Bioconjugate Chem. 1883, 4, 262-267
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Functional Peptide-Polyurethane Conjugates with Extended Circulatory Half-Lives James A. Braatz,*J Yoshiaki Yasuda,t Kenneth Olden,$ Kenneth M. Yamada,ll and Aaron H. Heifetzf W. R. Grace & Company-Conn., Research Division, Department of Mammalian Cell Research, 7379 Route 32, Columbia, Maryland 21044, Department of Anatomy, School of Medicine, Kumamoto University, Kumamoto 880, Japan, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, North Carolina 27709-2233, and Laboratory of Developmental Biology, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892. Received January 12, 1993
Peptides containing the RGD sequencewere covalently attached to an isocyanate-containingpolyurethane prepolymer and the biological properties of the complexes were evaluated. For the pentapeptide HzNTyr-Arg-Gly-Asp-Ser-OH (single-letter code, YRGDS), polymer conjugation lead to an increased halflife in the blood circulation of mice to >10 h. The effect of covalent attachment of polymer on biological Ala-Cys-OH (GRGDSPAC). activity was examined in a related peptide, HzN-Gly-Arg-Gly-Asp-Ser-ProInhibition of B16-FlO melanoma cell attachment and spreading on plates coated with fibronectin by an RGD-containing peptide and polymer-conjugated GRGDSPAC was observed to occur at similar concentrations. We conclude that the conjugation of peptides containing the Arg-Gly-Aspmotif to this polymer resolves previous problems of their rapid loss from the circulation, while still allowingretention of full biological inhibitory activity.
INTRODUCTION Synthetic polymers can be covalently attached to biological molecules, leading to an alteration of their biological properties which could render them useful for a variety of applications. For example, polymer conjugates with drugs have been described that retain activity at lower doses (Bamford et al., 1987). Deferoxamine (DFO),which is used clinically to chelate iron and aluminum, but with known toxic effects, has been coupled to dextran and starch (Hallaway et al., 1989). The DFO-polymer was shown to retain metal binding capacitywhile displaying an extended plasma half-life and lower toxicity. This lowering of drug toxicity while potency is maintained would be of great benefit for cancer chemotherapeutic agents. In one such study, daunomycin linked to acrylamide copolymers through a degradable linkage was more effective than the native drug in extending mean survival times in mice inoculated with L1210 leukemia cells (Duncan et al., 1988). Similar conjugation with adriamycin decreased its toxicity against hematopoietic precursors in bone marrow while generating a negligible antibody response to the conjugates (Rihova et al., 1989). The antitumor activity of 5-fluorouracil conjugates with chitin and chitosan was demonstrated against P388 lymphocytic leukemia in mice at doses which were not acutely toxic (Ouchi et al., 1989). The effect of polymer coupling to larger molecules has been examined. Polyethylene glycol (PEG) chains attached to enzymes modulate their activities in proportion to the extent of substitution, and the effect is also dependent on the molecular weight and functionality of the PEG used (Yoshinaga et al., 1987). The coupling chemistry also affected the activity of the conjugates (Yoshinaga et al., 1989). Modification of enzymes with PEG has also been found to render certain enzymessoluble + W.
R. Grace & Co.-Conn.
* Kumamoto University. 8 National Institute of 11 National Institute of
Environmental Health Science. Dental Research. 1043-1802/93/2904-0262$04.00/0
and active in a variety of organic solvents (Takahashi et al., 1985, 1986, Kodera et al., 1986). In addition, conjugation of water-soluble polymers, primarily polyethylene glycols, to proteins leads to alterations of their immunological properties (Abuchowski et al., 1977; Davis et al., 1979). There are numerous reports showing the effects of polymer conjugation on the clearance, immunogenicity, and activity of a variety of other proteins including immunoglobulins(Torchilin et al., 1987; Andersen et al., 1988), superoxide dismutase (Tamura et al., 1988; Beckman et al., 1988; Galcia-Alveset al., 1989), hemoglobin (Iwasaki et al., 1986), uricase (Tsuji et al., 1985; Fujita et ai., 1990), and interleukin-2 (Katre et al., 1987;Knaufet al., 1988). Clinical trials using polyethylene glycol-modifiedtherapeutic enzymes including asparaginase, superoxide dismutase, adenosine deaminase, and uricase are currently under evaluation (Fuertes and Abuchowski, 1990). This technology may prove useful in providingmore effectivetreatment for a number of diseases while at the same time reducing the adverse side effects associated with these therapies. The successful use of polyethylene glycols prompted us to evaluate a series of isocyanate-based poly(ether urethane) prepolymers for their effects on certain properties of RGD-containing peptides. It was previously demonstrated that RGD-containing peptides could inhibit pulmonary metastasis of B15-FlO melanoma cells when administered simultaneously with the tumor cells in the tail veins of mice (Humphries et al., 1986, 1988). The simultaneous administration was required, at least in part, because of the rapid rate of clearance of the small peptide from the circulation. Studies with radiolabeled peptides showed that 75 7% of the peptide GRGDS was eliminated from the circulation within 2 min after injection, while the remainder was cleared with a half-life of 8 min. I t would clearly be advantageous to have a more extended window in which to conduct studies such as these. The possible therapeutic potential of these peptides would also be improved considerably. The prepolymers used here have a polyether backbone and a branched structure terminating in multiple isocy0 1993 American Chemical Society
Bioconjugate Chem., Vol. 4, No. 4, 1993 203
Peptide-Polyurethane Conjugates
Chart I
R,NCO R,NH, CH3
I
dH3
/
CH3
anate groups, having an active group (NCO), functionality of 13. These trifunctional prepolymers appeared worthwhile to investigate in this context, as it has been shown that, in certain cases, enzymes retain more of their activity when modified with bifunctional as opposed to monofunctional PEGS (Bamford et al., 1987). These prepolymers could be used directly for modification of peptides, proteins, drugs, or other molecules which have an accessible group capable of reacting with isocyanates. We have found interesting effects on the properties of small peptides using these prepolymers in relatively straightforward coupling reactions. These effects are the subject of this report. EXPERIMENTAL PROCEDURES
Materials. HYPOL XP-5 (formerly BIOPOL XP-5, from Hampshire Chemical Co., Lexington, MA) is a member of a family of poly(ether urethane) prepolymers prepared by end capping a branched polyether poly01with a diisocyanate (Braatz et al., 1992). A representative structure for these prepolymers is given in Chart I. In this structure, R can be H or CH3. When R is H, the bracketed unit is ethylene oxide (EO), when R is CH3, it is propylene oxide (PO). The number average molecular weight of the polyether triol component of the prepolymer is approximately 7000, and is composed of 75% EO and 25 % PO units. Capping of the terminal hydroxyl groups of the polyether triol with isophorone diisocyanate (IPDI) produces the isocyanate-terminated prepolymer. However, during the capping reaction the isocyanate-terminated prepolymers can form dimer and higher oligomeric forms, producing a heterogenous prepolymer composition which is evident when analyzed by size-exclusion HPLC with dry organic solvent as mobile phase. The profile would appear nearly identical to that shown in Figure 1, panel A, between 12 and 22 min. The prepolymer is soluble in aqueous solution and reccta with water to form a hydrogel after a period of time that depends on the amount of polymer dissolved. The reaction with water proceeds as follows: 1 Abbreviations used: NCO, isocyanate; PEG, poly(ethy1ene glycol);PBS, 0.01 M sodium phosphate, pH 7.4, containing 0.15 M NaC1; THF, tetrahydrofuran.
+ H,O
+ R,NCO
-
R,NH,
+ CO, (slow)
R,-urea-R, (very fast)
(1) (2)
The first reaction, conversion of isocyanate to a primary amine, is relatively slow. However, once the amine is formed, it reacts rapidly with an isocyanate group to form a urea cross-link. As this reaction proceeds, a threedimensional cross-linked hydrogel structure will form. If, however, an amine-containing compound such as a peptide is included in the aqueous mixture, the following reaction can also occur:
+
-
R,NCO peptide-NH, R,NHCONH-peptide (3) in which the peptide forms a urea linkage to the polymer. Since the amines react with isocyanate much faster than water, peptide can preferentially react with polymer to cap the termini of the prepolymer and prevent polymerpolymer cross-linking. This is evident from Figure 1,panel A, since, as mentioned above, the profile of the reaction product between prepolymer and peptide/water resembles the prepolymer in composition. Peptides were obtained from Bachem Inc., Torrance, CA (YRGDS) and Peninsula Laboratories, Inc., Belmont, CA (GRGDSPAC). Iodine-125 (low pH) was obtained from New England Nuclear, Boston, MA. Disposable, prepacked Sephadex PD-10 columns were from Pharmacia LKB Biotechnology,Piscataway, NJ. Spectrapor dialysis tubing, 6000-8000 MW cutoff,was from Spectrum Medical Industries, Inc., Los Angeles CA. All chemicals used were the best grades available commercially. Methods. Preparation and Radiolabeling of PolymerTyramine. To 5.0 g of Hypo1 XP-5 dissolved in 5.0 g of tetrahydrofuran (THF) was added 0.51 g of tyramine base (2 mol/equiv of NCO) dissolved in 5.0 g of THF + 30 mL of 2-propanol. After 17 h at room temperature, approximately 43% of the added tyramine became associated with the polymer, as determined by size-exclusion HPLC analysis. A portion of this reaction mixture was prepared for radioiodination by diluting 0.1 mL of this solution with 1 mL of 0.01 M sodium phosphate, filtering through a 0.45-pm filter, evaporating to dryness under a stream of nitrogen, and then redissolving in 0.05 mL of water. This conjugate was radioiodinated using Na1251(Hunter and Greenwood, 1962) and the free and bound iodine were separated using a PD-10 column with PBS as the eluant. In a separate experiment the iodination reaction was repeated using nonradioactive iodine in order to characterize the product. Size-exclusion HPLC analysis of the iodination product demonstrated that no significant alteration of the product's composition resulted from iodination. Preparation and Radiolabeling of YRGDS-Polymer. A 5 mg/mL solution of YRGDS was prepared in PBS. To 0.3 mL of this solution (1.5 mg, 2.5 X 10-6 mol) was added 0.3 mL of 200 mg/mL HYPOL XP-5 (6.0 mg, 2.4 X lo4 equiv of NCO) in 2-propanol. Product formation was monitored by size-exclusion high-performance liquid chromatography on a system which included a 9.5 mm X 25 cm Zorbax GF-250 column, a Hewlett-Packard 1050 quaternary pump and a Waters 990 photodiode-array detector. The mobile phase was 0.05 M sodium phosphate, pH 7.0, containing 0.3 M NaC1, which was run at 30 OC and 0.5 mL/min. Conjugates were detected by their absorption at 210 nm. Iodination of the YRGDS-polymer and separation of free and bound iodine were performed as described above for the tyramine-polymer conjugate. Iodination of YRGDS was performed as described above,
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Bbmnjugate Chem., Vol. 4, No. 4, 1993
but the separation of free from bound iodine was done over a PD-10 column using water in place of PBS as the eluant. Clearance of Peptide-Polymer from the Circulation. C57BL6 mice were injected with 0.2 mL of labeled polymer conjugates and the rate of clearance from the circulation was determined as previously described (Humphries et al., 1988),but with frequent sampling of blood during the first distribution phase. The percent remaining at each time point was based on the injected dose. Preparation and Testing of GRGDSPAC-Polymer. To 80 mg of GRGDSPAC (1.05 X lV mol) in 4.0 mL water were added 0.025 mL of 0.35 g/mL N-methylimidazole (1.05 X 10" mol) and 2.0 mL of 115mg/mL HYPOL XP-5 (230 mg, 1.0 X lo4 equiv NCO) in 2-propanol. This solution was incubated for 6 h at room temperature then 17 h at 4 "C. The solution was dialyzed for 24 h at 4 "C vs 1 L of water with four changes. Ellman analysis was performed to measure free thiol before and after the conjugation reaction (Riddles et al., 1979). The activity of this conjugate was determined by measuring inhibition of B16-FlO melanoma cell spreading on fibronectin as previously described (Yamada and Kennedy, 1984,1987).
0.5
A
A.
0
.
10
20
TIME,
RESULTS
The conjugation of small peptides with an isocyanatecontaining polyurethane prepolymer can proceed in a buffered aqueous solution since nucleophilic groups on the peptide react much faster than water. However, since water is present at a much higher concentration, substantial side reaction with the solvent will occur. Figure 1,panel A, is an HPLC profile of the product of the reaction between YRGDS and prepolymer. The major peak at 24 min corresponds to free YRGDS peptide. The complex series of peaks between 14 and 20 min is a typical profile for the prepolymer, which consists of a monomeric species eluting at about 19 min preceded by several smaller peaks at earlier retention times that correspond to higher oligomeric forms of the polymer. This profile demonstrates the distribution of polymer-peptide conjugate and unreacted peptide in a reaction mixture which contained approximately equimolar amounts of reactive groups. From the relative areas of these peaks, the yield of the reaction was estimated to be 46 % The contaminating free peptide was removed by gel permeation on a PD-10 column. The HPLC profile of the partially purified conjugate is shown in panel B of Figure 1. In this case the free peptide at 24 min has been essentially eliminated. This conjugate was radioiodinated and the labeled free and polymer-conjugatedpeptides were separated on a PD10 column, as shown in panel C of Figure 1. The material excluded from the PD-10 column, fractions 3-5, Figure lC, was used as the labeled conjugate for subsequent experiments. The radiolabeled polymer-peptide conjugate was compared to the unmodified pentapeptide for their rates of clearance from the circulation. When injected iv into a mouse, YRGDS was cleared with a half-life of approximately 30 s in the first phase and 2.7 h in the second phase. The first- and second-phase clearance half-times for the polymer conjugate, on the other hand, were 0.5 and 11.3 h, respectively, as shown in Figure 2. In addition to the above clearance study, the behavior of the polymer labeled with a simpler reporter molecule was examined. Excess tyramine was reacted with the isocyanate groups of the prepolymer to provide a labeling center. The radioiodinated form of this product when injected iv into mice had a half-life of approximately 7 h. This clearance pattern is also shown in Figure 2.
t
.I
30
40
MINUTES
'-1
FRACTION
NUMBER
Figure 1. High-performancesize-exclusion chromatography of YRGDS-HYPOL XP-5 conjugate: (A) HPLC of product before dialysis, (B)HPLC of product after dialysis, (C)PD-10 column fractionation after radioiodination of material from panel B.
The coupling reaction with YRGDS likely occurred with the primary amine of the peptide. With another peptide, GRGDSPAC, reaction with isocyanate may likewise occur at sites other than the terminal amine. For this peptide it was desired to link the polymer on the carboxyl terminal end in order to preserve the structure around the RGD sequence for subsequent activity analysis. To accomplish this, N-methylimidazole was added to the reaction to facilitate attachment to the sulfhydryl group. An indication that this in fact occurred is given in Table I, which shows the loss of a sulfhydryl group after conjugation. As with the pentapeptide, HPLC analysis demonstrated incomplete conjugation of peptide (Figure 3, panel A, free peptide) to polymer (Figure 3, panel B, unreacted peptide at 24 min, peptide-polymer conjugate at 14-20 min). The estimated yield for this reaction based on relative area in the HPLC profile was 35%. However, as shown in Figure 3, panel C, the free peptide in this case could be removed effectively by dialysis. The effect of coupling of this polymer on the biological activity of this peptide is shown in Figure 4. The GRGDSPAC-polymer conjugate was found to be as effective, on a molar basis (see Discussion), as the unconjugated octapeptide in blocking peptide-mediated attachment of B16-FlO melanoma cells to a surface.
Bioconlugate Chem., Vol. 4, No. 4, 1003 265
Peptide-Polyurethane Conjugates
I
O
I
.
~
~
S
O
L
~
U
U
U
P
I
I
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HOURS AFTER INJECTION Figure 2. Circulatory clearance rates of native and polymerlinked DentaDeDtid.8: (0) . . PIlYRGDS, _ . to) PIIYRGDSpolyme;, (A)l"41tyramine-polymer.
Table I. Peptide Sulfhydryl Content before and after Attachment to Polymer SH content, sample
moVmol of peptide
GRGDSPAC GRGDSPAC-polymer
