Bioconjugafe Chem. 1995, 6, 459-465
459
Vitamin BIZMediated Oral Delivery Systems for Granulocyte-Colony Stimulating Factor and Erythropoietin G. J. Russell-Jones,*!*S. W. Westwood,$ a n d A. D. Habberfields Biotech Australia Pty Ltd., P.O. Box 20, Roseville, NSW 2069, Australia, and Amgen Inc., Amgen Center, Thousand Oaks, California 91320. Received March 17, 1995@
As a prelude to the development of orally active erythropoietin (EPO) and granulocyte-colony stimulating factor (G-CSF), conjugates have been formed between these molecules and vitamin B12. During the formation of these conjugates intramolecular cross-linking of the proteins was avoided by the use of hydrazidyl derivatives of vitamin B12. A potentially biodegradable linkage was formed between vitamin B12 and G-CSF by reaction of the buried thiol in G-CSF with a long chain dithiopyridyl derivative of vitamin B12. In vitro and in vivo testing of the conjugates showed that their bioactivity was substantially maintained and that they were actively transported in an intrinsic factor dependent fashion across CaCo-2 cells and from the intestine to the circulation in a biologically active form.
INTRODUCTION
G-CSF (granulocyte-colony stimulating factor) and EPO (erythropoietin) represent two of the most exciting molecules to emerge out of the recent developments in recombinant DNA technology. G-CSF has been shown to be a powerful stimulator of neutrophil production in humans and has found application in the stimulation of neutrophils in cancer patients, thereby reducing the period of neutropenia after conventional chemotherapy (Bronchud et al., 1987; Golde & Gasson, 1988; Morstyn & Burgess, 1988). It also has application in the treatment of chronic neutropenia. EPO, on the other hand, stimulates the maturation of erythroid progenitor cells into mature erythocytes, and is used for the treament of anemia in kidney dialysis patients (Krantz & Goldwasser, 1984). Despite the enormous therapeutic potential of these two proteins, their use is limited by the fact that they must be administered parenterally to patients, as the proteins are not active following oral administration. The inability of these molecules to be efficacious orally stems primarily from their inability to pass through the villous epithelium of the gastrointestinal tract (GIT). Therefore, even if methods could be found to protect these proteins from proteolysis within the GIT, they would be excluded from entering the circulation by the cell membrane of the intestinal enterocyte, which forms a n almost impenetrable barrier to the uptake of all but the smallest of molecules. Thus, in common with virtually all proteins, peptides, and other large bioactive molecules, there is currently no method for the oral delivery of either G-CSF or EPO. Recently, a delivery technology has been described
which overcomes the impenetrable barrier that the small intestinal ileocytes present and which could potentially enable orally administered compounds, such as G-CSF and EPO, to pass from the intestinal lumen, across the ileocyte, and into the circulation. This technology is based upon the natural uptake system for vitaminBlz (VB~Z), or cyanocobalamin (Cbl). VBlz itself is an unusually large vitamin (MW 1356),which is too big to be taken up from the intestine by means of simple diffusion. Instead VBlz is taken up from the intestinal lumen by receptor-mediated endocytosis. In this process VBlz must first be bound by intrinsic factor (IF) produced in the stomach. The [IF-VB121 complex then binds to an I F receptor located on the lumenal surface of the ileocyte, which stimulates internalization of the VBIZand subsequent transcytosis of the vitamin across the ileocyte (Robertson & Gallagher, 1985; Gallagher & Foley, 1971; Baillant et al., 1990; Simpson et al., 1993).Russell-Jones and co-workers (Russell-Jones & Aizpurua, 1988; RussellJones, 1994) have found that it is possible to covalently link peptides and proteins to VBlz and have demonstrated that these molecules are cotransported from the intestinal lumen to the circulation with V B l z following oral administration. In order for the transport system to be effective it is important that during the conjugation of V B l z to the peptides/proteins care is taken to preserve the bioactivity of both the peptide and the V B l z to which it is coupled. In this paper we describe methods for the conjugation of VBlz to the two protein therapeutics, G-CSF and EPO, in such a manner as to preserve the binding affinity of V B 1 2 for IF while preserving the biological activity of these two protein therapeutics. MATERIALS AND METHODS
* Corresponding author:
Biotech Australia Pty Ltd., P.O. Box 20,Roseville, NSW 2069,Australia. Biotech Australia Pty Ltd. 5 Amgen Inc. Abbreviations: G-CSF, granulocyte-colony stimulating factor; EPO, erythropoietin; GIT, gastrointestinal tract; Cbl, cyanocobalamin; V B 1 2 , vitamin BIZ; IF, intrinsic factor; EDAC. HC1, l-ethyl-3-[(dimethylamino)propyllcarbodiimide; SPDP, succinimidyl3-(2-pyridyldithio)propionate;RP-HPLC, reversedphase high-performance liquid chromatography; DTP, dithiopyridyl; DSS, disuccinimyl suberate; eVB12, e-carboxylate isomer of V B 1 2 ; PBS, phosphate-buffered saline. Abstract published in Advance ACS Abstracts, July 1,1995.
*
@
VBlz was obtained from Rousell-Uclaf (Paris, France). G-CSF and EPO were obtained from Amgen Inc. Manufacturing. l-Ethyl-3-[(dimethylamino)propyllcarbodiimideHC1 (EDAC-HC1)was obtained from Bio-Rad (Richmond, CAI. Succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and succinimidyl 6-[3-(2-pyridyldithio)propionamidolhexanoate (LC-SPDP) were obtained from Pierce Chemical Co. (Rockford, IL). All other reagents were obtained from Fluka (Buchs, Switzerland). Synthesis of V B l z Reagents and General ConjugatiodCharacterizationF’rotocols. (A) Production
1043-1802/95/2906-0459$09.00/0 0 1995 American Chemical Society
460 Bioconjugafe Chem., Vol. 6,No. 4, 1995
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Scheme 1
la lb
IC Id le 11
lg Ih li lj Ik 11
lm In 10
R = R = R = R = R = R = R = R I R =
R
I
R = R E R =
R = R =
Figure 1.
and purification of the e isomer of monocarboxy-VB,, (eVB12). Native V B l z ( l a ) (Figure 1)contains no suitable site for conjugation to either EPO or G-CSF. For conjugation, a carboxylic acid group can be introduced into the molecule by mild acid hydrolysis of one of the three propionimide side chains of the corrin ring. Following hydrolysis (0.4 M HCl, 72 h, RT), the e isomer of monocarboxy vitamin Blz (lb) (eVB12, e isomer; Anton and co-workers, 19801, was separated from the b and d isomers, also formed during acid hydrolysis, by a combination of Dowex AG 1-X2 (Bio-Rad) chromatography and semipreparative C-18 RP-HPLC (using a gradient of 5-100% acetonitrile in 0.1% TFA). (B) Production of Amino Derivatives of eVB12. Five amino derivatives of eVBlz were prepared by reacting the e isomer with 1,2-diaminoethane, 1,6-diaminohexane, 1,lPdiaminododecane, 1,3-diamin0-2-hydroxypropane, and 1,6-diamino-3,4-dithiahexane(a.k.a. cystamine) to give amino VBlz derivatives IC,Id, le, lf, and lg, respectively. All reactions were performed a t pH 6.5 using a 20-fold molar excess of the diamine over e isomer and a 20-fold molar excess of EDAC. In a typical reaction 135 mg of eVBl2 was dissolved in distilled water (6 mL) to which was added 1.2 mL of 1.0 M diamine, pH 6.5. Dry EDAC (270 mg) was then added, and the reaction mixture was left overnight a t room temperature. All amino derivatives were purified by reverse-phase chromatography on a semipreparative C-4 column using a gradient of 5-100% acetonitrile in 0.1% TFA. Eluted material was further purified by S-Sepharose chromatography. Non-amino VI312 derivatives were removed by washing the column with water, and the amino derivatives were subsequently eluted with 0.1 M HCl, followed by extraction into phenol, and back-extraction into water aRer the addition of dichloromethane to the phenol phase. The amino eVBlz derivatives were then recovered from the water phase by lyophilization. The derivatives were
pure (>98%)by analytical RP-HPLC analysis. Ion spray MS characterization data: (IC)obsd M+ 1398, calcd M+ 1398; (Id) obsd M+ 1454, calcd M+ 1454; (le) obsd M' 1598, calcd M' 1598; (If)obsd M' 1428, calcd M' 1428; (lg) obsd M+ 1490, calcd M' 1491. (C) Preparation of 3-(2-Pyridyldithio)propionamido Derivatives of Amino-eVBlz. Three dithiopyridyl (DTP) amino-eVBlz derivatives were prepared by reacting SPDP with 2-aminoethyl-eVB1z (IC),6-aminohexyl-eVBlz(Id),and 12-aminododecyl-eVB1z(le)to give the respective derivatives (lh),(li)and (lj). In a typical reaction the terminal amino-eVBlz was dissolved a t 50 mg/mL in 0.1 M Po4 buffer, pH 7.5, containing 0.1 M NaC1. SPDP was dissolved a t 50 mg/mL in acetone, and 800 pL of the solution was added to the amino-eVBlz. After reaction overnight a t room temperature the DTPamino-eVBlz product was purified by RP-HPLC on a semiprep C-4 column and then lyophilized. Ionspray ms characterization data: (lh)obsd M+ 1595, calcd M+ 1596; (li) obsd M+ 1652, calcd M' 1652; (lj) obsd M+ 1736, calcd M+ 1736. (D) Preparation of a Long-chain Analogue of the DTP-aminododecyl-eVB12Reagent. This spacer was prepared from 6-aminohexyl-eVBlz (Id) by sequential reaction with disuccinimidyl suberate (DSS) (to give [[(monosuccinimidyl)suberyllhexyll-eVBl~) and 1,12-diaminododecane to give [[(12-aminododecyl)suberyllhexylleVBlz (lk). The resultant spacer, which is more than twice the length of le, was derivatized a t the terminal amino group with SPDP, purified on RP-HPLC, and lyophilized to give derivative 11. The synthesis is outlined in Scheme 1. Ionspray ms characterization data: (lk) obsd M+ 1793, calcd M+ 1793; (11) obsd M+ 1990, calcd M' 1990. (E)Production of Hydrazide Derivatives of eVBlz Carboxylate. Three hydrazide derivatives of eVBlz carboxylate were prepared for conjugation to carboxyl groups of G-CSF by reaction with EDAC. The hydrazide derivatives used, and their (shorthand) chemical structures, were hydrazido-eVB12 (eVB12-CONHNH2) (lm), Cys-hydrazidoaVBlz(eVBlz-CONHcCHz)zSS-(CH2)2NHCO(CH2)zCONHNHz) (In), and (adipyl hydrazido)-eVBlz (~VB~~-CONHNHCO(CHZ)~CONHNHZ) (lo). HydrazidoeVBlz (lm)was prepared by a two-step synthesis involving the coupling of tert-butyl carbazate to carbox-
Conjugation of Vitamin BI2 to G-CSF and EPO Scheme 2
1
1. HzNNHBOC / EDAC 2.TFA
9
ylate and subsequent removal of the t-Boc group to generate the free hydrazide. Cys-hydrazido-eVBlz was synthesized from eVBlz cystamine (lg). The conversion of this material to eVBlz Cys-hydrazide (In) proceeded by succinylation of the eVBlz cystamine and subsequent conversion of the resultant terminal carboxyl group to a hydrazide by the procedure outlined above for eVB12 hydrazide. This synthesis is outlined in Scheme 2. The (adipylhydrazido)-eVBlz reagent (lo) was readily prepared in one step from eVBlz carboxylate and a 20-fold excess of adipylhydrazide by the addition of EDAC. Ionspray MS characterization data: (lm)obsd M+ 1370, calcd M+ 1370; (In) obsd M+ 1604, calcd M+ 1604; (lo) obsd M+ 1512, calcd M+ 1512. (F) Determination of VBl2-Protein Substitution Ratios. The U V absorbance of a n aqueous conjugate solution was measured at 361 and 278 nm. Absorbance a t 361 nm is only due to V B 1 2 . The concentration of cobalamin in solution was calculated from the literature value for its 361-nm absorbance, assuming that the cobalamin conjugated to protein has the same absorbance as native V B ~ Z . Protein concentrations were subsequently calculated from the 278-nm absorption after subtraction of the contribution to the 278-nm absorption due to cobalamin. Amino acid analysis of all V B l z conjugates was performed on a n Applied Biosystems amino acid analyzer. Protein concentrations measured from W absorbance agreed (&lo%)with the concentrations determined by amino acid analysis. (G) Analysis of VB12Conjugates via SDS-PAGE and Western Blotting. Both EPO and G-CSF conjugates were analyzed by SDS-PAGE according to the method of Laemmli (1970) using 12.5% and 17-20% SDS-PAGE minigels, respectively (ISS Daiichi minigels). A method was developed a t Amgen for the detection of VBlz in western blots using a modification of the method of Russell-Jones and co-workers (Russell-Jones & Gotschlich, 1984; Blake et al., 1984). Briefly, proteins were transferred from gels to Millipore Immobilon-P membranes (Millipore, Bedford, MA). ARer blocking with 10% goat serum in PBS, the VBlz-containing bands were developed using a primary monoclonal antibody of mouse anti-VBlz(Sigma, St. Louis, MO), followed by a goat antimouse biotinylated second antibody (Sigma). An Extravidin alkaline phosphatase conjugate (Sigma) was then added, after which the VBlz-containing. bands were visualized using 5-bromo-4-chloroindolyl phospate and nitroblue tetrazolium. Formation of VB12-G-CSF Complexes: General Protocol for Purification of IVB12-G-CSFl coqjugates. [VBIZ-G-CSFI conjugates were separated from
Bioconjugate Chem., Vol. 6, No. 4, 1995 461
unreacted VBlz and other reagents by size-exclusion chromatography on Sephadex G-50 in 2.5% acetic acid. Fractions containing [VBlZ-G-CSFl were pooled, concentrated by membrane filtration (Amicon YMlO membrane), and dialyzed (4 "C) for a t least 20 h against sterile distilled water. Aliquots were removed for amino acid analysis, IF assay, and spectroscopic and HPLC analysis. The structures of the various conjugates are shown in Table 1. Synthesis of disulfide-linked G-CSF conjugates. (A) Conjugation of D"P-amino-eVBlzDerivatives to G-CSF. In preliminary experiments with G-CSF it was found that it was not possible to modify the free cysteine (Cys-17)in G-CSF with standard thiol-modifying agents. Initial experiments with DTP-ethyl-eVBlz, however, showed that it was possible to achieve some 20% substitution of G C S F with the VBlz in the absence of guanidine and that this level rose to '80% in the presence of 4 M guanidine. It was therefore decided that it might be possible to access the free thiol with DTP-amino-eVBlz in the absence of guanidine if a longer spacer was used for the conjugation. In a second series of experiments G-CSF was reacted with DTP-aminoethyl-, DTP-aminohexyl- and DTP-aminododecyl-eVB12 in the presence or absence of 4 M guanidine in 0.1 M sodium acetate buffer, pH 4.0. The degree of substitution of G-CSF by various DTP-amino-eVBlz analogues is shown in Table 3. As it was possible to conjugate to G-CSF using DTP-dodecyleVBlz in the absence of guanidine, the reaction was scaled up as follows: To 2.5 mL of G-CSF (6 mg/mL; 15 mg) was added 1.6 mL of DTP-dodecyl-eVBlz (10 mg/mL in 2.5% acetic acid). The reaction was allowed to proceed for 48 h a t 4 "C, after which the unreacted V B l z was separated from the conjugate by the standard purification protocol. The resultant conjugate, GBC-1, was stored at 4 "C prior to analysis in bioactivity studies. Another disulfide-linked eVB1z-G-CSF conjugate containing a long-chain hydrocarbon spacer (Table 1) between VBlz and G-CSF was formed by the reaction of G-CSF with DTP-[(12-dodecylsuberyl)hexyll-eVBl~ (prepared as described above) using the method described. The resultant conjugate, LC-GBC1, was purified in the usual fashion. (B) Synthesis of Amide-Linked G-CSF Conjugates. In order to prepare conjugates between aminoeVBlz derivatives and GCSF, it was necessary to perform the conjugation reaction a t an acid pH, as G-CSF was found to aggregate quickly as the pH of the reaction mixture was raised above pH 7.0. Two amide-linked VB1z-G-CSF conjugates were prepared: GBC-2, by reaction of cystaminyl-eVBlz with G-CSF; and GBC-3, by with G-CSF. reaction of 3-amino-2-hydroxypropyl-eVBl~ Typically a solution of amino-eVBl2 (26.5 mg, 18 pmol) in 2 mL of G-CSF (6 mg/mL, 0.63 pmol) was cooled to 4 "C. An aliquot of freshly prepared EDAC solution (100 mg/mL, 120 pl, 63 pmol) was added. After 24 h at 4 "C a second aliquot of freshly prepared EDAC solution was added. The reaction was allowed to proceed for a total of 48 h a t 4 "C, after which the unconjugated amino-eVBlz derivative was separated from the conjugate and aggregate by chromatography on Sephadex G50 in 2.5% acetic acid. (C) Synthesis of Acyl Hydrazide-Linked G-CSF Conjugates. In order to reduce the level of aggregation of the G-CSF found when amino-eVBlz derivatives were coupled to G-CSF using EDAC, it was decided to use the more reactive hydrazidyl derivatives of V B 1 2 , which would enable the reaction to be carried out a t a lower pH and with lower quantities of the carbodiimide. Three VBlz-hydrazido-G-CSF conjugates were synthesized:
462 Bioconjugafe Chem., Vol. 6,No. 4, 1995
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Table 1. Structures of VBlz-G-CSFand VBla-EPO Conjugates
Conjugate
SPACER
Name GBC-1
LCGBCl
GBC2
GBC3
GBC4
GBCS
I
0
GBC6 0
GBC-4, by reaction of hydrazido-VBlz with G-CSF; GBC5, by reaction of (cystaminylhydrazidol-VBlz with G-CSF; and GBC-6, by reaction of (adipylhydrazido)-eVBlz with G-CSF. In a typical synthesis a solution of the VBIZhydrazide (10 mg) in 4 mL of G-CSF solution (4 mg/mL, 0.84 pmol) was cooled to 4 "C and a n aliquot of EDAC solution (50 mg/mL, 40 mL, 10 pmol) was added. After 5 h an identical aliquot of fresh EDAC solution was added, and the reaction mixture was left overnight a t 4 "C. Conjugate was removed from unreacted VBlz and other reagents by chromatography on Sephadex G-50 in 2.5% acetic acid. Preparation of VB1z-EPO Complexes. VB12-EP0 complexes were prepared by conjugation via an amide linkage, formed by EDAC-mediated coupling of an aminoeVBlz derivative to carboxyl groups on EPO such as the C-terminus of EPO, the carboxylate side chains of the Asp/Glu residues, or the sialic acid residues of the carbohydrate portion of EPO. Second, conjugates were formed via an acyl hydrazide linkage, between hydrazidoeVBlz and the carboxylate side chains of the AspIGlu residues of EPO or the carboxylate groups of the sialic acid residues of the carbohydrate portion of EPO. Finally an attempt was made to form a hydrazone linkage between a hydrazido-eVB12derivative and an aldehyde group generated by periodate oxidation of the carbohydrate residues of EPO. (A) Synthesis of Amide-Linked VB12-EPO Conjugates. Two amide-linked conjugates between aminowere formed: EBC-1, by reaction of 2-aminoethyl-
0
VBIZ with EPO; and EBC-2, by reaction of (6-aminodithiahexyl)-eVBlz with EPO. In a typical reaction, a mixture of amino-eVBlz (8 mg, 5.7 pmol) and EPO (27 mg/mL, 200 pL, 0.18 pmol) was cooled to 4 "C and a n aliquot of EDAC solution (10 mg/mL, 100 pL, 5 pmol) was added. The reaction mixture was left for 64 h a t 4 "C and finally purified by size-exclusion chromatography on a Superdex-75 column. Elution with a buffer consisting of Tris (pH 7.5, 10 mM)/NaCl (100 mM) afforded the purified VB12-EPO complex. (B) EDAC-Mediated Conjugation of eVBlz-hy. drazides to EPO. Two VBl2 -hydrazide-EPO conjugates were synthesized; EBC-3 by reaction of hydrazidoeVBlz with EPO; and EBC-4, by reaction of (adipylhydrazido)-eVBlZ with EPO. Briefly, a solution of the eVBlz hydrazide (10 mg, 7.3 pmol) in 7 mL of EPO solution (2.6 mg/mL, 0.6 pmol) was cooled to 4 "C and a n aliquot of EDAC solution (20 mg/mL, 50 pL, 5 pmol) was added. After 5 h a second aliquot of fresh EDAC solution (10 mg/mL, 25 pL, 1.3pmol) was added, and the reaction mixture was left overnight a t 4 "C. The conjugate was purified by size-exclusionchromatography on a Sephadex G50 column. Elution with a buffer consisting of Tris (pH 7 5 1 0 mM)/NaCI(lOO mM) afforded the purified EPOVBlz complex. RESULTS AND DISCUSSION
The most desirable linkage between V B l z and G-CSF would be a linkage that could be cleaved in serum to produce native G-CSF once it had been transported from the intestine into the circulation. Such a linkage could
Conjugation of Vitamin BI2 to G-CSF and EPO
Bioconjugate Chem., Vol. 6, No. 4, 1995 463
Table 2. Structures of EPO Conjugates
Conjugate
I
SPACER
Name
I
EBc-l
0
Table 3. Substitution of G-CSF with DTP-eVB12 Derivatives* spacer DTP-aminoethyl DTP-aminohexyl DTP-aminododecyl
-guanidine (%) 37.5 45.5 100.0
+guanidine (%) 89.3 95.2 100.0
a G-CSF was reacted with DTP-aminoethyl-, DTP-aminohexyland DTP-dodecyl-eVBl2 in the presence or absence of 4 M guanidine in 0.1 M sodium acetate buffer, pH 4.0. After 24 h the degree of substitution of G-CSF by various DTP-amino-eVBlz analogues was determined following RP-HPLC of the conjugates.
be achieved by the formation of a disulfide bond with the free (but buried) thiol of Cys-17 in G-CSF. The disulfide bond would potentially be cleaved in serum to regenerate the native G-CSF and free thiolated VBIZthrough the reducing action of serum glutathione. Initial attempts to modify the Cys-17 thiol using standard thiol-modifying agents such a s Ellman's reagent proved unsuccessful. Subsequent experiments, using various DTP derivatives of VBlz in the presence or absence of guanidine, showed that it was in fact possible to achieve significant levels of modification of this thiol even in the absence of guanidine when a suitably long hydrophobic spacer was attached to the V B l z (Table 3). It can be seen that the initial attempts to conjugate to the free thiol group in G-CSF using the DTP-aminoethyl derivative of eVBlz resulted in a small degree of conjugation, around 2040% in the absence of guanidine. The addition of 4 M guanidine (final concentration) raised the conjugation efficiency to over 80% (Table 3). Preparation of a longer, more hydrophobic derivative of VBIZ,DTP-dodecyl-eVBlz, resulted in 100% substitution of G-CSF after 24 h a t 4 "C,without the need for the addition of guanidine. The use of the thiol interchange chemistry in this reaction proved advantageous, as the VBIZ conjugation was surprisingly successful a t the pHs required to minimize the extent to which G-CSF undergoes spontaneous aggregation. The recent publication of Arakawa and co-workers (1993) has subsequently shown that Cys-17 is partially solvent-exposed and shows differential reactivity with sulfhydryl-modifying reagents. One of the most elegant aspects of this chemistry was the observation that the thiol insertion reaction which was required to form the disulfide-linked conjugate between G-CSF and VBIZ(to form GBC-1) could be performed a t high efficiencies a t
Table 4. IF A f f h i t y and VBl2-Substution of VBlz-G-CSF Conjugates In vitro in vivo VB12:G-CSF IF a f h i t y bioactivity bioactivity* conjugate ratio (%I (%I (%I GBC-1 0.97:l 2.6 11.2 61 LC-GBC-1 1:l 23 8.5 66 GBC-2 0.8:l 2.8 31 85 GBC-3 0.8:l NDc ND 29 3.4:l 4 18 85 GBC-4 GBC-5 ND 15 78 100 GBC-6 1.6:l 4 24 ND a The in vitro bioactivity was assessed using the stimulation of mitogenesis, as assayed by the incorporation of L3H1thymidine into primary cell cultures of mouse bone marrow cells (JensenPippo, 1995). The in vivo bioactivity of the VB12-G-CSF conjugates was assessed in Syrian hamsters. Briefly, vehicle and two doses of G-CSF or VB12-G-CSF conjugate (20 and 100 p g k g ) were administered as single S.C. injections. Blood samples were take a t 12, 24, 48, and 72 h, and the total white blood cell count was determined at each point. The percent activity of the conjugates was calculated from the 48-h median area under the curve analysis. The affinity of IF for the various [VBlz-protein] conjugates compared to the affinity of IF for VBl2 was determined in a competitive binding assay. Dilutions of the conjugate were mixed with 1ng of 57C0VBlz (Amersham). One IU of IF was then added and the mixture was incubated for 20 min. at room temperature before the addition of a suspension of 5% activated charcoal in 0.1% BSA (IF and V B ~ f r e eSigma). , Samples were centrifugated and the relative number of counts in the supernatant (IF bound) and pellet (free 57C0-VB12)were used to determine the relative affinity of the material tested for IF. ND Not determined.
*
very low pH (pH 2-3) thereby enabling the G-CSF to remain fully soluble during conjugation and workup. The resultant conjugate (GBC-1)had good in vivo bioactivity but low IF binding ability (2-3% of native) (Table 4).The loss in IF affinity of the VB12-G-CSF conjugate was presumably due to the close proximity of the VBIZ molecule to G-CSF, thus sterically interfering with the ability of IF to bind to V B 1 2 (Table 4). In order to increase the IF affinity of the disulfide-linked conjugate, a conjugate with increased IF affinity was prepared by synthesizing a longer chain analogue of the DTP-(dodecylamino)-eVBlz. The synthesis of this analogue, LC-GBC 1, used the same SPDP chemistry to conjugate through the cysteine of G-CSF; however a longer spacer arm was attached to the V B 1 2 . By increasing the length of the cross-linker still further, another thiol-linked conjugate,
464 Bioconjugate Chem., Vol. 6,No. 4, 1995
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Table 5. IF Affinity and V B l z Substution of VBlz-EPO Conjugates conjugate EBC-1 EBC-2 EBC-3 EBC-4
VB~Z:ECSF ratio 1.14:l 0.7:l 1.6:l 1.8:l
IF affinity (%'a) 3.2 NDb 5.9 11.4
in vivo bioactivity (%) 3.4 ND 22 17
aThe in vivo bioactivity of the VBlz-EPO conjugates was assessed in the exhypoxic polycythemic mouse model of Cotes and Bangham (1961). Briefly, female BDFl mice were maintained under hypobaric conditions of 0.4 atm for 18-24 hiday for a total of 14 days. Following the hypobaric exposure the mice were brought up ro ambient pressure for 72 h prior to testing with the VBlZ-EPO conjugates. EPO or VBlZ-EPO conjugates were injected i.p. at 0 and 24 h. At 48 h the mice were injected via the tail vein with 200 pL of 0.9% NaC1, 0.3% trisodium citrate, and 0.5-1.0 pCi of 59FeC13.After a further 48 h the mice were sacrificed with COz and weighed. Blood was collected to determine haematocrit volume and percent incorporation of 59FeC13into erythrocytes. ND, not determined.
LC-GBC1, was formed which showed a 10-fold increase in IF affinity (Table 4). Thus, increasing the length of the spacer joining the V B l z to the G-CSF from 6.8 to 15.6 produced a conjugate in which the i n vivo bioactivity (65%) of the G-CSF was preserved, but with a higher afinity for IF (23%)(eVB12,25%)than GBC-1(2.3%).The functional group most commonly used for formation of protein-protein conjugates is the amino group. Studies a t Amgen (unpublished observations) had previously shown that modification of amino groups on G-CSF quickly leads to inactivation of the molecule. Thus, crosslinking with traditional amino-reactive spacer molecules was not attempted. Initially amide-linked conjugates were prepared between G-CSF and VI312 by activating carboxyl groups on G-CSF using the carbodiimide, EDAC, and reacting the activated ester with cystamido- and [(hydroxypropyl)amido]-eVB12derivatives. The two conjugates so formed (GBC-2 and GBC-3, respectively) showed variable i n vivo bioactivity (85 and 29.5%, respectively); however, the yields of the conjugates were low due to unacceptably high levels of aggregation of the G-CSF during the conjugation. Conjugates were therefore prepared using hydrazido-eVB12 derivatives. Three conjugates were prepared between G-CSF and hydrazidoem12 derivatives. EDAC-mediated coupling of hydrazidoeVBlz analogues to the carboxylate side-chains of G-CSF proceeded more readily, and required significantly lower amounts of VBlz derivative and EDAC, than conjugations of the corresponding amino VBlz derivatives to G-CSF. This is readily explainable in terms of the relative 2.6) in comparison with basicity of hydrazides (pK, amines (pK, 8-9). Thus at the pH a t which the G-CSF coupling takes place (-4-5) a hydrazido V B l z derivative would be primarily in the reactive, non-protonated form, while an amino VBlz derivative will be primarily in the nonreactive, protonated form. The hydrazido derivatives (GBC-4, hydrazido-; GBC-5, Cys-hydrazido and GBC-6; adipyl-hydrazido-1 proved to be much more reactive a t the low pHs required for the maintenance of G-CSF solubility; therefore much less EDAC could be used during conjugate formation, thereby reducing the level of dimer and trimer formation during conjugation. GBC-4 and GBC-5 were both found to have good i n vivo bioactivity with moderate affinity for intrinsic factor (4% and 15%respectively; Table 3). The slight increase in spacer length from GBC-4 to GBC-6 did not result in any significant improvement in IF affinity (4%), although it did increase the in vitro bioactivity of the conjugate slightly (from 18% to 24%; not significant in this assay). GBC-1, GBC-2, and GBC-4 were incubated with IF and
a
-
-
tested in transport studies in CaCo-2 cell cultures. All three were transported in an IF-dependent fashion. Several of the VB12-G-CSF conjugates, namely, GBC-1, LC-GBC-1, and GBC-4, were also tested in rat duodenal uptake studies and found to be actively transported from the duodenum to the circulation. (Habberfield et al., 1995). Two classes of VBIZ-EPO conjugates were prepared by the reaction of the carbodiimide, EDAC, with the carboxyl groups of the C-terminus of EPO, the Asp/ Glu residues, or the sialic acid residues of the carbohydrate portion of EPO. Two amide-linked conjugates, EBC-1 and EBC-2 were prepared using 2-aminoethylrespectively. eVBlz and (6-amino-3,4-dithiahexyl)-eVB1~, The use of the amino derivatives during this conjugation strategy required high levels of EDAC for efficient conjugation and thus resulted in considerable dimer formation. Although the mono and di forms of EBC-1 and EBC-2 were separable by SEC, it was decided to prepare similar conjugates using hydrazido derivatives of VBlz rather than the amine derivatives. Two conjugates, EBC-3 and EBC-4, were prepared using hydrazido and adipylhydrazido derivatives of VB12. Conjugates formed in this fashion were vastely superior to those formed with the amino derivatives of VB12, as they had low levels of dimer formation and good bioactivity in the hypoxic mouse model and maintained good IF binding activity (Table 4). EBC-4 has subsequently been found to be actively transported into serum following intraduodenal infusion in rats (Habberfield et al., 1995). Deglyocosylation of the VBlz-EPO conjugates showed that in all cases the VBIZwas linked to Asp/Glu residues and not to the sialic acid groups. SUMMARY
For the successful formation of bioconjugates between two molecules of disparate functional activities, care must be taken to maintain the bioactivities of each of the molecules within the complex. Procedures are described for the formation of stable conjugates between V B l z either and G-CSF or EPO which have the potential to be taken up from the intestine following oral administration by the normal VBl2-uptake mechanism. Through the choice of appropriate chemistries and spacers it was found that it was possible to form conjugates which maintained significant affinity for intrinsic factor, while maintaining substantial bioactivity of G-CSF and EPO when tested i n vivo. Successful conjugation was achieved between V B l z and a buried thiol within G-CSF. The linkage so formed had the potential to regenerate the intact, unaltered protein in serum. Other studies have shown that several of the conjugates were transported in an intrinsic factor dependent fashion across CaCo-2 cells, and also from the intestine to the circulation in rats in a biologically active form. EPO and G-CSF which were not conjugated to V B l z were not transported in these systems. ACKNOWLEDGMENT
The authors would like to thank Angela Jarvis a t BA for her many IF assays and Angela Phillips, also a t BA, for the synthesis of many of the v B l 2 derivatives used in the conjugate preparations. The authors would also like to thank Kathleen Jensen-Pippo and LLoyd Ralph for their characterization of the conjugates a t Amgen. LITERATURE CITED Anton, D. L., Hogenkamp, H. P. C., Walker, T. E., and Matwiyoff, N. A. (1980) Carbon-13 nuclear magnetic studies of the
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