Albumin−Insulin Conjugate Releasing Insulin Slowly under

Jul 2, 2005 - on covalent conjugation to the amino group of insulin and the Cys-34 side ... 3-maleimidopropionate)-fluorene-N-hydroxysuccinimide; MIB-...
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Bioconjugate Chem. 2005, 16, 913−920

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Albumin-Insulin Conjugate Releasing Insulin Slowly under Physiological Conditions: A New Concept for Long-Acting Insulin Yoram Shechter,*,‡ Marina Mironchik,‡ Sara Rubinraut,§ Ayala Saul,‡,§ Haim Tsubery,‡,§ and Mati Fridkin*,§ Departments of Biological Chemistry and Organic Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel. Received February 27, 2005; Revised Manuscript Received April 26, 2005

The covalent linkage of peptides or protein drugs to human serum albumin (HSA) greatly prolongs their lifetime in vivo but is pharmacologically irrelevant when it irreversibly inactivates them. We retain drug bioactivity by synthesizing a heterobifunctional reagent (MAL-Fmoc-OSu, 9-hydroxymethyl2-(amino-3-maleimidopropionate)-fluorene-N-hydroxysuccinimide) that generates HSA-Fmoc-insulin on covalent conjugation to the amino group of insulin and the Cys-34 side chain of HSA. HSA-Fmocinsulin is water-soluble and, upon incubation in aqueous buffers reflecting normal human serum conditions, slowly, spontaneously, and homogeneously hydrolyzes to release unmodified insulin with a t1/2 of 25 ( 2 h. A single subcutaneous or intraperitoneal administration of HSA-Fmoc-insulin to diabetic rodents lowers circulating glucose levels for about 4 times longer than an equipotent dose of Zn2+-free insulin. Following subcutaneous administration, onset of the glucose-lowering effect is delayed 0.5-1 h and persists for 12 h. Thus, we present a prototype insulin formulation possessing three desirable parameters: high aqueous solubility, delayed action following subcutaneous administration, and prolonged therapeutic effect.

INTRODUCTION

Most polypeptide drugs, in particular nonglycosylated proteins of molecular mass less than 50 kDa, are shortlived species in vivo, having circulatory half-lives of 5-20 min (1). The short lifetimes of proteins in vivo are attributed to several mechanisms, including glomerular filtration in the kidney and proteolysis at several levels (2). Insulin is degraded primarily in the liver through a mechanism defined as receptor-mediated endocytosis (3, 4). This mechanism is an efficient route for terminating the action of the hormone after the levels of glucose and other nutrients have been normalized. Chemically modified insulins with negligible receptor binding affinities are therefore longer-lived species within the circulation; however, they are biologically ineffective (5). IDDM1 patients receive multiple daily subcutaneous administrations of “rapid” and long-acting insulins (2, 6-8). Prolonged long-acting insulin preparations are needed to supply low basal levels of circulating insulin between meals and overnight, this being a physiological * To whom correspondence should be addressed. YS: Tel 9728-9344530; fax 972-8-9344118; e-mail [email protected]. MF: Tel 972-8-9342505; fax 972-8-9344142; e-mail mati.fridkin@ weizmann.ac.il. ‡ Department of Biological Chemistry. § Department of Organic Chemistry. 1 Abbreviations used: BSA, bovine serum albumin; DCC, N,N′-dicyclohexylcarboimide; DCU, N,N′-dicyclohexylurea; DMF, N,N′-dimethylformamide; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); HPLC, high-performance liquid chromatography; HSA, human serum albumin; HSA-Fmoc-insulin, a conjugate of human serum albumin and insulin; IDDM, insulin-dependent diabetes mellitus; MAL-Fmoc-OSu, 9-hydroxymethyl-2-(amino3-maleimidopropionate)-fluorene-N-hydroxysuccinimide; MIBNHS, m-maleimido benzoic acid-N-hydroxysuccinimide ester; STZ, streptozocin; TCA, trichloroacetic acid; TFA, trifluoroacetic acid; THF, tetrahydrofurane.

requirement for reducing triglyceride breakdown and suppressing hepatic glucose output under “resting” conditions (9-11). Most long-acting insulins currently in use are suspensions of crystals, produced either by Zn2+ ions or by the addition of basic protamine. Such injected suspensions have decreased rates of absorption from the subcutaneous compartment to the circulatory system. Slow dissolution at the site of injection brings about the protracted effect (2, 12, 13). The application of subcutaneous infusion pumps was also introduced as a measure for controlled continuous supply of short-acting insulin (reviewed in ref 14). In recent years, major efforts have been devoted to prolonging the action of insulins that are soluble in aqueous buffered media. In one such innovative approach, two arginine moieties were covalently linked to insulin to raise the isoelectric point of the hormone. The derivative thus obtained (i.e., insulin-glargine) is formulated in a soluble form in a slightly acidic media and is crystallized and precipitated immediately after injection at the physiological neutral pH of the subcutaneous space (15-17). Many versatile polymeric prodrugs have been developed for controlled drug delivery. Thus, conjugates of different drugs with polymeric carriers employing various chemical anchoring modes were developed, for example, hydrazones, esters carbamates, citraconyl, amides and acetals (reviewed in refs 18 and 19). Noteworthy among the polymeric carriers are derivatives of poly(ethylene glycol) (20). Albumin is long-lived in vivo. Similarly, drugs and endogenous substances that associate tightly with albumin have lower clearance rates than do the unbound substances and exhibit prolonged lifetime profiles in vivo (21). Long-chain-free fatty acids bind tightly to albumin (Ka ) 108 M-1) (22), a fact that provided the impetus for

10.1021/bc050055w CCC: $30.25 © 2005 American Chemical Society Published on Web 07/02/2005

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preparation of soluble fatty acid acylated insulins that could bind to serum albumin. However, the affinity of the insulin conjugates for albumin (Ka ≈ 105 M-1) was considerably less than that of the unbound long-chainfree fatty acids, and the conjugates exhibited only moderately prolonged actions in vivo (23-25). A series of HSA-peptide conjugates have been recently prepared through formation of a stable covalent bond between maleimido-derivatives of biologically active peptides (e.g., atrial nutriuretic peptide (26), dynorphine (27), the Kringle 5 region of plasminogen (28)) and the mercapto moiety of the Cys-34 of HSA. This was undertaken with the aim of increasing the half-life of the peptide in circulation by reducing elimination through the kidney and concomitantly protecting it from proteolysis by plasma enzymes. The activities of the HSA-bound peptides, though significantly reduced as compared to the parent peptides, were found to be rather substantial. Moreover, the conjugates proved to be long-acting species. Therapeutically, insulin differs from many other polypeptide drugs in the sense that overdosing may lead to severe hypoglycemia (29-31). To avoid that, only a limited dosage of insulin can be administered each time. Such a dosage is often insufficient to maintain the basal nighttime insulin level needed to avoid hyperglycemia at dawn (6-8). This therapeutic drawback can be overcome by engineering an insulin prodrug that is biologically inactive at the time of administration. In this study, we aim to link insulin to HSA via a covalently stable bond. We expect such a nondissociable molecule to share the longevity of albumin in vivo. Because an extended surface area of the insulin molecule is engaged in receptor binding (32, 33), we initially thought it unlikely that such a conjugate would be biologically (and therapeutically) active. Nevertheless, even an inactive, insulin-HSA chimera could be advantageous if the conjugate is capable of generating the active unmodified parent insulin with a desirable pharmacokinetic pattern. Our efforts in this latter direction are brought here in detail. METHODS

Materials. Human (Zn2+-free) insulin was donated by Novo Nordisk (Bagsvaerd, Denmark) and by Biotechnology General (Rehovot, Israel) and was used without further purification. D-[U-14C]glucose (4-7 mCi/mol) was obtained from DuPont-NEN (Boston, MA). Collagenase, type I (134 U/mg), was purchased from Worthington (Freehold, NJ). 9-Fluorenylmethoxycarbonyl-N-hydroxysuccinimide (Fmoc-OSu) and di-tert-butyldicarbonate (t-Boc)2O were obtained from Novabiochem (Lau¨felfingen, Switzerland). MIB-NHS was obtained from SigmaAldrich (Rehovot, Israel). HSA was acquired from Omrix Biopharmaceutical Ltd. (Weizmann Science Park, NesZiona, Israel). The protein was treated with 1 equiv of dithiothreitol for 20 min at pH 6.0 and then extensively dialyzed. This treatment removes any mixed disulfide form and any other removable protection from cysteine34 of HSA (34). HSA prepared by this procedure contains 0.76 ( 0.03 mol of SH per mol of HSA as determined with DTNB (35). All other materials used in this study were of analytical grade. HPLC analyses were performed using a SpectraPhysics SP-8800 liquid chromatography system with an Applied Biosystems 757 variable wavelength absorbance detector and a Spectra-SYSTEM and P2000 liquid chromatography system with a Spectra-SYSTEM AS100 autosampler and Spectra-SYSTEM UV1000, all controlled by a ThermoQuest chromatography data system

Shechter et al.

(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 Performance RP-18e column (4.6 mm × 100 mm, Merck KGaA, Darmstadt, Germany). The column was eluted with a binary gradient established between solution A (0.1% TFA in H2O) and solution B (0.1% TFA in acetonitrile/H2O; 3:1 v/v). Preparative separations were performed with a prepacked Vydac, RP-18, or RP-4 column (250 mm × 22 mm, Hesperia, CA). Biological Methods and Procedures. Iodination of insulin and of HSA using [125I] iodine was performed using the chloramine-T method (36). Rat adipocytes were prepared from the fat pads of male Wistar rats (100200 g) by collagenase digestion (37). Lipogenesis (during which [U-14C]glucose was incorporated into the lipids) was performed using the procedure of Moody et al. (38). Diabetes was induced by a single intravenous injection of freshly prepared streptozocin (STZ) solution (55 mg/ kg of body weight) as per the method of Meyerovitch et al. (39). Rats were maintained at 24 °C under conditions of controlled lighting and were fed ad libitum. Blood samples for the analysis of blood glucose were taken from the tail veins and measured with a glucose analyzer (Beckman Instruments, Fullerton, CA) by the glucose oxidase method. Groups consisted of five Wistar rats weighing 170 ( 5 g. Data are presented as means ( SE. Synthesis of 9-Hydroxymethyl-2-(amino-3-maleimidopropionate)-fluorene-N-hydroxy-succinimide (2). 9-Hydroxymethyl-2-(amino-3-maleimidopropionate)fluorene (1). 3-Maleimidopropionic acid (1 g, 5.9 mmol) and DCC (0.69 g, 2.95 mmol) were dissolved in 10 mL of DMF and incubated for 4 h. N,N′-Dicyclohexylurea (DCU) was filtered off, and the anhydride thus formed (2.95 mmol) was dissolved in DMF (12 mL) and added to a suspension of 9-hydroxymethyl-2-amino fluorine (36; 0.4 g, 1.9 mmol) and NaHCO3 (0.74 g, 8.85 mmol) in water. The homogeneous reaction mixture was stirred for 40 min, and product formation was monitored by analytical HPLC (Chromolith column, Rt ) 4.46 min, 10-100% B over 10 min with a flow rate of 3 mL/min). The crude product was purified using preparative HPLC (RP-18 column, 10-100% B over 60 min with a flow rate of 12 mL/min). Yield, 57%, 1.08 mmol, 0.39 g. ESMS (calcd) 362.38 Da; ESMS (found) 362.42 Da (electrospray MS analyses were carried out with the Micromass Platform LCZ 4000). 9-Hydroxymethyl-2-(amino-3-maleimidopropionate)fluorene-N-hydroxysuccinimide (2) was prepared according to the procedure of Albericio et. al. (40). Pyridine (0.17 mL, 2 mmol) was added dropwise to a stirred solution of 9-hydroxymethyl-2-(amino-3-maleimidopropionate)fluorene (0.37 g, 1.02 mmol) and triphosgene (0.425 g, 1.43 mmol, 4.2 equiv) in dry THF (10 mL). After 20 min, the precipitated pyridine hydrochloride salt was filtered off, and the THF was removed under vacuum. The oil obtained was dissolved in 10 mL of dry THF containing N-hydroxysuccinimide (0.61 g, 5.3 mmol). Pyridine (0.26 mL, 3.2 mmol) was then added, and the solution was stirred for 20 min. The precipitated pyridine hydrochloride salt was filtered off, and the THF was removed by vacuum. The oil obtained was dissolved in chloroform (100 mL) and washed with an aqueous NaHCO3 solution (0.5 N, 3 × 50 mL), HCl (0.1 N, 3 × 50 mL), water (2 × 50 mL), and brine. The organic phase was dried over MgSO4 and evaporated to give the desired product with a yield of 89% (0.9 mmol, 0.45 g). Analytical HPLC (Rt ) 5.7 min, 10-100% B over 10 min with a flow rate of 3 mL/min) was used to ascertain the purity of the product.

Albumin−Insulin Conjugate Releasing Insulin Slowly

ESMS (calcd) 503.13 Da; ESMS (found) for [M + Na]+ ) 526.38 Da and for [M + K]+ ) 542.30 Da. Preparation of HSA-Fmoc-Insulin. To a stirred solution of Zn2+-free insulin (5.9 mg, 1 µmol in 1.0 mL of 0.1 M phosphate buffer at pH 7.2) was added 505 µg (1 equiv) of MAL-Fmoc-OSu (50.5 µL from a fresh solution of MAL-Fmoc-OSu in DMF, 10 mg/mL). The reaction was carried out for 20 min at 25 °C. HSA (0.37 mL from a solution of 1.6 mM) was then added to a final concentration of 0.42 mM (0.6 mol/mol of insulin). The reaction was carried out for 2 h, and the mixture was then dialyzed overnight against H2O at 7 °C. HSA-Fmoc-insulin was purified from unreacted insulin and from insulin-FmocMAL that had not reacted with HSA by preparative HPLC (RP-4 column, Hesperia CA. 20-100% B over 60 min with a flow rate of 10 mL/min) The fractions corresponding to HSA-Fmoc-insulin, coeluted with HSA, were collected and lyophilized. Preparation of HSA-BENZ-Insulin. This (irreversible) HSA-insulin conjugate was prepared under identical conditions to those used for HSA-Fmoc-insulin except that the hetrobifunctional agent used was m-maleimido-benzoate-N-hydroxysuccinimide ester (MIBNHS). The reagent (32 µL from a fresh solution of MIBNHS at 10 mg/mL) was reacted with insulin for 20 min at 25 °C prior to the addition of HSA (see above). HPLCpurified HSA-BENZ-insulin was characterized by MALDI-TOF MS. A mass of 72.432 kDa was found (calculated mass for 1:1 conjugate is 72.570 kDa). RESULTS

Pharmacological Pattern of Subcutaneously Administered HSA in the Rat. 125I-labeled HSA was administered subcutaneously to rats and the pharmacological pattern as a function of time was constructed by withdrawing blood aliquots at various time points and determining the TCA-precipitable counts for each aliquot. Subcutaneously administered HSA is a long-lived species in the rat circulatory system in vivo. It reached a peak value of 14 ( 1 h after administration and then declined with a t1/2 of 41 ( 2 h (see appendix in Supporting Information). For comparison, we subcutaneously administered 125I-insulin under similar experimental conditions. Insulin reached its peak value at 1 ( 0.1 h and then declined with a t1/2 of 9.0 ( 1.0 h. Thus, subcutaneously administered HSA exhibits a substantially prolonged lifetime in vivo compared to insulin or any other nonglycosylated peptide or protein of molecular mass lower than 17 kDa (reviewed in ref 41). Engineering an HSA-Insulin Conjugate that Releases Insulin under Physiological Conditions. The covalent linking of insulin to HSA with MIB-NHS resulted in a nondissociable HSA-insulin conjugate having negligible biological potencies in vitro (subsequent paragraphs). We therefore designed and synthesized a heterobifunctional agent, consisting of an Fmoc-OSu derivative in which a maleimide group was attached to the flurenyl backbone. The steps involved in the synthesis of MAL-Fmoc-OSu and its structure are illustrated in Figure 1. Using MAL-Fmoc-OSu enabled us to link proteins via their amino side chains to the single cysteinyl residue of HSA. Previously we have found that Fmoc moieties linked to the amino side chains of peptides and proteins undergo slow hydrolysis in aqueous solutions under physiological conditions, generating the unmodified parent peptides and proteins (41, 42). The procedure described in detail in the Methods section was found optimal for coupling equimolar amounts

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Figure 1. Schematic illustration of the synthesis and the structure of 9-hydroxymethyl-2-(amino-3-maleimido-propionate)fluorene-N-hydroxysuccinimide (MAL-Fmoc-OSu). This heterobifunctional agent was used to conjugate HSA to insulin in the preparation of HSA-Fmoc-insulin.

of insulin to HSA. In brief, MAL-Fmoc-OSu is first reacted stoichiometrically with insulin for 20 min at pH 7.2, this being a pH value at which MAL remains chemically stable for several hours (43). Albumin is then added with a stoichiometry of 0.6 mol per mol of derivatized insulin in an attempt to reach a quantitative coupling of insulin-Fmoc-MAL to the cysteinyl-34 of this carrier protein. Unreacted insulin and insulinFmoc-MAL are removed by a semipreparative HPLC procedure. General Features of HSA-Fmoc-Insulin. Table 1 summarizes several characteristic features of the HPLCpurified HSA-Fmoc-insulin that we prepared. It is a highly water-soluble derivative (>200 mg/mL) in buffered near-neutral solutions (pH 6-7) owing to the extreme solubility of the carrier protein (44). MALDI-TOF MS Table 1. Chemophysical Features of HSA-Fmoc-Insulin covalently linked insulina solubility in aqueous buffer (pH 7.0) HPLC analysis retention timea MALDI-TOF mass spectrum analysisb(m/z) molar extinction coefficient

24 ( 3 µg/mg of HSA >200 mg/mL calcd found calcdc found

8.6 min 72.650 kDa 73.189 kDa 280 ) 51.328 0.1% 280 ) 0.706 280 ) 54.400 0.1% 280 ) 0.744

a Determined by HPLC analysis following incubation of the conjugate in 0.1 M Na2CO3 (pH 10.3) for 4 h at 25 °C. Analytical HPLC procedures were carried out under the experimental conditions specified in the legend to Figure 3. Under these conditions, insulin elutes with Rt ) 6.91 min and has a surface area of 187 000 ( 9000 mav/µg of insulin, and HSA (either free or linked to insulin) elutes with Rt ) 8.1 min and has a surface area of 156 000 ( 7000 mav/µg of HSA. b MALDI-TOF MS analyses were carried out with the Bruker-Reflex-Reflection model. The molecular weight of HSA is calculated according to the amino acid composition of this carrier protein (ref 44). c Molar extinction coefficient for HSA-Fmocinsulin was calculated by combining the 280 values of HSA (280 ) 35 280, ref 37), insulin (280 ) 5800), and Fmoc (280 ) 10 250).

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Figure 2. HPLC analysis of purified HSA-Fmoc-insulin before and after the release of insulin by hydrolysis: (A) purified HSA-Fmoc-insulin (100 µg loaded); (B) purified HSA-Fmocinsulin following 4 h hydrolysis through incubation at pH 10.3, 25 °C. HPLC was conducted with a linear gradient from 0 to 100% of 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, using a Chromolith RP 18e (100 mm × 4 mm) column at a rate of 3 mL/min. The effluent was monitored at 220 nm. Under the same experimental conditions, insulin elutes with Rt ) 6.91 min and has a surface area of 187 000 ( 9000 mav/µg of insulin.

analysis revealed a molecular mass of 73.189 kDa (calculated mass for the 1:1 conjugate is 72.650 kDa). Analytical HPLC revealed that HSA-Fmoc-insulin coemerges with HSA as a single symmetric peak (Figure 2; retention time 8.6 min). A molar extinction coefficient of 280 ) 54 400 (0.1% 280 0.744) was found (calculated for 1:1 conjugate 280 ) 51 328). The HSA-Fmoc-insulin thus obtained contains 24 ( 3 µg of covalently linked insulin per mg of HSA as judged by HPLC analysis following the release of the covalently linked insulin from the conjugate by incubation at pH 10.3 for 4 h at 25 °C (Figure 2). In theory, 61 µg of covalently linked insulin per mg of conjugate is expected, based on 0.76 mol of SH per mol of HSA (experimental part). Thus our preparation of HSA-Fmoc-insulin appears to contain also HSA that is not linked to insulin. Biological Potency of HSA-Fmoc-Insulin. Figure 3 shows the dose-response curve for native insulin and for HSA-Fmoc-insulin in a lipogenic assay in rat adipocytes. Based on the value of 24 ( 3 µg of covalently

Shechter et al.

linked insulin per mg HSA in the conjugate (Table 1), HSA-Fmoc-insulin has 12.5% ( 3% of the biological potency of the native hormone (ED50 value ) 2.4 ( 0.1 ng/mL versus ED50 ) 0.3 ( 0.01 ng/mL for insulin, Figure 3). The covalent linking of insulin to HSA using the nonreversible agent MIB-NHS under similar experimental conditions (see Methods) yielded a conjugate having negligible potency (ED50 ) 130 ( 10 ng/mL, being ∼0.2% of the biological potency of insulin). HSA-Fmoc-Insulin Releases Insulin upon Incubation under Physiological Conditions. Incubation of HSA-Fmoc-insulin at pH 10.3 for 4 h at 25 °C causes the covalently linked insulin to be released quantitatively from the conjugate (Figure 2B). In Figure 4A, HSAFmoc-insulin was incubated in 0.1 M phosphate buffer (pH 8.5, 37 °C), and the amount of insulin released from the conjugate as a function of time was quantified by HPLC analysis. At pH 8.5, the rate of Fmoc hydrolysis from Fmoc-protein conjugates is nearly identical to that obtained in normal human serum at 37 °C (41, 42). As shown in the figure, insulin is released from the conjugate in a slow homogeneous fashion, having a half-life of 24 ( 3 h. After 80 and 150 h, the cumulative amount of free insulin reached 71% and 100%, respectively, of the initial HSA-Fmoc-insulin level. Reactivation of HSA-Fmoc-Insulin upon Incubation. In Figure 4B, aliquots were withdrawn from incubated HSA-Fmoc-insulin (pH 8.5, 37 °C) at different time points and analyzed for their biological potencies in a lipogenic assay in rat adipocytes. As shown in the figure, the conjugate undergoes reactivation upon incubation in a nearly linear fashion. Thus, starting from 12% ( 3% at time 0, lipogenic potency is elevated to 31% ( 3%, 43% ( 4%, and 59% ( 5% following 10, 20, and 40 h of incubation, respectively. Upon 150 h of incubation, HSA-Fmoc-insulin regains its full biological potency (Figure 4B). A Single Intraperitoneal or Subcutaneous Administration of HSA-Fmoc-Insulin Facilitates a Prolonged Glucose-Lowering Pattern in Mice. In Figure 5, we compare the glucose-lowering pattern of HSA-Fmoc-insulin (0.4 mg/mouse, corresponding to 9.6 µg of covalently linked insulin) to that of Zn2+-free insulin (3 µg/mouse) following a single administration. For Figure 5A, administration was intraperitoneal, while in Figure 5B, it was subcutaneous. As shown in Figure 5A, HSA-Fmoc-insulin facilitates a prolonged and stable glucose-lowering pattern over a period of 14 h, exceeding

Figure 3. Dosage-dependent stimulation of lipogenesis in rat adipocytes. Lipogenesis was carried out for 2 h at 37 °C in plastic vials containing 0.5 mL of fat cell suspension (1.5 × 105 cells) and 0.2 mM [U-14C]glucose in the presence or absence of the indicated concentrations of native insulin or HSA-insulin conjugates. Results are expressed as a percentage of maximal stimulation. Insulin (100 ng/mL) stimulated lipogenesis 4-5-fold above basal levels. HSA-Fmoc-insulin (1 mg/mL) was taken as containing 24 ( 3 µg of insulin per mg of HSA in this assay (see Table 1). The ED50 values for native insulin (0.3 ng/mL), for HSA-Fmoc-insulin (2.4 ng/mL), and for HSA-BENZ-insulin (130 ng/mL) are indicated with arrows on the figure.

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Albumin−Insulin Conjugate Releasing Insulin Slowly

Figure 4. The rate of insulin release from HSA-Fmoc-insulin and reactivation of the conjugate upon incubation at pH 8.5, 37 °C. A solution of HSA-Fmoc-insulin (1 mg/mL) was incubated in 0.1 M phosphate buffer at pH 8.5, 37 °C. At the indicated time points, aliquots (100 µL) were analyzed by HPLC for the amount of released insulin (A) and for biological potency in rat adipocytes (B). Results are expressed as the amount of insulin released per mg of HSA-Fmoc-insulin. An aliquot of HSAFmoc-insulin exhibiting ED50 value ) 3.0 ng/mL in a lipogenic assay was considered to have 10% of the native biological potency.

by about 4 times the duration obtained by the native hormone. Nearly the same glucose-lowering patterns were obtained following subcutaneous administration (Figure 5B). Again, the conjugate produced a prolonged and stable glucose-lowering pattern over many hours. A noticeable difference, however, is a delay of about 0.5 h until the fall in glucose level following subcutaneous

administration of the conjugate commences (Figure 5B). This suggests that the rate of conjugate diffusion from the subcutaneous compartment into the circulatory system is considerably slower than that from the peritoneum. Indeed, diffusion and transportation rates of subcutaneously administered proteins across capillary membranes are known to decrease in proportion with increasing size of molecular radius (21, 45). Glucose-Lowering Pattern of HSA-Fmoc-Insulin in STZ-Rats. Figure 6 shows the glucose-lowering pattern of the conjugate after a single subcutaneous administration in streptozocin-treated hyperglycemic rats. Here we have compared a conjugate dose (7 mg/ STZ-rat) that is equipotent to the administered dose of Zn2+-free insulin (20 µg/STZ-rat) at the time of administration. The dosage calculation was based on 24 ( 3 µg of covalently linked insulin per mg of HSA having 12% of the biological potency of the free hormone (Table 1, Figure 3). As shown in Figure 6, the glucose-lowering effect of HSA-Fmoc-insulin is about 2.6 times greater than that of the native hormone. A small decrease is seen shortly after administration (i.e., at 0.5 h). Circulating glucose levels then fall gradually, reaching a maximal decrease at 2-3 h after administration (190 ( 20 mg/ dL). Hyperglycemia then reoccurs (t1/2 ) 4.7 h). The area under the curve of the saline-treated group, following HSA-Fmoc-insulin administration exceeds, by about four times, that obtained with native hormone (integrated from Figure 6). Thus, HSA-Fmoc-insulin is considerably more effective than insulin in lowering circulating glucose levels in insulin-deficient diabetic rats. Figure 6 also shows the glucose-lowering pattern obtained by subcutaneous administration of insulin-glargine. Since the latter insulin derivative was documented to be less potent than the native hormone (15), a higher dose (40 µg/STZ-rat) was administered. As shown, this dosage facilitates a glucose-lowering pattern that is nearly similar to that obtained following administration of HSA-Fmoc-insulin (Figure 6). DISCUSSION

Long fatty-acid acylated insulins that are noncovalently associated with albumin, even with moderate affinity (Ka ) 105 M-1), are long-acting species in vivo (23-25). We wondered whether this would be equally valid if an insulin molecule were to be covalently linked to the carrier protein, particularly since HSA-peptide conjugates have been found to retain substantial parentpeptide activity (26-28). Since the linkage of insulin to HSA through the nonreversible linker MIB-NHS yielded

Figure 5. Circulating glucose levels in mice following a single subcutaneous or intraperitoneal administration of HSA-Fmocinsulin. Mice were injected intraperitoneally (A) or subcutaneously (B) with Zn2+-free insulin (3 µg/mouse in 0.2 mL saline) or HSAFmoc-insulin (0.4 mg/mouse). Blood glucose levels were determined at the indicated time points. Food was removed during the experiment. Each point is the arithmetic mean of n ) 5 mice ( SE.

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Figure 6. Effect of a single subcutaneous administration of HSA-Fmoc-insulin and of insulin-glargine on blood glucose levels in STZ-rats. STZ-rats received saline solution (control group), Zn2+-free insulin (20 µg/STZ rat), HSA-Fmoc-insulin (7 mg/STZ-rat), and insulin-glargine (40 µg/STZ-rat). Blood glucose levels were determined at the time points indicated in the figure. Each point represents the arithmetic mean of the blood glucose levels of n ) 5 rats ( SE. Insulin-glargine was diluted to a concentration of 0.2 mg/mL with 1 mM hydrochloric acid prior to injection. Each STZ-rat has received 0.2 mL subcutaneously.

an inactive conjugate (HSA-BENZ-insulin, Figure 3), we designed and synthesized the Fmoc-containing heterobifunctional reagent MAL-Fmoc-OSu (Figure 1). This development enabled us to link insulin (and potentially any other peptide or protein drug) via its amino groups to cysteine-34 of HSA. HSA is likely to be linked to insulin through the R-amino side chain of PheB1 (under investigation). The latter is considerably more exposed to alkylation, than the R-amino side chain of GlyA1 (32). At the low pH used for the conjugation (pH 7.2, methods), the -amino group of LysB29 is fully protonated. Since the linkage between HSA and insulin is fully reversible and the rates of Fmocdetachment from R- and -amino groups are similar as found in our previous studies (41, 42), we expect that each new preparation of HSA-Fmoc-insulin would be similar with respect to its pharmacokinetic behavior. In previous studies, we found that Fmoc moieties linked to proteins are detached in a slow and spontaneous fashion under aqueous physiological conditions generating the unmodified parental proteins (41, 42). The rate of Fmoc hydrolysis is dictated exclusively by the pH, temperature, and protein concentration of the serum, three parameters that are maintained in mammals in strict homeostasis (46). We therefore anticipated that HSA-Fmoc-insulin would yield insulin in a rather similar manner. HSA-Fmoc-insulin is an extremely water-soluble conjugate in which insulin has about 12% of the biological potency of the native hormone (Figure 3) in lipogenesis assay. We had expected it to have negligible activity given that an extended surface area of the insulin molecule is required for receptor binding (32, 33). This activity, however, may stem from release of insulin from its conjugate during the assay, that is, incubation for 2 h at 37 °C in the presence of 20 mg/mL BSA. Indeed, such an explanation would be consistent with the inactivity of the stable covalent conjugate HSA-BENZ-insulin. Yet, upon incubation in aqueous solutions (i.e., pH 8.5, 37 °C),

the covalently linked insulin is released from the HSAFmoc-insulin conjugate in a nearly homogeneous fashion over a prolonged period (t1/2 ) 24 ( 3 h) with the concomitant regeneration of insulin possessing full biological potency (Figure 4A,B). HSA-Fmoc-insulin facilitates prolonged glucose-lowering patterns in mice and in STZ-rats following a single subcutaneous or intraperitoneal administration (Figures 5 and 6). Based on administration of equipotent doses of insulin and of the conjugate at time 0, the glucose-lowering potency of HSA-Fmoc-insulin exceeds 3-4 times that facilitated by insulin in terms of longevity as well as efficacy, as judged by the t1/2 values and the areas under the curves of the saline-treated rodents. Lente and ultralente insulins, as well as insulinglargine, are long-acting formulations following subcutaneous administration, because they are in a crystal form or crystallize in the subcutaneous tissue immediately after injection (2, 14, 15). Slow dissolution at the site of injection brings about the protracted effect. Our HSA-Fmoc-insulin is a soluble insulin preparation that exerts its prolonged glucose-lowering effect by a different principle, namely, by decreasing the clearance rate of the conjugates through the kidneys as well as by protecting the covalently linked insulin from proteolysis prior to its detachment from the conjugate. Therefore, the comparison to suspended formulations of insulin may be only partially representative. Nevertheless, we have also included in Figure 6 the glucose-lowering pattern of subcutaneously administered insulin-glargine, showing its nearly similar pattern to that obtained with HSAFmoc-insulin in STZ-rats (Figure 6). However, it is questionable whether the rat subcutis resembles the human subcutaneous tissue with regard to dissolution and diffusion of insulin-glargine from the subcutaneous compartment to the circulatory system. The issue of potential immunogenicity of HSA-Fmocinsulin deserves special consideration. Previously we found that an insulin derivative containing two rigid,

Albumin−Insulin Conjugate Releasing Insulin Slowly

aromatic Fmoc moieties (Fmoc2-insulin) is less immunogenic than the native hormone (42). Also, multiple injections of Fmoc-KLH conjugate to rabbits failed to yield antibodies against Fmoc (unpublished observations). It therefore appears that Fmoc is a poor antigen. However, because the HSA-insulin chimera is a new species, albeit combining two endogenous detachable proteins, its antigenicity should be carefully evaluated in future clinical studies. In summary, taking insulin as a model, we have presented here the first prototype of an HSA-polypeptide/protein conjugate unique in its capability to release the covalently bound peptide/protein drug with a desirable pharmacokinetic pattern. Short-lived protein/peptide drugs can be turned into conjugates of molecular weight higher than 67 kDa and so resist clearance by kidney filtration (1). The covalently linked peptide/protein drug is then released by Fmoc hydrolysis, which returns it to its active state at a slow rate determined solely by the stable pH, temperature, and protein levels of the circulatory system. This new development is expected to extend the lifetime, bioavailability, and efficacy of existing peptide drugs and to extend the same in peptide drug candidates that may yet be discovered by genomics and proteomics. ACKNOWLEDGMENT

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. We thank Alexandra Singer for editing the manuscript. This research was supported by the Levine Institute of Applied Science and the Howard M. Siegler grant for Diabetes research. Supporting Information Available: Pharmacokinetic profiles of subcutaneously administered radiolabeled insulin and human serum albumin in rats. This material is available free of charge via the Internet at http:// pubs.acs.org. LITERATURE CITED (1) Goodman, L., and Gilman, A. G. (1995) The Pharmacological Basis of Therapeutics, McGraw-Hill, New York. (2) Kahn, C. R., and Shechter, Y. (1990) Insulin, oral hypoglycemic agents and the pharmacology of the endocrine pancreas. In Goodman and Gilman Handbook of Pharmacology (Goodman, A. G., Ed.) pp 1463-1495, Pergamon Press, New York/ Oxford. (3) Duckworth, W. C. (1988) Insulin-degradation: Mechanisms, products, and significance. Endocr. Rev. 9, 319-345. (4) Benzi, I., Cechetti, P., Ciccarone, A., Pilo, A., Di, C. G., and Navalesi, R. (1994) Insulin degradation in vitro and in vivo: A comparative study in men. Evidence that immunoprecipitable, partially rebindable degradation products are released from cells and circulate in blood. Diabetes 43, 297-304. (5) Chap, Z., Ishida, T., Chou, J., Hartley, C. J., Entman, M. L., and Entman, D. (1987) First-pass hepatic extraction and metabolic effects of insulin and insulin analogues. Am. J. Physiol. 252, 209-217. (6) Albert, K. G. M. M. (1981) Insulin treatment and diabetes; half a century of therapeutic misadventure? Advanced Medicine, pp 1-13, Pitman, Bath, United Kingdom. (7) Thow, J. C., and Johnston, A. B. (1989) Effect of raising injection site temperature on isophane insulin crystal dissociation. Diabetes Care 12, 432-434. (8) Lauritzen, T., Pramming, S., Gale, E., Deckert, T., and Binder, C. (1982) Absorption of isophane (NPH) insulin and its clinical implications. Br. Med. J. 285, 159-162.

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