Newly Designed Modifier Prolongs the Action of Short-Lived Peptides

Jul 3, 2012 - ... Prolongs the Action of Short-Lived. Peptides and Proteins by Allowing Their Binding to Serum Albumin. Yoram Shechter,*. ,†. Keren ...
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Newly Designed Modifier Prolongs the Action of Short-Lived Peptides and Proteins by Allowing Their Binding to Serum Albumin Yoram Shechter,*,† Keren Sasson,† Vered Lev-Goldman,‡ Sara Rubinraut,‡ Menachem Rubinstein,§ and Mati Fridkin*,‡ †

Departments of Biological Chemistry, ‡Organic Chemistry and §Molecular Genetics, The Weizmann Institute of Science, Rehovot, 76100, Israel ABSTRACT: We found that human serum albumin (HSA) contains a single binding domain for derivatives of long-chain fatty acid (LCFA)-like molecules in which the carboxylate is replaced by sulfonate. Accordingly, we have synthesized 16-sulfo-hexadecanoic acid-N-hydroxysuccinimide ester [HO3S-(CH2)15-CONHS], an agent that reacts selectively with the amino side chains of peptides and proteins. A macromolecule containing a single 16-sulfohexadecanoate moiety associating with albumin with a Ka value of 0.83 ± 0.08 × 106 M−1, a sufficient affinity to extend the actions in vivo of such short-lived peptides and proteins. Subcutaneous administration of insulin-NHCO-(CH2)15-SO3− into mice facilitated a glucose-lowering effect 4.3 times in duration and 6.6 times in area under the curve (AUC) as compared to an in vitro equipotent amount of Zn2+-free insulin. Similarly, subcutaneous and intravenous administration of exendin-4-NHCO-(CH2)15-SO3− to mice yielded prolonged and stable reduction in glucose level, 5−9-fold longer than that of exendin-4. Also, a single subcutaneous administration of human interferon-α2-[NH-CO-(CH2)15-SO3−]3 to mice yielded circulating antiviral activity over a period of 40 h. In conclusion, a simple, hydrophilic reagent has been engineered, synthesized, and studied. Its linkage to peptides and proteins in a monomodified fashion yielded hydrophilic, prolonged acting derivatives, due to their acquired ability to associate with serum albumin after administration.

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INTRODUCTION Extending the actions of short-lived peptide/protein drugs can be accomplished by endowing them with affinity to serum albumin. This has been achieved by the covalent linking of ligands that bind albumin such as long-chain fatty acids (LCFA) or bile acids.1−5 This leads to significant elevation in the hydrophobic character of those peptide/protein derivatives and a modest elevation in their affinity toward serum albumin. Bile acids have low association affinity to albumin6 and have considerably lower affinity after linkage to a peptide or a protein. Long-chain fatty acids (LCFA) associate tightly with albumin, but negligibly if covalently linked via their carboxylate moiety.7 These findings were the impetus for designing insulin-detemir and insulin degludec, two human insulin derivatives in which an LCFA-like probe was integrated into the insulin molecule.1,2,8 In both cases, ThrB30 was removed to obtain a free carboxylate anion, in close vicinity to the long methylene chain located on lysineB29. In this study, we examined the option of engineering a new version of reagents containing an albumin binding probe, which combines the following features: (i) specificity to the α or ε amino side-chain moieties thereby permitting linkage to essentially any peptide or a protein; (ii) preservation of high affinity to albumin, following linkage to a peptide or a protein, in a monomodified fashion; and (iii) minimization of increased hydrophobicity and potential antigenicity of the resultant derivatives.9,10 © 2012 American Chemical Society

LCFAs such as palmitic acid [H3C-(CH2)14-COOH] associate tightly with albumin.7,11 Here, we wished to investigate whether the carboxylate anion, at the end of the methylene-chain, can be replaced with the highly hydrophilic sulfonate anion [-(CH2)nSO3−] to generate considerably more hydrophilic peptide/ protein derivatives. A key question to be answered was whether a methylene chain, ending with a sulfonate anion, would maintain the efficacy of LCFA to associate with albumin, and would retain sufficient affinity after linkage to a peptide or a protein to prolong significantly conjugate stability and residence time in vivo. We present here our studies in this direction.

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EXPERIMENTAL SECTION

Materials. Human (Zn+2-f ree) insulin was donated by Novo Nordisk, Bagsvalrd, Denmark or by Biotechnology General (Rehovot, Israel). Insulin-detemir, a Novo Nordisk product, was extensively dialyzed against 0.01 M NaHCO3 and stored at 7 °C until use. The concentration of this derivative was determined by its absorbance at 279 nm (ε279 = 5800) and/or by acid hydrolyzing an aliquot (in 6 M HCl for 22 h at 110 °C) followed by quantitative amino acid analysis. Received: February 20, 2012 Revised: May 21, 2012 Published: July 3, 2012 1577

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D-[U-14C] glucose (4−7 mCi/mol) was obtained from Du Pont-NEN (Boston, Ma). Type 1 collagenase (134 U/mg) was purchased from Worthington (Freehold, NY). Exendin-4 (HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH2) was synthesized by the solid-phase method using a multiple peptide synthesizer, AMS 422 (Abimed Analyzer Technik, GmbH). IFNα2 was prepared as described in detail by Piehler and Schreiber.12 11-Mercaptoundecanoic acid and 16-mercaptohexadecanoic acid were purchased from Aldrich (Saint-Louis, MO). All other materials used in this study were of analytical grade. Chemical Syntheses. HO3S-(CH2)15-COOH was prepared by performic acid oxidation of 16-mercaptohexadecanoic acid. The latter compound (432 mg, 1.5 mmol) was suspended in 14 mL formic acid. Hydrogen peroxide (1.0 mL) was then added (5.86 mmol), and the reaction was carried out for 14 h at 25 °C. Formic acid and residual H2O2 were removed by evaporation. The product obtained was suspended in H2O and lyophilized. This procedure was repeated 3 times. The calculated mass of HO3S-(CH2)15-COOH is 336.4858 Da. Found ESMS M = 335.16 Da. The product was obtained in 94% yield. HO3S-(CH2)10-COOH was prepared by performic acid oxidation of 11-mercaptoundecanoic acid under the same synthesizing conditions applied above. The calculated mass of HO3S-(CH2)10-COOH is 266.3518 Da. Found ESMS M = 264.96 Da. Synthesis of HO3S-(CH2)15-CONHS. This was carried out in 1.0 mL DMF containing 34 mg HO3S-(CH2)15-COOH (0.1 mmol) and 38 mg of N-hydroxysuccinimide (0.3 mmol). N-Ethyldiisopropylamine (DIPEA) (5.2 μL) was then added to obtain a DIPEA concentration of 30 mM. Solid EDC (40 mg) were next added (0.2 mmol). The reaction was carried out at 25 °C with stirring over a period of 2 h. Hydrochloric acid (3 mL, 0.02 M) was then added, and the precipitated product was dialyzed overnight against 0.01 M HCl at 7 °C to dialyze out DMF, DIPEA, EDC, and NHS, which did not participate in the coupling procedure. The suspended product thus obtained was lyophilized. The calculated mass of HO3S-(CH2)15-CONHS is 433.559 Da. Found ESMS M = 432.18 Da. Synthesis of HO3S-(CH2)10-CONHS. This procedure was carried out under the same synthesizing conditions applied for HO3S-(CH2)15-CONHS, starting from HO3S-(CH2)10-COOH. The calculated mass is 363.425 Da. Found ESMS M = 362.05 Da. Preparation of PEG40-NH-(CH2)3-NH2. PEG40-OSu (m-PEG2N-hydroxysuccinimide ester, Shearwater product) was dissolved at a concentration of 20 mg/mL in 0.1 M NaHCO3, containing 1 M of 1.3 diaminopropane dihydrochloride (Aldrich). The reaction was carried out for 1 h at 25 °C. The product was extensively dialyzed against H2O, lyophilized and kept at 7 °C until use. Preparation of PEG40-(NH2)2. PEG40-OSu (Batch Nof, Sunbright, GL2−400TS) was dissolved at a concentration of 20 mg/ mL in an aqueous solution of 0.5 M L-glutamic acid in 0.1 M NaHCO3 (pH 8.5). Reaction was carried out for 2 h at 0 °C. The product thus obtained [PEG40-Glu-(COOH)2] was dialyzed overnight against H2O, lyophilized, and dissolved in 0.5 M dicystamine-diHCl in H2O (pH 6.0). Solid EDC was then added (10 mg; 100 molar excess over PEG40-Glu-(COOH)2), and the reaction was carried out for 2 h at 25 °C. The product thus obtained was dialyzed extensively against H2O and lyophilized. Preparation of PEG40-NHCO-(CH2)15,10-SO3‑] and PEG40[NH-(CH2)15-SO3‑]2. PEG40-NH2 or PEG40-(NH2)2 (10 mg/

mL of each in 0.1 M NaHCO3) were treated with 20 molar excess of −O3S-(CH2)10 CONHS or −O3S-(CH2)15 CONHS (predissolved in 0.1 mL DMF) for 2 h at 25 °C, followed by dialysis for 24 h against 0.01 M Na2CO3 (pH = 10.3) and 24 h against H2O and then lyophilized. The products thus obtained were TNBS negative, indicating that the amino side chains were fully derivatized. Preparation of Monomodif ied Exendin-4 [Exendin-4-NHCO(CH2)15-SO3‑]. Exendin-4 (4.2 mg, 1 μmol) dissolved in 0.1 M Hepes buffer, pH 7.4. −O3S-(CH2)15-CONHS (2.2 mg, 5 μmol dissolved in 30 μL DMF) was then added and the reaction was carried out for 3 h with stirring at 0 °C. The reaction mixture was then dialyzed to remove low-molecular-weight materials and the remaining protein function was lyophilized. Monomodified derivative of exendin-4 linked to ∼CO-(CH2)15-SO3− was purified from unreacted exendin-4 and from residual bismodified derivative using semipreperative HPLC on RP-4 column. The derivative was obtained in an overall yield of 34 ± 3%. It migrates on an analytical HPLC column as a single symmetric peak with an Rt value of 9.22 ± 0.04 min. Native exendin-4 migrates under the same running conditions with an Rt value of 8.2 ± 0.1 min. The calculated mass of Exendin-4-NHCO-(CH2)15-SO3H is 4505 Da, found by ESMS-analysis, M = 4504.44 ± 0.77 Da. Preparation of Monomodif ied Insulin [Insulin-NHCO-(CH2)15SO3‑]. To a stirred solution of Zn2+-free insulin (29 mg in 4.8 mL 0.1 M Hepes buffer, pH 7.4, 5 μmol) was added 6.5 mg −O3S(CH2)15-CONHS dissolved in 0.2 mL DMF (3 molar excess over insulin). The reaction was carried out for 4 h at 0 °C, dialyzed extensively against H2O, and lyophilized. Monomodified derivative of insulin linked to ∼CO-(CH2)15-SO3− was obtained in 29 ± 2% yield following removal of unreacted and bismodified insulin by semipreparative HPLC, using RP-4 column. Preparation of IFNα2-[NHCO-(CH2)15-SO3‑]3. IFNα2 (0.5 μmol/mL) in 0.1 M Hepes buffer (pH = 7.4) was treated with 8 molar excess of −O3S-(CH2)15-CONHS (1.73 mg, 4 μmol, dissolved in 40 μL DMF). The reaction was carried out for 1 h with stirring at 0 °C. The product was extensively dialyzed overnight against 0.01 M Na2CO3 and then for an additional 12 h against H2O, and lyophilized. This derivative contains 3.1 ± 0.3 mol HO3S-(CH2)15-CO∼ per mole IFNα2 as determined by quantitating the remaining amino side chains of this derivative with TNBS. IFNα2-[NHCO-(CH2)15-SO3−]3 has 20 ± 4% of the antiviral potency of the native cytokine as judged by its potency to protect human WISH cells from vesicular stomatitis virus (VSV) induced cytopathic effects in vitro. An I.C.50 value of 1.5 ± 0.2 pM was obtained for this derivative. Chemophysical Procedures. Ultraviolet spectra were obtained by a Beckman DU 7500 spectrophotometer in 1-cm path length UV cuvettes. Mass spectra were determined using ESMS technique (Bruker-Reflex-Reflectron model and VGplatform-II electrospray single-quadrupole mass spectrometer). HPLC analyses were performed using a Spectra-Physics SP8800 liquid chromatography system equipped with an Applied Biosystems 757 variable wavelength absorbance detector and a Spectra-SYSTEM P2000 liquid chromatography system equipped with a Spectra-SYSTEM AS100 autosampler and a SpectraSYSTEM UV1000, all controlled by a ThermoQuest chromatography data system (ThermoQuest Inc., San Jose, CA). The column effluents were monitored by UV absorbance at 220 nm. Analytical reverse-phase HPLC was performed using a prepacked Chromolith column (4.6 × 100 mm, Merck, Darmstadt, Germany). The column was eluted with a binary gradient of 10−100% solution B over 10 min with a flow rate of 3 1578

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contain a C-terminal sulfonate rather than a carboxylate anion are not available commercially. We therefore converted 11mercapto-undecanoic acid [HOOC-(CH2)10-SH] and 16mercaptohexadecanoic acid [HOOC-(CH2)15-SH] to the corresponding sulfonated derivatives using performic acid oxidation (Experimental Section and a schematic illustration in Figure 1A). Those were subsequently turned into the active

mL/min (solution A was 0.1% trifluoroacetic acid in H2O and solution B was 0.1% trifluoroacetic acid in acetonitrile:H2O; 3:1, v/v). Preparative separations were performed with prepacked Vydac RP-18 or RP-4 columns (250 × 22 mm). The column was eluted with 10−100% solution B over 60 min (12 mL/min). Isothermal scanning calorimetry measurements were performed with ITC200 microcalorimeter (Micro Cal LLC, Northampton, MA 01060, USA). Experimental details were carried out according to the ITC200 microcalorimeter users’ manual with the modifications specified in the legend of Figure 2. Biological Procedures. Rat adipocytes were prepared from fat pads of male Wistar rats (100−200 g) by collagenase digestion.13 Lipogenesis (during which [U−14C] glucose was incorporated into lipids) was carried out by the procedure of Moody et.al.14 The fat pads of three rats were cut into small pieces with scissors and suspended in 3 mL of KRB-buffer (0.7% BSA) or in 3 mL of KRP-buffer (0.7% BSA) (pH 7.4). KRB Krebs Ringer bicarbonate buffer consists of the following: NaCl, 110 mM; NaHCO3, 25 mM; KCl, 5 mM; KH2PO4, 1.2 mM; CaCl2, 1.3 mM; MgSO4, 1.3 mM. KRP buffer consists of Hepes, 10 mM; NaCl, 120 mM; KCl, 5.2 mM; CaCl2, 1.4 mM; MgSO4, 1.2 mM; and NaH2PO4/Na2HPO4, 25 mM, containing collagenase, 1 mg/ mL. The digestion was performed in a 25 mL flexible plastic bottle under an atmosphere of carbogen (95% O2, 5% CO2) for 40 min at 37 °C with vigorous shaking. Five milliliters of buffer was then added, and the cells were squeezed through a mesh screen. The cells were then allowed to stand for several minutes (in a 15 mL plastic test tube at room temperature, floating) and the buffer underneath was removed. This procedure (suspension, floating, and removal of buffer underneath) is repeated three times. The fat cell suspensions (3 × 105 cells/mL) were divided into plastic vials (0.5 mL per vial) and incubated for 60 min at 37 °C under an atmosphere of 95% O2, 5% CO2, with 0.2 mM [U−14C]glucose, in either the presence or absence of insulin (100 ng/mL) and its derivatives. The reaction was terminated by adding toluene-based scintillation fluid (1.0 mL per vial) and the extracted lipids were counted. Insulin-stimulated lipogenesis was 4- to 5-fold higher than basal; basal ∼2000 c.p.m. per 3 × 105 cells/h; Vinsulin 8000−10 000 cpm per 3 × 105 cells/h. Blood glucose levels were determined at varying time points following administration of insulin, exendin-4, and their derivatives in blood aliquots taken from the tail vein. Glucose analyzer (Beckman Instruments Fullerton, CA) was used. Groups consisted of six mice each. Data are presented as means ± SE. Antiviral activity. Antiviral activity of IFNα2, its derivatives, and serum samples containing IFNα2 was determined by the capacity of the cytokine to protect human amnion WISH cells against vesicular stomatitis virus (VSV)-induced cytopathic effects.15 WISH cells (4.5 × 105 cells/ml) were seeded in a 96-well plate (100 μL/well) and incubated with 2-fold serial dilutions of IFNα2, its derivative, or serum samples, measuring the absorbance of crystal violet stained cells in an ELISA plate. In this assay, native IFNα2 shows 50% protection of VSV-induced WISH cells at a concentration of 0.3 ± 0.04 pM (5.8 ± 0.75 pg/ mL).

Figure 1. Schematic illustration of the synthesis and the structure of 11sulfo-undecanoic-N-hydroxysuccinimide [HO3S-(CH2)10-CONHS] and of 16-sulfohexa-decanoic-N-hydroxysuccinimide [HO3S-(CH2)15CONHS].

esters by linking N-hydroxysuccinimide to their carboxylate moieties (Figure 1B). The structures of the intermediates and the final products were validated by mass spectroscopy. Both compounds, − O 3 S-(CH 2 ) 10 -CONHS and − O 3 S-(CH 2 ) 15 CONHS, were obtained in >80% overall yields. Associating Affinity of Macromolecules Containing ∼∼ (CH2)10-SO3− and ∼∼ (CH2)15-SO3− with Human Serum Albumin. Figure 2 shows the associating affinities of macromolecules containing a single or a pair of covalently linked ∼OC(CH2)10/15-SO3− toward HSA using isothermal scanning calorimetry.16 As a model for a macromolecule, we used a poly(ethylene glycol) chain of 40 kDa. PEG40-NH-CO-(CH2)10SO3− and PEG40-NH-CO-(CH2)15-SO3− associate with HSA. The Ka values observed for these macromolecules were 0.195 ± 0.0274 and 0.829 ± 0.0821 × 106 M−1, respectively (Figure 2A,B). PEG40 containing a pair of the longer moieties showed a Ka value of 0.803 ± 0.0495 × 106 M−1 (Figure 2C). Thus, a methylene chain composed of C10 or C15 atoms ending with a C-terminal sulfonate anion, which has been linked to a macromolecule, binds to HSA with Ka values in the range of 0.2−0.8 × 106 M−1. Chemical and Biological Features of Insulin-NHCO(CH2)15-SO3−. Table 1 summarizes the characteristic features of the HPLC-purified insulin derivative relevant to this study. Insulin-NHCO-(CH2)15-SO3H is a monomodified derivative having molecular weight of 6124.98 ± 0.27 Da (calculated value is 6126 Da), as verified by mass spectroscopy. The derivative is soluble in aqueous buffers (pH 7.5−8.0) at a concentration of >10 mg/mL. It has the absorbance of the native hormone (ε279 = 5800). Insulin-NHCO-(CH2)15-SO3− emerged as a symmetric peak on analytical HPLC column with Rt = 7.97 ± 0.04 min (Figure 3). It activates lipogenesis in rat adipocytes at about 20% the efficacy of insulin, yielding a half-maximal effect (ED50) at a concentration of 0.5 ± 0.5 nM (summarized in Table 1). HPLC purified insulin-NHCO-(CH2)15-SO3− is derivatized exclusively on the α-amino moiety of PheB1, as determined from N-terminal protein sequence analysis of this derivative (Figure 4, and summarized in Table 1). The sequence obtained was GlyIleu-Val-Glu, identical to that of the A-chain of human insulin. Insulin-NHCO-(CH2)15-SO3− is Relatively Hydrophilic. Figure 5 shows the retention times (Rt values) of insulin, insulinNHCO-(CH2)15-SO3−, and insulin-detemir loaded on analyticalHPLC-chromolith R.P-18e column. This approach of hydrophobic chromatography yields an estimate of the overall

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RESULTS Engineering and Synthesizing Sulfonyl-Containing Albumin Binding Probes for the Covalent Attachment to Peptides and Proteins. Derivatives resembling LCFAs that 1579

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Figure 2. Simulating binding isotherms for the association of PEG40-NHCO-(CH2)10-SO3−, PEG40-NHCO-(CH2)15-SO3−, and PEG40-[NHCO(CH2)10-SO3−]2 with human serum albumin as determined by ITC-200. The raw data were obtained for 50 injections each of 0.3 μL at intervals of 1 min. The sample cell contained the ligands at a concentration of 20 μM, and the injection syringe contained 400 μM HSA. Both components dissolved in 0.1 M Hepes buffer, pH 7.4. The experiments were conducted at 23 °C.

Table 1. Chemical and Biological Features of HPLC-Purified Insulin-NHCO-(CH2)15-SO3− characteristic

Ins-NHCO-(CH2)15-SO3−

amino acid composition absorbance at 279 nma analytical HPLC; a single peak, retention time (Rt) mass spectroscopy (electrospray ionization) calcd (m/z) found ES− found ES+ solubility in aqueous buffer (pH 7.5−8.0) lipogenic potency in rat adipocytes

identical to insulin ε279 = 5800, identical to insulin 7.97 ± 0.04 minb

site of derivatization

6126 Dac 6124.98 ± 0.27 Da 6126.82 ± 0.50 Da > 10 mg/mL ED50 = 0.5 ± 0.03 nM (∼20%)d amino side chain of PheB1e

Figure 3. HPLC analysis of purified insulin-NHCO-(CH2)15-SO3−. The derivative (30 μg) was loaded onto a chromolith Rp-18e (100 mm × 4 mm) column and run with a linear gradient from 0% to 100% solution A (0.1% TFA) to solution B (acetonitrile−H2O 75:25 in 0.1% TFA) over 10 min and then 4 min in solution B at a rate of 3 mL/min. The effluent was monitored at 220 nm.

a

Derivative with known absorbance at 279 nm was quantitated by hydrolyzing an aliquot in 6 N HCl (110 °C, 22 h). bUnder the same running conditions, insulin elutes with Rt = 7.30 ± 0.01 min. c Calculated mass is the molecular weight of human insulin (5807 Da) combined with that of ∼∼OC-(CH2)15-SO3H which amounted to 319 Da. dIn this assay, insulin stimulates lipogenesis 4−5 times above basal level, with ED50 value of 0.1 ± 0.02 nM (0.58 ± 0.06 ng/mL). e Determined by N-terminal protein sequence analysis (Figure 4).

mouse was compared to a dose of 0.13 nmol/mouse of the native hormone. Insulin-NHCO-(CH2)15-SO3− showed a prolonged glucose lowering pattern (t1/2 = 3 ± 0.4 h), 4.3 times in duration and 6.6 times the area under the curve (AUC) of that manifested by administering an equipotent amount of Zn2+-free insulin as determined by the in vitro assay. It should be noted, however, that AUC is not a sufficient parameter for determining glucodynamic potency of insulin and derivatives in vivo.19 Additional measurements, such as glucose-clamps,20,21 are to be applied in the future as well. High-Bioavailability of Insulin-NHCO-(CH2)15-SO3− Following Subcutaneous Administration. To evaluate if there is any reduction in the diffusion rate of Insulin-NHCO-(CH2)15SO3− from the subcutis to the circulatory system, in Figure 7 we have compared the glucose-lowering pattern of intravenous versus subcutaneous administered Insulin-NHCO-(CH2)15SO3− to that of Zn2+-free insulin (Figure 7A,B). The areas

lipophilicity of peptides analogues.17 Insulin, insulin-NHCO(CH2)15-SO3−, and insulin-detemir emerged from the column with Rt values of 7.31, 7.94, and 9.10 min, respectively. Thus, Insulin-NHCO-(CH2)15-SO3− is quite hydrophilic in nature. This is particularly valid in comparison to insulin-detemir. The latter derivative contains a single C-14 fatty acid chain covalently linked to lysine B29.18 Glucose Lowering Pattern of Insulin-NH-CO-(CH2)15SO3−. In Figure 6, we compared the glucose-lowering pattern obtained after a single subcutaneous administration of insulinNH-CO-(CH2)15-SO3− to that of Zn2+- free insulin. Since insulin-NH-CO-(CH2)15-SO3− has ∼20% the potency of insulin to stimulate lipogenesis in vitro (Table 1), a dose of 0.65 nmol/ 1580

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Figure 4. Insulin-NHCO-(CH2)15-SO3− is derivatized exclusively at the α-amino group of phenylalanine B1. HPLC purified Insulin-NHCO-(CH2)15SO3− (190 pmol) was subjected to four cycles of N-terminal protein sequence analysis. The sequence obtained was Gly-Isoleu-Val-Glu, corresponding to the A-chain N-terminal portion of human insulin. Sequence analysis of native insulin yields as well Phe-Val-Asn-Glu on cycles 1 to 4, respectively (not shown).

times those obtained following intravenous administrations

under the curves (AUC) were integrated (Figure 7C). The AUC of subcutaneous administered Zn2+-free insulin and of InsulinNHCO-(CH2)15-SO3− exceeded by 1.23 ± 0.06 and 1.43 ± 0.1

(Figure 7C). 1581

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injected never falls below a threshold level, which in CD1 mice amounted to 70 ± 7 mg/dl.22,23 Following subcutaneous administration of Exendin-4-NHCO(CH2)15-SO3− glucose level fell to 70 ± 5 mg/dl and remained at low level over a period of ∼24 h, before slowly returning to the level of the saline-injected CD1 mice. The half-life of the glucose lowering response was 32 ± 2 h, which is 5.5 times longer than that obtained by the same dose of the native hormone. The AUC of subcutaneously administered exendin-4-NHCO-(CH2)15SO3H exceeded 7 ± 0.1 times that obtained by a similar subcutaneously administered dose of the native hormone (Figure 8). Glucose Lowering Pattern of Exendin-4-NHCO-(CH2)15SO3− Following Intravenous Administration. The prolonged-acting feature of subcutaneous administered Ex-4NHCO-(CH2)15-SO3− may be partly due to association with albumin present in the subcutaneous interstitial fluid, and therefore, this derivative may have slower, more prolonged rate of diffusion from the subcutis to the circulatory system. In Figure 9, we bypassed the subcutaneous compartment by administering Exendin-4 and Exendin-4-NHCO-(CH2)15-SO3− intravenously. Intravenously administered Ex-4-NHCO-(CH2)15-SO3− yielded a prolonged glucose-lowering pattern having a t1/2 value of 28 ± 2 h, exceeding that of exendin-4 by 9−10-fold (Figure 9). BGL was still low 48 h after administration, which was the last time point measured. Thus, the prolonged-acting feature of Ex-4-NHCO(CH2)15-SO3− appears to be predominantly the outcome of its association with circulating serum albumin. IFNα2-[NH-CO-(CH2)15-SO3−]3 is a Long-Lived Species in Vivo. Either native or the tris-modified IFNα2 derivative were administered at equal antiviral units to mice by a single

Figure 5. Relative hydrophilicity of insulin-NH-(CH2)15-SO3− as determined by hydrophobic chromatography. A mixture of Zn2+-free insulin, HPLC-purified monomodified insulin-NHCO-(CH2)15-SO3−, and insulin-detemir (40 μg each) were loaded on to a chromolith Rp18e (100 mm × 4 mm) column and run under the same running conditions specified in detail in the legend to Figure 3.

Exendin-4-NHCO-(CH2)15-SO3− Facilitates Prolonged Glucose-Lowering Effect in CD1 Mice Following Subcutaneous Administration. Figure 8 shows the glucoselowering profile of subcutaneously administered native exendin4 and of HPLC-purified exendin-4-NHCO-(CH2)15-SO3−, both administered at a dose of 0.25 nmol/CD1 mice. Previously, we reported that in this strain of mice the action of this glucagon-like peptide-1 agonist resembled the pharmacodynamic pattern obtained in db/db mice. Also, as has been observed in Type II diabetic patients, circulating BGL at any dosage of exendin-4

Figure 6. Circulating glucose levels in mice following a single subcutaneous administration of insulin-NHCO-(CH2)15-SO3−. CD1-mice were injected subcutaneously with saline (0.2 mL/mouse), Zn2+-free insulin (0.13 nmol/mouse in 0.2 mL saline), or with insulin-NHCO-(CH2)15-SO3− (0.65 nmol/ mouse in 0.2 mL saline). Blood glucose levels were determined at the indicated time points. Each point is the arithmetic mean of n = 6 mice ± SE. 1582

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Figure 7. Intravenous versus subcutaneous glucose lowering patterns of Insulin-NHCO-(CH2)15-SO3−. Comparison to Zn2+-free insulin. Groups of CD1-mice (n = 6 per group) have received intravenously or subcutaneously Zn2+-free insulin (0.35 nmol/mouse, A) or Insulin-NHCO-(CH2)15-SO3− (1.3 nmol/mouse, B). Circulating glucose levels were then monitored. Results are expressed as percent decrease in plasma glucose concentrations relative to those found in the saline-treated groups, measured at the same time point during the day. (C) AUC values integrated from (A) and (B).

Figure 8. Glucose lowering pattern of Exendin-4-NHCO-(CH2)15-SO3− following a single subcutaneous administration to CD1-mice. Three groups of CD1-mice (n = 6 per group) underwent one subcutaneous administration of saline, native exendin-4 (0.25 nmol/mouse), or exendin-4-NHCO(CH2)15-SO3− (0.25 nmol/mouse). Circulating glucose levels were monitored at the time points indicated. Each point is the arithmetic mean of n = 6 mice ± SE.

subcutaneous injection. Figure 10 shows the concentration of IFNα2 activity in the circulatory system of these mice. The circulating levels of native IFNα2 activity reached a plateau at 1 h, declining with a half-life of 2.5 ± 03 h. No detectable levels of antiviral activity were observed 10 h after administration (limit of detection is 100 IU/mL; Figure 10A and inset). Following administration of the tris-modified IFNα2-derivative, circulating IFNα2 activity was gradually elevated over a period of 2 h, suggesting the association of this derivative with subcutis albumin. The antiviral activity of the modified IFNα2 persisted

much longer than that of the native IFNα2, exhibiting a half-life of 6.5 ± 0.4 h (Figure 10B). Antiviral activity at a concentration sufficient to fully suppress viral infection (≥100 IU/mL) was present in the mouse circulation for over 40 h (Figure 10B, and inset).

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DISCUSSION In this study, we aimed to design new reagents containing albumin-binding probes capable of converting short-lived peptide/protein drugs into long-lived species in vivo. We have 1583

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Bioconjugate Chemistry

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Figure 9. Glucose lowering pattern of Exendin-4-NHCO-(CH2)15-SO3− following a single intravenous administration to CD1-mice. Three groups of CD1-mice (n = 6 per group) underwent one intravenous administration of saline, native exendin-4 (0.25 nmol/mouse), or exendin-4-NHCO-(CH2)15SO3− (0.25 nmol/mouse). Circulating glucose levels were then monitored. Results are expressed as percent decrease in plasma glucose concentration in the group treated with exendin-4 or Exendin-4-NHCO-(CH2)15-SO3−, relative to that found in the saline treated group, measured at the same time point during the day.

sulfonate anion. The latter is highly hydrophilic, but the absence of LCFA-like sulfonated derivatives prevented the option of determining albumin association. Notably, sulfonate is a highly hydrated anion, surrounded by several molecules of water, and its molecular radius significantly exceeds that of the carboxylate anion.25,26 We have converted 11-mercaptoundecanoic acid [HOOC(CH2)10-SH] and 16-mercaptohexadecanoic acid [HOOC(CH2)15-SH] to the corresponding LCFA-like sulfonated derivatives by performic acid oxidation (Figure 1A). The products were obtained in high yield in spite of the negligible solubility of the parent molecules in formic acid (experimental procedure). Those were turned into active esters by linking Nhydroxysuccinimide to the carboxylate moieties using EDC (Figure 2B), thereby turning them into reagents that react selectively with amino side chain moieties of peptides and proteins. Initially, we linked those derivatives to PEG40-NH2 in a monomodified fashion and investigated the association with HSA using isothermal scanning calorimetry. Both PEG40NHCO-(CH2)10-SO3− and PEG40-NHCO-(CH2)15-SO3− do associate with HSA; the shorter version yielded a Ka value of 0.195 × 106 M−1 and the longer version had a Ka value of 0.829 × 106 M−1 (Figure 2A,B). Thus, HSA has a binding domain for LCFA-like sulfonated molecules as well. There was no increase in affinity toward HSA applying PEG40 containing two such probes [PEG40-(NHCO-(CH2)15-SO3−)2 ] (Figure 2C), suggesting that HSA contains a single binding domain for a sulfonated LCFAlike molecule if the given spacing allows a bivalent type of association with this carrier protein. As observed here, the covalent introduction of a single ∼OC(CH2)15-SO3− moiety to PEG molecule of 40 kDa yielded a macromolecule that associates with HSA with a Ka of 0.829 × 106 M−1 (Figure 2A). This value exceeds by about 4.3 times the

Figure 10. Subcutaneously administered IFNα2-[NH-CO-(CH2)15SO3−]3 in mice facilitates prolonged circulating antiviral activity. Mice received subcutaneously 10 μg of native IFNα2 (circles) or 50 μg of IFNα2-[NH-CO-(CH2)15-SO3−]3 (triangles). At time-0 (3 min after administration), and at later time points, three mice were sacrificed and serum IFNα was determined by the antiviral activity assay. Each value is the arithmetic mean of three mice.

searched for molecules containing neither ring structures nor bulky elements that are hydrophilic in nature and preserve sufficient affinity to albumin after covalent attachment of one such probe to a peptide or a protein. By analogy to LCFA, we hypothesized that a long methylene chain, containing no double bonds, would be an appropriate spacer element.24 In order to significantly elevate hydrophilicity, we tested whether the carboxylate anion could be replaced by a 1584

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Bioconjugate Chemistry documented affinity of insulin-detemir to this carrier protein.18 We therefore envisioned that such associating affinity would be sufficient to significantly extend the actions of short-lived peptides and proteins in vivo. Indeed, derivatives of insulin and exendin-4 containing a single such probe are long-lived in vivo. The glucose-lowering effect of insulin was increased about 4.3fold and that of exendin-4 about 5−10-fold (Figures 6−9). Thus, HO3S-(CH2)15-CONHS is a simple, hydrophilic reagent, containing no ring nor hydrophobic elements, which turns short-lived peptide drugs into long-lived species in vivo. Conjugates are expected therefore to be neither toxic nor immunogenic. It should be noted that this approach is restricted to peptides and proteins, which do not significantly lose their biological/ therapeutic activity upon introduction of this probe to the drug molecule. Proteins in general can undergo a considerable degree of lysine modifications before being substantially inactivated, and this is valid also to monomodified derivatives of insulin and exendin-4.4,5,27 However, this may not be the case for lowmolecular-weight drugs such as amino acid derivatives, catecholamines, and aminoglycosides. With this drug category, a strategy of introducing a slowly hydrolyzable albumin-binding probe, previously developed in our laboratories, is recommended.28 Previously, we found that we can covalently link up to seven bulky groups to the amino side chains of IFNα2 with relatively little loss in its antiviral potency.29 In view of that, we studied here an IFNα2-derivative containing three covalently linked∼OC(CH2)15-SO3− moieties per one IFNα2 molecule. This trismodified derivative showed an IC50 value of 1.5 pM, representing 20% of the native antiviral potency. Such derivatization yielded a dramatically prolonged pharmacodynamic profile in mice (Figure 10B). Since albumin has a single binding domain for a protein-linked ∼CO-(CH2)15-SO3− residue (Figure 2), we currently study whether this tris-modified IFNα2 derivative can accommodate more than one molecule of albumin and whether such a complex displays a further increase in residence time in vivo. Finally, we stress that modification with HO3S-(CH2)15CONHS can be simply applied as the last step of solid-phase peptide synthesis, linking it selectively to the α-amino side-chain, prior to deprotecting the lysine moieties. This approach can turn any peptide that can therapeutically tolerate an additional molecule at its N-terminal position into an albumin binding species, with prolonged residence time in vivo, as well as resistance to degradation by serum amino peptidases.

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ABBREVIATIONS USED

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REFERENCES

Article

HSA, human serum albumin; HPLC, high-performance liquid chromatography; HO3S-(CH2)10-COOH, 11-sulfo undecanoic acid; HO3S-(CH2)15-COOH, 16-sulfo hexadecanoic acid; HO3S-(CH2)10-CONHS, 11-sulfo undecanoic-N-hydroxysuccinimide ester; HO3S-(CH2)15-CONHS, 16-sulfo hexadecanoicN-hydroxysuccinimide ester; EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; ESMS, electrospray single quadrupole mass spectroscopy; DMF, dimethylforamide; NHS, N-hydroxysuccinimide; AUC, area under the curve; BGL, blood glucose level; LCFA, long-chain fatty acid; Ex-4-NH-CO-(CH2)15-SO3−, monomodified exendin-4 containing -CO(CH2)15-SO3− moiety; Ins-NH-CO(CH2)15-SO3−, monomodified insulin containing -CO(CH2)15-SO3− moiety; IFNα2, interferon-α2; IFNα2[NHCO(CH2)15-SO3−]3, interferon-α2 containing 3 mol/mol of -CO(CH2)15-SO3−; PEG, poly(ethylene glycol); PEG40-NHCO(CH2)10-SO3−, poly(ethylene glycol) chain of 40 kDa containing a moiety of -CO(CH2)10-SO3−; PEG40-NH-CO(CH2)15-SO3−, poly(ethylene glycol) chain of 40 kDa containing a moiety of -CO(CH2)15-SO3−; PEG40-[NH-CO(CH2)15SO3−]2, PEG chain of 40 kDa containing two moieties of -CO(CH2)15-SO3−; ITC, isothermal scanning calorimetry; TNBS, trinitrobenzenesulfonic acid

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AUTHOR INFORMATION

Corresponding Author

*Y. S.: Tel. 972-8-9344530, Fax 972-8-9344118; e-mail: yoram. [email protected]. M. F.: Tel. 972-8-9342505, Fax 972-89344142; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Y.S. is the incumbent of the C.H. Hollenberg Chair in Metabolic and Diabetes Research established by the friends and associates of Dr. C. H. Hollenberg of Toronto, Canada. M.F. is the Lester Pearson Professor of Protein Chemistry. M.R. is the Edna & Maurice Weiss Professor of Cytokine Research. 1585

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