Amphiphilic Conjugates of Human Brain Natriuretic Peptide Designed

Bioconjugate Chem. 2006, 17, 267−274

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ARTICLES Amphiphilic Conjugates of Human Brain Natriuretic Peptide Designed for Oral Delivery: In Vitro Activity Screening Mark A. Miller,† Navdeep B. Malkar,† Diana Severynse-Stevens, Kevin G. Yarbrough, Mark J. Bednarcik, Robert E. Dugdell, Monica E. Puskas, Radha Krishnan, and Kenneth D. James* Nobex Corporation, 617 Davis Drive, Suite 100, Durham, North Carolina 27713. Received March 31, 2005; Revised Manuscript Received January 20, 2006

Congestive heart failure (CHF) is a complex syndrome involving altered neurohormonal levels and impaired cardiac and renal function. In recent years, intravenous administration of exogenous human brain-type natriuretic peptide (hBNP) has become an important therapy in treating patients with acutely decompensated CHF. However, reports during the past year suggest that hBNP could play a prominent role in the chronic treatment of CHF patients as well. We are currently developing conjugates of hBNP suitable for oral delivery to provide a patientfriendly treatment option for chronic heart failure patients. In this report, we present in vitro activity results obtained from hBNP conjugates featuring a variety of rationally designed amphiphilic oligomers. Mapping studies revealed that the hydrophobic/hydrophilic balance of the oligomer impacted the regioselectivity of conjugation. Additionally, the regiochemistry and extent of conjugation had a significant impact on activity. Many monoconjugates retained activity comparable to native peptide and are currently under evaluation in subsequent in vivo screens.

INTRODUCTION Cardiovascular diseases constitute the leading cause of death in the United States regardless of gender or ethnicity. Of these diseases, congestive heart failure (CHF) is the only one that is increasing in prevalence (1, 2). Hospital expenses for the treatment of CHF are more than double those for all forms of cancer combined (3). Approximately 78% of CHF patients are being hospitalized at least twice per year, accounting for nearly 6 million hospital days in the United States alone (4). Despite the prevalence of this disease, there is still no curative therapeutic approach (5). Multiple hospitalizations and inadequate therapeutics define the current situation faced by those who suffer from CHF. In August 2001, recombinant hBNP (human brain-type natriuretic peptide; Scios, Inc.) was approved by the FDA for the treatment of patients with acutely decompensated CHF with dyspnea at rest or with minimal activity. Although the current use of hBNP is limited to acute intravenous infusion, recent studies in patients with moderate to severe heart failure indicate that hBNP could play an important role as a chronically administered therapeutic (6-8). BNP is one of a family of peptides that are involved in cardiovascular, renal, and endocrine homeostasis (9). It has natriuretic, diuretic, vasorelaxant, lusitropic, anti-aldosterone, and anti-fibrotic properties (10). It was discovered in 1988 (11), almost a decade after the discovery of atrial natriuretic peptide (ANP). First isolated from porcine brain, it is known for its activity at receptors in cardiac, renal, vascular smooth muscle, and endothelial cells. BNP binds to the * Corresponding author. Kenneth D. James, 617 Davis Drive, Suite 100, Durham, NC 27713. Phone: (919) 474-0507. Fax (919) 474-9407. E-mail: [email protected]. † Equal contributions to this work.

natriuretic peptide receptor A (NPR-A), a membrane-bound protein on the cell surface. The binding event triggers the synthesis of cGMP in the cytosol by guanylate cyclase. It is through this secondary messenger that BNP mediates its physiological effects. Our objective has been to generate an amphiphilic conjugate of hBNP that would be suitable for oral administration and the chronic treatment of CHF as part of an outpatient or homebased therapy. The amphiphilic oligomers that were used are composed of both hydrophilic and hydrophobic moieties. The hydrophilic portion is composed of poly(ethylene glycol) (PEG), the attachment of which has been demonstrated to increase circulating half-life and reduce immunogenicity (12-14). The hydrophobic portion is composed of an alkyl group and is intended to provide amphiphilic balance, a feature that is an essential characteristic of oral drugs (15). While the conjugation of amphiphilic oligomers can be an important tool to improve the pharmacokinetics and pharmacodynamics of a drug, it is important that the agonist activity of the resultant conjugate is not severely compromised. In this report we describe the impact of various oligomers and the site of conjugation to hBNP on the in vitro production of cGMP by human aortic endothelial cells (HAEC). The lead conjugates from this activity screen are currently being tested in healthy dogs after oral administration. Preliminary results from these studies have recently been published (16).

EXPERIMENTAL PROCEDURES General. Unless otherwise mentioned, all reagents were purchased from Aldrich (Milwaukee, WI). PEGs and MPEGs were purchased from TCI America (Portland, OR). Lysyl endopeptidase was purchased from Wako BioProducts (Richmond, VA). HPLC columns were purchased from Phenomenex

10.1021/bc0501000 CCC: $33.50 © 2006 American Chemical Society Published on Web 02/24/2006

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(Torrance, CA). ELISA kits were purchased from Molecular Devices (Sunnyvale, CA). Primary human aortic endothelial cells (HAEC) were purchased from Cambrex (Clonetics; East Rutherford, NJ). Chemical shifts are reported in reference to TMS internal standard. General Procedure for Conjugation of Amphiphilic Oligomers to hBNP. Method 1: Preparation of Monoconjugates. hBNP (1 equiv) was dissolved in DMSO (1 mL/35 mg of hBNP). The activated oligomer (1.1 equiv) was dissolved in a minimal amount of THF and added to the solution. The progress of the reaction was monitored by HPLC: 50 µL aliquots were diluted in 500 µL of H2O containing 0.1% TFA. Reactions were typically complete within 45 min. Method 2: Preparation of Di-, Tri-, and Tetraconjugates. hBNP (1 equiv) was dissolved in DMSO (1 mL/35 mg of hBNP). Triethylamine (120 equiv) was added. After 5 min, the activated oligomer (2.2 equiv for diconjugate, 4 equiv for triconjugate, 5 equiv for tetraconjugate) was dissolved in a minimal amount of THF and added to the solution. The progress of the reaction was monitored by HPLC: 50 µL aliquots were diluted in 500 µL of H2O containing 0.1% TFA. Reactions were typically complete within 45 min. General Procedure for Purification of hBNP Conjugates. The conjugates prepared by Methods 1 and 2 were isolated using reversed-phase HPLC (C18, 1.0 cm. i.d. × 25 cm length). A single gradient system was sufficient for purification of most of the conjugates (A: H2O with 0.1% TFA; B: acetonitrile with 0.1% TFA; %B: 25-75 over 120 min). Each conjugate was obtained as a white powder after rotary evaporation and lyophilization. Activation of Oligomers. Method 1. The acid (1.0 equiv) and N-hydroxysuccinimide (1.1 equiv) were dissolved in dry CH2Cl2 (0.2 M with respect to acid). Ethyl dimethylaminopropyl carbodiimide hydrochloride (EDC, 1.0 equiv) was added. After being stirred at room-temperature overnight, the reaction mixture was diluted with CH2Cl2 and was washed with water (2×). The organic layer was dried (MgSO4) and concentrated to a constant weight. The products were typically colorless oils. Activation of Oligomers. Method 2. The alkyl-PEG (1.0 equiv) was dissolved in acetonitrile (0.1-0.2 M with respect to alkyl-PEG). After the addition of disuccinimidyl carbonate (DSC, 1.5 equiv), triethylamine (1.5 equiv) was added dropwise over the course of 10 min. After being stirred overnight at room temperature, the crude reaction mixture was concentrated, dissolved in sat. NaHCO3, and extracted with ethyl acetate (2×). The organic layer was dried (MgSO4) and concentrated. Purification by silica gel chromatography (EtOAc/MeOH, 10: 1) afforded the activated products. Activation of Oligomers. Method 3. The alkyl-PEG (1.0 equiv) dissolved in toluene was added dropwise to a stirring 20% phosgene solution in toluene at -10 °C (0.2 M with respect to alkyl-PEG). After 30 min, the reaction mixture was allowed to warm to room-temperature overnight. Phosgene and toluene were removed by vacuum distillation. The crude acid chloride was dissolved in CH2Cl2 (0.4 M with respect to acid chloride), and N-hydroxysuccinimide (1.1 equiv) was added. Triethylamine (1.1 equiv) was added dropwise over the course of 10 min. After being stirred overnight at room temperature, the crude reaction mixture was concentrated, dissolved in sat. NaHCO3 (20 mL), and extracted with ethyl acetate (2×). The organic layer was dried (MgSO4) and concentrated. Purification by silica gel chromatography (EtOAc) afforded the activated product. MPEG7C16 Succinimidyl Ester (4). To a solution of the acid 21 (15.3 g, 45 mmol) in ethanol (300 mL) was added H2SO4 (1.5 mL, 31.25 mmol). After being stirred for 48 h, the crude reaction mixture was diluted with water and extracted with CH2Cl2 (2 × 300 mL). The organic layer was washed with H2O (2

Miller et al.

× 300 mL) and sat. NaHCO3 (2 × 300 mL). The organic layer was then dried (MgSO4) and evaporated to dryness to afford an off-white solid (16.03 g, 98%): MS (ESI+) 364 (M + 1). To a solution of heptaethylene glycol monomethyl ether (8.51 g, 25 mmol) in THF (250 mL) was added potassium tertbutoxide (3.1 g, 27.5 mmol) in small portions over 30 min. The reaction mixture was then stirred for an additional 1 h. The ester (10 g, 27.5 mol in 90 mL THF) was added dropwise over 30 min, and the reaction mixture was stirred overnight. The crude reaction mixture was filtered through Celite and concentrated. The crude oil was purified by silica gel chromatography (25% MeOH in CHCl3) to yield 24 as a clear yellow oil (2.48 g, 16%): MS (ESI+) 623 (M + 1), 645 (M + Na+). To a solution of the ester 24 (2.22 g, 3.56 mmol) in alcohol (1:1 MeOH:EtOH, 50 mL) was added 1 N NaOH (50.0 mL). After being stirred for 24 h, the reaction mixture was concentrated, acidified to pH 2 with 1 M HCl, saturated with NaCl, and extracted with CH2Cl2 (3 × 75 mL). The organic layers were combined, washed with sat. NaCl, dried (MgSO4), and concentrated. Purification of the crude solid by silica gel chromatography (ethyl acetate) provided the amphiphilic acid as a white powder (858 mg, 40%): MS (ESI+) 617 (M + Na+). The acid (324 mg, 544 mmol) was activated according to Activation Method 1. The product 4 was obtained as a clear oil (290 mg, 77%): 1H NMR (400 MHz, CDCl3, δ): 1.27 (m, 18H), 1.40 (m, 2H), 1.58 (m, 2H), 1.73 (m, 4H), 2.60 (t, 2H), 2.84 (m, 4H), 3.38 (s, 3H), 3.45 (t, 2H), 3.56 (m, 6H), 3.62 (m, 22H); MS (ESI+) 714 (M + Na+). MPEG3C3 Succinimidyl Ester (1). Methyl triethylene glycol (3.84 g, 23.4 mmol) and tert-butyl acrylate (1.71 mL, 11.7 mmol) were dissolved in dry THF (10 mL). Sodium metal (27 mg, 0.117 mmol) was added to the solution. After being stirred for 4 h at room temperature, the reaction mixture was quenched by the addition of 1 M HCl (30 mL). The quenched reaction mixture was then extracted with CH2Cl2 (1 × 100 mL, 1 × 50 mL). The organic layer was dried (MgSO4) and concentrated. After purification by silica gel chromatography (ethyl acetate), the product was obtained as an oil (2.94 g, 86%): MS (ESI+) 315 (M + Na+). The tert-butyl ester 25 (1.00 g, 3.42 mmol) was deprotected by stirring at room temperature in trifluoroacetic acid (5.0 mL). The contents were then concentrated to a constant weight (0.708 g, 88%): MS (ESI+) 236 (M+), 239 (M + Na+). The acid (0.705 g, 2.99 mmol) was activated according to Activation Method 1. The product 1 was an oil (0.848 g, 85%): 1H NMR (400 MHz, CDCl3, δ): 2.81 (m, 4H), 2.87 (t, 2H), 3.35 (s, 3H), 3.52 (m, 2H), 3.61 (m, 10H), 3.82 (t, 2H); MS (ESI+) 356 (M + Na+). C6PEG7 Succinimidyl Carbonate (5). Triethylene glycol (30 g, 0.2 mol) was dissolved in 8 mL of 100% NaOH and stirred for 10 min. Benzyl chloride (7.8 g, 61.6 mmol) was added, and the reaction was stirred at reflux overnight. The reaction mixture was diluted with sat. NaCl (500 mL) and extracted with CH2Cl2 (2 × 400 mL). Organic layers were combined, washed sat. NaCl (800 mL), dried (MgSO4), and evaporated to dryness. Silica gel chromatography (ethyl acetate) provided the protected PEG3 as a yellowish oil (9.84 g, 66%): MS (FAB+) 241 (M + 1). Triethylamine (7.1 mL, 0.054 mol) was added to a solution of benzyl PEG3 (9.84 g, 0.041 mol) in CH2Cl2 (50 mL). The solution was cooled to 0 °C, and methanesulfonyl chloride (3.9 mL, 0.049 mol), dissolved in CH2Cl2 (10 mL), was added via addition funnel. The reaction was stirred for 0.5 h at 0 °C and then 4 h at room temperature. The reaction mixture was filtered through Celite and diluted with CH2Cl2 (150 mL). The filtrate was then washed with H2O (200 mL), sat. NaHCO3 (200 mL), and H2O (200 mL). The organic layer was dried (MgSO4) and

In Vitro Activity of Amphiphilic Conjugates of BNP

concentrated to a constant weight, affording the mesylate as a yellowish oil (10.85 g, 83%): MS (FAB+) 319 (M + 1). NaH (0.45 g, 0.019 mol) was added portionwise over 0.5 h to a solution of tetraethylene glycol (7.32 g, 0.038 mol) in tetrahydrofuran (140 mL). The mixture was stirred for 1 h. Then the benzyl PEG3 mesylate (6.0 g, 0.019 mol), dissolved in tetrahydrofuran (20 mL), was added dropwise via addition funnel. After being stirred overnight at room temperature, the reaction mixture was filtered through Celite and evaporated to dryness. The resultant oil was dissolved in CH2Cl2 (150 mL) and washed with H2O (150 mL), sat. NaHCO3 (150 mL), and H2O (150 mL). The organic layer was dried (MgSO4) and concentrated. Silica gel chromatography (ethyl acetate/methanol, 10:1) afforded benzyl PEG7 (26) as a clear oil (3.83 g, 49%): MS (FAB+) 417 (M + 1). To a solution of 26 (5.45 g, 0.013 mol) in tetrahydrofuran (160 mL) was added potassium tert-butoxide (1.6 g, 0.014 mol), and the reaction mixture was stirred for 1 h. Then hexylmethanesulfonate (2.59 g, 0.014 mol), dissolved in tetrahydrofuran (20 mL), was added via addition funnel, and the reaction was stirred overnight at room temperature. The reaction mixture was filtered through Celite and concentrated. The resultant oil was dissolved in ethyl acetate (150 mL), washed with H2O (2 × 150 mL), dried (MgSO4), and concentrated. Silica gel chromatography (ethyl acetate) afforded the protected alkyl PEG7 (27) as a clear oil (2.40 g, 36%): MS (FAB+) 501 (M + 1). To a solution of 27 (2.40 g, 4.8 mmol) in ethyl acetate (16 mL) was added palladium on activated carbon (1 g, 10wt % on carbon). The reaction vessel was stirred under H2 atmosphere overnight at room temperature. The reaction mixture was then filtered through Celite and concentrated to afford the alkyl-PEG7OH as a clear oil (1.61 g, 82%): MS (FAB+) 411 (M + 1). The alkyl-PEG7OH (1.60 g, 3.9 mmol) was activated according to Activation Method 3. The product was a clear oil (1.06 g, 53%): 1H NMR (400 MHz, CDCl3, δ): 0.88 (m, 3H), 1.29 (m, 4H), 1.58 (m, 2H), 2.84 (m, 4H), 3.45 (t, 2H), 3.58 (m, 2H), 3.65 (m, 22H), 3.79 (m, 2H), 4.47 (m, 2H); MS (FAB+) 552 (M + 1). (Branched MPEG4) Urethanyl C6 Succinimidyl Ester (11). Tetraethylene glycol monomethyl ether (14.0 g, 67 mmol) was dissolved in tetrahydrofuran (90 mL). NaH (1.77 g, 74 mmol) was added portionwise, and the reaction was stirred for 2 h. Then epichlorohydrin (26.3 mL, 0.34 mol) was added dropwise and the reaction was stirred at room temperature for 48 h. The crude reaction mixture was filtered through Celite and washed CH2Cl2 (250 mL). The filtrate was washed H2O (2 × 250 mL), dried (MgSO4), and evaporated to dryness. Column chromatography (silica, ethyl acetate) afforded 28 a clear oil (10.15 g, 57%): MS (ESI+) 287 (M + Na+). Tetraethylene glycol monomethyl ether (7.96 g, 0.038 mol) and 28 (10.1 g, 0.038 mol) were dissolved in CH2Cl2 (100 mL). BF3‚OEt2 (0.48 mL, 0.0038 mol) was added and the reaction mixture was stirred overnight at room temperature. The crude mix was diluted with CH2Cl2 (200 mL), washed with sat. NaHCO3 (300 mL) and H2O (300 mL), dried (MgSO4), and evaporated to dryness. Column chromatography (silica, ethyl acetate/MeOH, 10:1) afforded 29 a clear oil (4.5 g, 25%): MS (ESI+) 495 (M + Na+). 4-Nitrochloroformate (2.87 g, 14.3 mmol) and 29 (4.5 g, 9.5 mmol) were dissolved in CH2Cl2 (45 mL). After the reaction mixture was stirred for 10 min, TEA (2.1 mL, 15 mmol) was added and reaction stirred overnight at room temperature. The crude mix was diluted with CH2Cl2 (130 mL), washed with 1M HCl (175 mL) and H2O (175 mL), dried (MgSO4), and evaporated to dryness. Column chromatography (silica, ethyl

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acetate/MeOH, 15:1) afforded 30 a yellowish oil (2.38 g, 40%): MS (ESI+) 660 (M + Na+). 6-Aminocaproic acid (0.126 g, 0.96 mmol) and K2CO3 (0.221 g, 1.6 mmol) were dissolved in H2O (DI, 5 mL). Then 30 (0.5 g, 0.8 mmol) was dissolved in THF (0.7 mL) and added dropwise. After being stirred overnight at room temperature, the reaction mixture was diluted with H2O (20 mL), acidified to pH ∼ 1 with HCl, and extracted with CH2Cl2 (2 × 25 mL). The organic layers were combined, dried (MgSO4), and evaporated to dryness. Column chromatograpy (silica, CHCl3/ MeOH, 15:1) afforded 31 a clear oil (0.428 g, 85%): MS (ESI+) 652 (M + Na+). The acid was activated using Method 1:31 (0.40 g, 0.64 mmol), N-hydroxysuccinimide (0.088 g, 0.77 mmol), EDC (0.160 g, 0.83 mmol), and CH2Cl2 (5 mL). Column chromatography (silica, ethyl acetate/MeOH, 10:1) afforded 11 a clear oil (0.320 g, 69%): 1H NMR (400 MHz, CDCl3, δ): 1.45 (m, 2H), 1.53 (m, 2H), 1.77 (m, 2H), 1.87 (br s, 1H), 2.62 (t, 2H), 2.84 (m, 4H), 3.16 (m, 2H), 3.38 (s, 6H), 3.55 (m, 6H), 3.65 (m, 38H), 5.01 (m, 1H); MS (ESI+) 749 (M + Na+). Peptide Mapping. Either native hBNP or a conjugate (1.0 mg) and lysyl endopeptidase (2.0 ng) were incubated in 1.0 mL Tris buffer (0.1 M, pH 9) at 30 °C for 1 h. Reactions were quenched by the addition of 50% acetic acid (50 µL) to bring the solutions to pH ≈ 4. As a negative control, hBNP was also incubated at 30 °C without addition of the lysyl endopeptidase. All hBNP and conjugate solutions were analyzed by HPLC prior to incubation to determine purity and concentration. The incubated samples were analyzed by HPLC to reveal the fragment profile. Sites of conjugation were determined after subsequent isolation and mass spectral analyses of the peptide fragments. cGMP Assay. Primary human aortic endothelial cells (HAEC) were thawed and placed in a T75 flask prior to use in an experiment. Cells were grown for 2 days until they reached 7080% confluence and were then plated into 12-well plates at 2.5 × 104 cells/well. The next day the media was removed, and cells were preincubated for 10 min at 37 °C with 0.5 mM IBMX to inhibit phosphodiesterases. Conjugates to be screened were added to the cells for an additional 60 min at 37 °C. Incubation was stopped by lysing cells using Cell Lysis Solution (Molecular Devices; Sunnyvale, CA). The released intracellular cGMP was then acetylated using a 2:1 mixture of triethylamine to acetic anhydride. Acetylation of the cGMP lysate was found to increase the sensitivity of this assay 5-fold. Acetylated cGMP was then measured using an ELISA-based system (CatchPoint-cyclic GMP Fluorescent Assay Kit), which quantitates cGMP via a competitive immunoassay in 96-well format. Lysates were added to the coated microplate followed by the addition of an anticGMP antibody and a horseradish peroxidase (HRP)-cGMP conjugate. Plates were incubated for 2 h at room temperature, followed by four washes. A substrate solution was added and the fluorescent intensity of each well was quantitated. The fluorescent signal intensity decreased with increasing levels of cGMP. Native hBNP was tested in each experiment as a positive control. Statistical analyses were performed with SigmaStat using a one-way ANOVA and a post-hoc Dunnett’s test with significance set at p < 0.05.

RESULTS AND DISCUSSION Sites of Conjugation. The hormone hBNP is expressed as the inactive prepro-peptide (17-19), which is cleaved after translation by the enzyme Corin (20) to the mature peptide shown in Figure 1. It has four prominent sites for conjugation: Ser1, Lys3, Lys14, and Lys27. Because binding to the NPR-A is believed to occur mostly in the loop region, we expected that activity would likely be compromised upon conjugation to Lys14 and perhaps Lys27. Thus, we anticipated that mono- or

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Figure 1. Primary structure of mature hBNP. It is a 32-amino acid peptide with a loop region composed of 17 residues, which is the characteristic loop size for the various natriuretic peptides. hBNP has four sites suitable for conjugation with these oligomers: the free N-terminus and three lysine residues (shown in red).

diconjugates composed only of Ser1 and Lys3 conjugates would have little adverse effect on activity, whereas mono-, di-, or triconjugates involving one or both of the Lys14 and Lys27 positions may reduce or eliminate activity. However, the verity of these assumptions and the extent of impact of conjugation at these sites had not yet been demonstrated. Conjugation reactions performed in aqueous phosphate buffer (10 mM; pH 7.4 and 9.5) led to undesirable mixtures of products with little indication of selectivity. In contrast, reactions performed in polar organic solvents (DMSO and DMF) resulted in much cleaner product profiles. The sites of attachment of the oligomers were determined by peptide mapping studies using lysyl endopeptidase, followed by analyses of the fragments by HPLC and mass spectrometry. Characteristic results from these studies are presented in Table 1. The six potential fragments are listed in the table in addition to the intact hBNP or conjugate. The presence of specific fragments were verified by retention time (Rf) and molecular weight. Because the conjugated lysines are resistant to lysyl endopeptidase, the presence of particular fragments indicates the lysine residues that were not conjugated. Conversely, the absence of fragments indicates conjugation at a specific site. These indications are verified by the molecular weights of the fragments. Predictably, unconjugated hBNP was fragmented into the peptides expected from cleavage at each of the three lysine residues. Cleavage at Lys14 (for native hBNP or a conjugate) results in the peptide fragments that remain covalently attached through the disulfide linkage rather than through a peptide bond. The tri- and tetraconjugates were

completely resistant to proteolysis with lysyl endopeptidase, resulting in no fragmentation. The site preference for attachment of the oligomers in either DMSO or DMF followed the trend Lys3 > Lys14 > Lys27 . Ser1 (Figure 2a). Conditions could be adjusted so that the Lys3 monoconjugate was the major product. Diconjugates tended to form only transiently. Rather than accumulating, the diconjugates were excellent substrates for conjugation and rapidly proceeded to the triconjugate, a phenomenon we have occasionally observed with other peptides. The N-terminus was so unreactive that the triconjugate could be obtained as the exclusive product. Nonetheless, the tetraconjugate could be formed by the addition of excess oligomer and extension of reaction times. The stated trend held true for the majority, but not all, of the oligomers that were tested. Oligomers that were very nonpolar overall or that consisted of a nonpolar moiety distal from the site of conjugation resulted in a different trend: Lys14 > Lys3 > Lys27 . Ser1 (Figure 2b). Because other reaction conditions were unchanged, presumably the switch in selectivity is due to nonpolar interactions bringing the active ester of the hydrophobic oligomers proximal to the lysine of the loop region. Oligomer Design. The objective through these conjugations is to maximize the benefits imparted to the peptide while minimizing perturbation and adverse effects on activity. The effects of the site and extent of conjugation on activity were first addressed using oligomer 2, a linear oligomer of moderate length and balanced amphiphilicity that has been used effectively with insulin (21-23). Reactions with this oligomer led to the generation of the mono-, di-, tri-, and tetraconjugates. Oligomer 2 is representative of the Class I (nonhydrolyzable) oligomers, which are designed to remain intact from dosing to target. A series of linear and branched non-hydrolyzable oligomers are shown in Figure 3. Because the tri- and tetraconjugates using 2 were virtually inactive, we did not know whether the higher conjugates were forbidden or if smaller oligomers would enable the higher conjugates to retain partial activity. To address this question, a series of micropegylated conjugates, which utilize Class II oligomers, were designed. These oligomers retain the amphiphilic properties of the Class I oligomers initially, but are intended to shed the hydrophobic portion before they reach the receptor (Figure 4). To mimic the activity of micropegylated conjugates after hydrolysis in vivo, a series of higher conjugates were prepared using oligomers of minimal size, ranging from one to three PEG units. Figure 5 illustrates the Class II oligomers that were used to make the various micropegylated conjugates. Also depicted are the corresponding oligomers that were intended to model the Class II oligomers after hydrolysis. We do note that the model oligomers end with a methoxy group rather than a hydroxy group. Nevertheless, both the Class II oligomers posthydrolysis and the model oligomers remove the charge of the lysines and contain the same number of ethylene

Table 1. Mapping Results from the Incubation of hBNP or hBNP Conjugates with Lysyl Endopeptidasea compound fragment present? (Rf/MW)

hBNP

C1

C2

C3

C8

1-3 28-32 4-14 + 15-27b 1-14 + 15-27 + oligomerb 4-14 + 15-32 + oligomerb 1-27 + 2x oligomer intact BNP or conjugate

4.2/330 8.4/680 13.7/2508 15.4/3464

8.4/680 16.4/3257 17.2/3901

18.6/4775

19.3/5212

8.4/680 18.5/3675 18.2/4355

a Each of the conjugates depicted was made using oligomer 2. If a fragment was present after incubation, its retention time and molecular weight are listed. The corresponding values for the parent compound are provided for comparison. b The backbone was cleaved at Lys14, but the fragments are linked by a disulfide bond.

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Figure 2. Chromatograms illustrating differing selectivity based on the amphiphilic balance of the oligomer. Detection was set at 220 nm. a. Hydrophilic oligomers react first at Lys3. b. Hydrophobic oligomers react preferentially at Lys14.

Figure 3. Linear and branched Class 1 oligomers.

glycol units. We therefore regarded them as acceptable models at this stage in screening. Both the tri- and tetraconjugates were inactive even with the smallest of oligomers, prompting a focus on Lys3 monoconjugates. Even larger Class I oligomers attached at Lys3 resulted in active conjugates; therefore, Class II oligomers offered no advantage for this peptide. Syntheses of Oligomers. The linear Class 1 oligomers were mostly prepared by similar methods, varying only according to the orientation of the polyetheylene glycol (PEG) and alkyl moieties (Scheme 1 A-C). For oligomers featuring the alkyl

moiety inside, the substituted alkanoic acid was first esterified. The monomethoxy PEG (MPEG) was then attached. Depending on availability, either the terminal bromide or hydroxyl (activated as the mesylate) was used. Ester hydrolysis and activation as the succinimidyl ester led to the desired oligomers. The one exception to this group is the oligomer 1. In this case, the MPEG moiety was coupled to tert-butyl acrylate in the presence of sodium catalyst. After deprotection of 25 in neat TFA, the oligomer was activated as previously described. The oligomers featuring the PEG moiety inside were made by different means. Briefly, the PEG diol was singly protected

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Figure 4. Different categories of oligomers used in the study. Class 1 oligomers had either the PEG or the alkyl moiety proximal to the peptide. Class 2 oligomers all featured the PEG portion proximal to the peptide.

Figure 5. Class 2 oligomers and their corresponding model oligomers.

with a benzyl group. After coupling to an alkyl mesylate, the benzyl group was removed by hydrogenolysis. Subsequent activation afforded the desired succinimidyl carbonate. The Class 2 oligomers were prepared in like manner. The benzyl PEG was first coupled with the appropriate alkanoic acid chloride. Hydrogenolysis followed by activation with disuccinimidyl carbonate completed the syntheses. Many of the Class 1 oligomers featuring branched PEGs utilized glycerol as the source of branching. Rather than starting with a poor nucleophile like glycerol itself or a protected glycerol, we found it advantageous to use a good electrophile

Miller et al.

such as epichlorohydrin (Scheme 2). Our initial attempts to go directly from epichlorohydrin to the branched PEG required reflux conditions and led to undesirable product mixtures. In the stepwise approach, the first epoxide opening proceeded smoothly, but the second epoxide opening using catalytic NaH and refluxing conditions produced low yields and likewise resulted in a mixture of products. However, better yields and cleaner product distributions were attained at ambient temperatures when a Lewis acid catalyst was used for the second epoxide opening. This method worked well with short PEGs, but yields dropped with increasing PEG length. Activation of the secondary alcohol was accomplished with p-nitrophenyl chloroformate, a reagent that proved more suitable than either phosgene or N,N′-disuccinimidyl carbonate. At this stage, the oligomer could either be conjugated to the peptide directly or be attached to a spacer, such as an -amino acid. Addition of the amino acid in an aqueous or aqueous/ethanol solution of potassium carbonate afforded the carbamate in good yield. Activation as the succinimidyl ester resulted in the branched amphiphilic oligomer, which could be conjugated to peptides. In Vitro Activity. To evaluate the activity of each conjugate at the natriuretic peptide receptor A (NPR-A), conjugates were screened using a human aortic endothelial cell (HAEC) system. HAEC cultures contain normal primary aortic endothelial cells and when exposed to hBNP, produce cGMP in a dose-dependent manner. The activity of each of the conjugates was measured by assay of the extracellular levels of cGMP. The impact of the site and the extent of conjugation is well demonstrated by the various conjugates that were made with oligomer 2 (Figure 6). Conjugate C1 (a Lys3 monoconjugate) retained good activity and potency in comparison to the unconjugated peptide. By contrast, C7 (a Lys14 monoconjugate) exhibited a partial loss of both activity and potency. When both the Lys14 and Lys 27 positions were conjugated (C2 and C3), the compounds did not induce cGMP production above background levels. The results for conjugates utilizing Class 1 and Class 2 oligomers are listed in Table 2. In general, the Lys3 monoconjugates consistently performed well in the assay in terms of both potency and activity. The two exceptions were C18 and C21. These conjugates share the trait of having a branched oligomer with hydrophobic portions distal from the site of attachment. It is possible that such oligomers induced a conformational change or aggregation that made them weaker agonists, but this question was not investigated. With those exceptions, oligomers of great variety of structure and size were conjugated to Lys3 while retaining activity comparable to the unconjugated peptide.

Scheme 1. Routes for Preparation of Linear Oligomersa

a A. (i) EtOH, H2SO4, 98%; (ii) MPEGnOH, KOtBu, THF, 4 h, 16%; (iii) 1 N NaOH, 40%; (iv) Method 1, 77%. B. (i) MPEG3OH, Na0, THF, 4 h, 86%; (ii) TFA Neat, 88%; (iii) Method 1, 85%. C. (i) Hexylmethanesulfonate, KOtBu, THF, 36%; (ii) H2, Pd/C, 82%; (iii) Method 3, 53%.

Bioconjugate Chem., Vol. 17, No. 2, 2006 273

In Vitro Activity of Amphiphilic Conjugates of BNP Scheme 2 a

a (i) NaH, THF, 57%, (ii) BF3‚OEt2, CH2Cl2, 25%, (iii) 4-nitrophenyl chloroformate, TEA, CH2Cl2, 40%, (iv) K2CO3, H2O, 85%, (v) N-hydroxysuccinimide, EDC, CH2Cl2, 69%.

Table 2. In Vitro Activity Results of Various Conjugates of hBNPa compound

oligomer

position of conjugation

calculated mass

observed mass

average EC50 ( SD (nM)

average Emax ( SD

hBNP C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 C18 C19 C20 C21 C22 C23 C24 C25 C26 C27 C28

2 2 2 14 14 15 2 2 18 17 17 16 13 11 5 3 3 10 10 6 8 8 9 4 7 12 6 1

3 3, 14, 27 1, 3, 14, 27 3, 14, 27 1, 3, 14, 27 1, 3, 14, 27 14 3, 14 1, 3, 14, 27 3, 14, 27 1, 3, 14, 27 1, 3, 14, 27 1, 3, 14, 27 3 3 3 3, 14 3 14 14 3 1 3/14 3/14 3 3 3/14 3

3464 3900 4772 5208 4406 4720 4560 3900 4336 4224 3902 4048 3872 4208 4075 3900 3984 4504 4215 4215 3996 3833 3833 4295 4040 4227 4590 3996 3682

3465 3901 4775 5211 4404 4722 4563 3901 4360 4224 3904 4049 3873 4209 4075 3900 3985 4505 4216 4216 3997 3834 3834 4296 4040 NA NA 3997 NA

235 ( 110 444 ( 30 >10000 >10000 >10000 >10000 >10000 1110 ( 330 1710 ( 250 >10000 >10000 >10000 >10000 >10000 369 ( 220 281 ( 81 321 ( 180 >10000 >10000 >10000 126 ( 47 358 ( 180 146 ( 95 245 ( 120 815 ( 550 171 ( 52 556 ( 240 212 ( 10 265 ( 74

100.0 ( 0.0 101.0 ( 6.8