]-hGRF(l-29)-Gly-Gly-Cys(S-CH -CH -CO-Nle-NHPEG5ooo)-NH in 75% methanol-H 0. 1

2

15

2

2

2

1

2

2

2

2


16.


FELIX

235

increase in the/n vivo biological activity of these peptides is unrelated to conformation. Site-specific PEGylation of β-amyloids


Alzheimer's Disease is a progressive dementia that is characterized by the presence of proteinaceous deposits causing lesions in the brain. These deposits are composed mainly of a hydrophobic family of peptides (28) known as the β-amyloids which are derived from a larger amyloid β-protein. Both the hydrophobic COOH-terminus (29) and the hydrophobic region between residues 17-20 (30) have been reported to be responsible for the amyloid properties of amyloid β-protein. Recent studies have shown that a structural transition may be required for the conversion of β-amyloid peptide into amyloid plaque(ii). βΑΡ(1-40)-ΝΗ2, 15, forms stable amyloid fibers and its deposition is reported to proceed by a nucleation-dependent mechanism (32). We have prepared COOHterrninal PEGylated derivatives of βΑΡ(1-40)-ΝΗ2 and evaluated the rate of aggregation using turbidity measurements modeled after those previously reported (32). Figure 10 shows the rate of aggregation of βΑΡ(1-40)-ΝΗ2, 15, and βΑΡ(140)-Orn(PEG5ooo-CH2-CO)-NH2, 15a. It was observed that the aggregation of βΑΡ(1-40)-ΝΗ2 began almost immediately, but the PEGylated analog, 15a, required a substantial period of time before aggregation was even detectable. These studies demonstrate the advantages of PEGylation of β-amyloid peptides since this will enable us to carry out more detailed studies, including conformational studies, on the structural transitions that are proposed during the formation of amyloid plaque. Conclusions Conditions for the site-specific PEGylation of peptides have been developed. The solid phase PEGylation procedures that are described for NH2-terminal and internal PEGylation are convenient since they can be incorporated into a standard solid phase peptide synthesis by simply removing an aliquot of peptide-resin at the appropriate cycle. The solution phase PEGylation that is described enables one to carry out postsynthesis site-specific PEGylation at a Cys residue at any position in the peptide. Both the solid phase and the solution phase PEGylation procedures are compatible with poly(ethyleneglycol) derivatives of varying molecular weight (i.e. PEG750, PEG2000, EG5ooo and PEG10000). The incorporation of two or more PEG units by solid phase synthesis (multiple PEGylation) can best be carried out with poly(ethyleneglycol) derivatives of lower molecular weight (i.e. PEG750, PEG2000). PEGylated peptides should be purified by a 2-stage process using dialysis and preparative HPLC. PEGylated peptides should be held to the same analytical standards as are currently required for free peptides. Homogeneity can be demonstrated by analytical H P L C and complete characterization includes amino acid analysis, laser desorption mass spectrometry and ÏH-NMR spectroscopy, in special cases sequence analysis can also be carried out. p

Our studies have demonstrated PEGylation by a site-specific procedure is of paramount importance. This was clearly demonstrated in the structure-activity studies that were carried out on the human growth hormone-releasing factor system. PEGylation at the NH2- terminus and at positions 21 and 25 had no significant effect on in vitro biological activity compared to the non-PEGylated peptide. However, PEGylation at positions 8 and 12 drastically reduced the in vitro potency of the peptide. Finally, the observation that PEGylation at the COOH-terminus resulted in


236



0.8

0

25

50

75

100

125

150

175

200

TIME, hours Figure 10. Time course formation of amyloid fibrils: Rate of aggregation of non-pegylated and pegylated β-amyloid peptides: · βΑΡ(1-40)-ΝΗ2 and • pAP(l-40)-Orn(PEG5ooo-CH2-CO)-NH . Concentration: 20uM. Solutions were prepared in DMSO (50uL) and aqueous buffer (950uL) and turbidity measured at 400 nm as previously described (reference 33). 2


16.

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237

an increase of the in vivo potency provides strong evidence of the importance of sitespecificity. Abbreviations used: Standard abbreviations are used for the amino acids and protecting groups as recommended by the IUPAC-IUB Commission on Biochemical Nomenclature: J.Biol.Chenu 1985, 260, p. 14. Other abbreviations are as follows: BOP, benzotriazol-1yloxytris(dimethylamino)phosphonium hexafluorophosphate; βΑΡ, β-amyloid peptide; CD, circular dichroism; DEEA, diisopropylethylamine; Fmoc, 9-floiirenylmethyloxycarbonyl; hGRF, human growth hormone-releasing factor; HIV, human immunodeficient virus; HPLC, high performance liquid chromatography; PEG, monomethoxypoly(ethyleneglycol); SPDP, N-succinimidyl 3-(2-pyridyldithio)propionate; tBu, tertbutyl; TFA, trifluoroacetic acid. Downloaded by FORDHAM UNIV on August 15, 2014 | http://pubs.acs.org Publication Date: August 5, 1997 | doi: 10.1021/bk-1997-0680.ch016

Acknowledgments. The author thanks Dr. Robert M . Campbell and his associates for carrying out the biological studies on the PEGylated G R F analogs and Dr. John Maggio and his associates for carrying out the biological studies on the β-amyloid PEGylated peptides. The author also thanks Dr. Y . - A . Lu and Dr. E. Heimer and his associates for carrying out the synthesis of the PEGylated peptides. The author also acknowledges Dr. Y . - C . Pan and Dr. H . Michel for the laser desorption ionization mass spectrometric measurements reported in this manuscript. References. 1. Abuchowski, A. and Davis, F. F. in Enymes as Drugs; Hosenberg, J. and Roberts, J., Eds; J. Wiley: New York, NY, 1981, p 367. 2. Abuchowski, Α., Kafkewitz, A. D. and Davis, F. F. Prep. Biochem. 1979, 9, pp. 205-211. 3. Rajagopaiain, S., Gonias, L. and Pizzo, S. V. J. Clin. Invest. 1985, 75, pp. 413-419. 4. Katre, Ν. V., Knaug, M. J. and Laird, W. J. Proc. Natl. Acad. Sci. USA 84, pp. 1487-1491. 5. Ho, D. H., Brown, N. S., Yen, Α., Holmes, Y. R., Keating, M., Abuchowski, Α., Newman, R. A. and Krakoff, I. H. Drug Metab. Disp. 1986, 14, pp. 349352. 6. Nucci, C. L., Shorr, R. and Abuchowski, A. Adv. Drug Delivery Res. 1991, 6, pp. 133-151. 7. Sartore, L., Caliceti, P., Schiavon, O. and Veronese, F. M. Appl. Biochem. Biotech. 1991, 27, pp. 45-54. 8. Sartore, L., Caliceti, P., Schiavon, O., Monfardini, C. and Veronese, F. M. Appl. Biochem. Biotech. 1991, 31, pp. 213-222. 9. Lu, Y.-A. and Felix, A.M. Pept. Res. 1993, 6, pp. 140-146. 10. Fournier, Α., Wang, C.-T. and Felix, A. M. Int. J. Pept. Prot. Res. 1988, 31, pp. 86-97. 11. Kaiser, E., Colescott, R. L., Bossinger, C. D. and Cook, P. I. Anal. Biochem. 1970, 34, pp. 595-599. 12. Wang, S.-S. J. Am. Chem. Soc. 1973, 95, pp. 1328-1333. 13. Rink, H. Tetrahedron Lett. 1987, 28, pp. 3787-3790. 14. Albericio, F., Kneib-Cordonier, N., Gera, L., Hammer, R. P., Hudson, D. and Barany, G. in Peptides: Chemistry and Biology; Proceedings of the Tenth American Peptide Symposium; Escom: Leiden, The Netherlands, 1988, pp. 159-161.



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15. Albericio, F., Kneir-Cordonier, N., Biancalana, S., Gera, L., Masada, R. I., Hudson, D. and Barany, G. J. Org. Chem. 1990, 55, pp. 3730-3743. 16. Mutter, M., Oppliger, H. and Zier, A. Makromol. Chem., Rapid Commun. 1992, 13, pp. 151-157. 17. Lu, Y.-A. and Felix, A.M. Int. J. Pept. Prot. Res. 1994, 43, pp. 127-138. 18. Lu, Y.-A. and Felix, A. M. Reactive Polymers 1994, 22, pp. 221-229. 19. Campbell, R. M., Lee, Y., Mowles, T. F.,McIntyre,K. W., Ahmad, M., Felix, A. M. and Heimer, E. P. Peptides 1992, 13, pp. 787-793. 20. Campbell, R. M., Ahmad, M., Heimer, E. P., Lambros, T., Lee, Y., Miller, R., Stricker, P. and Felix, A. M. Int. J. Pept. Prot. Res. (in press). 21. Campbell, R. M. and Scanes, C. G. Growth Regulation 1992, 2, pp. 175-191. 22. Felix, A. M., Heimer, E. P., Mowles, T. F., Eisenbeis, H., Leung, P., Lambros, T., Ahmad, M., Wang, C.-T. and Brazeau, P. Proc. 19th European Peptide Symposium; Walter de Gruyter, Berlin, 1987, pp. 481-484. 23. Felix, A. M., Lu, Y . - A and Campbell, R.M. Int. J. Pept. Prot. Res. 1995, 46, pp. 253-264. 24. Campbell, R. M., Bongers, J. and Felix, A. M. Biopolymers 1995, 37, pp. 6788. 25. Frohman, L. Α., Downs, T. R., Williams, T.C.,Heimer, E. P., Pan, Y.-C. and Felix, A. M. J. Clin. Invest. 1986, 78, pp. 906-913. 26. Heimer, E. P. Bongers, J., Ahmad, M., Lambros, T., Campbell, R. M. and Felix, A. M. in Peptides: Chemistry and Biology; Proceedings of the Twelfth American Peptide Symposium; Escom: Leiden, The Netherlands, 1992, pp. 8081. 27. Campbell, R. M.,Stricker,P., Miller, R., Bongers, J., Heimer, E. P. and Felix, A. M. in Peptides: Chemistry Structure and Biology; Proceedings of the Thirtennth American Peptide Symposium; Escom: Leiden, The Netherlands, 1994, pp. 378-380. 28. Glenner, G. G. and Wong, C.-W. Biochem. Biophys. Res. Commun. 1984, 122, pp. 1131-1135. 29. Jarrett, J. T., Berger, Ε. P. and Lansbury, P. T., Jr. Biochemistry 1993, 32, pp. 4693-4697. 30. Hilbich, C., Kister-Woike, B., Reed, J., Masters, C. L. and Beyreuther, K. J. Mol. Biol. 1992, 220, pp. 460-473. 31. Lee, J. P., Stimson, E. R., Ghilardi, J. R., Mantyh, P. W., Lu, Y . - A , Felix, A. M., Llanos, W., Behbin, Α., Cummings, M., Van Criekinge, M., Timms, W. and Maggio, J. E. Biochemistry 1995, 34, pp. 5191-5200. 32. Jarrett, J. T. and Lansbury, P. T., Jr. Biochemistry 1992, 31, pp. 1234512352.


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