Biopharmaceutical Properties of Uricase Conjugated to Neutral and

poly(N-vinylpyrrolidone) (Mw 6000 Da); (d) poly(N-acryloilmorpholine) (Mw 6000 Da). By intravenous administration to Balb/c mice, the conjugates displ...
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Bioconjugate Chem. 1999, 10, 638−646

Biopharmaceutical Properties of Uricase Conjugated to Neutral and Amphiphilic Polymers Paolo Caliceti,* Oddone Schiavon, and Francesco M. Veronese Department of Pharmaceutical Sciences, University of Padova, Via F. Marzolo 5, 35131 Padova, Italy. Received December 29, 1998; Revised Manuscript Received March 30, 1999

A comparative pharmacokinetic and biodistribution investigation of polymer-protein conjugates prepared with various amphiphilic polymers was carried out using uricase as a model. Four polymeruricase derivatives have been obtained by covalent binding of a similar number of polymer chains of (a) linear poly(ethylene glycol) (Mw 5000 Da); (b) branched poly(ethylene glycol) (Mw 10 000 Da); (c) poly(N-vinylpyrrolidone) (Mw 6000 Da); (d) poly(N-acryloilmorpholine) (Mw 6000 Da). By intravenous administration to Balb/c mice, the conjugates displayed different pharmacokinetic and organ distribution behaviors. (1) The unmodified enzyme and the poly(N-vinylpyrrolidone) conjugate were the enzyme forms with the shortest and the longest permanence in blood respectively (mean residence time 45 and 4378 min). (2) Native uricase was found to localize soon after administration significantly in heart, lungs, and liver from where it was also rapidly cleared. (3) The poly(N-acryloilmorpholine) derivative showed the highest concentration levels in liver (up to 25.5% of the dose) and considerable accumulation took also place in the other considered organs. (4) Poly(N-vinylpyrrolidone)-uricase displayed a relevant tropism for liver but low uptake indexes were found for the other organs. (5) The branched poly(ethylene glycol) derivative accumulated preferentially in liver and spleen. (6) The linear poly(ethylene glycol) conjugate was, among the various uricase forms, the species with the lowest distribution levels in all the examined organs. (7) Finally, all the enzyme forms slowly disposed in kidneys with higher levels for the poly(N-acryloilmorpholine) derivative (15% after 2880 min) and unmodified uricase (14% after 1440 min).

INTRODUCTION

The modification of protein and peptide drugs with soluble biocompatible polymers largely demonstrated to be a successful method to enhance their therapeutic performance. A number of studies report, in fact, that polymer-conjugated proteins usually present, as compared to the native counterparts, prolonged permanence in blood, lowered immunogenicity, improved physical, chemical, and enzymatic stability, and increased solubility. To date, monomethoxypoly(ethylene glycol) (PEG) is receiving the lion’s share in protein modification (1-3) and some PEG-protein conjugates are already in clinical use. However, despite the impressive results obtained with PEG, new synthetic and natural polymers are currently investigated for protein derivatization (3-11) in order to provide for macromolecules that, since their chemical and physical character, may dictate new properties and fate of the conjugate in the body. Indeed the polymeric portion, which often represents the major part of the construct, is more exposed than the protein to the biological environment and, therefore, can influence the interaction with biological structures and dictate body targeting and disposition. Besides polymers designed for a selective recognition of biological receptors such as polysaccharides, antibodies, and neoglycoproteins (12-14), most of the macromolecules devised for protein modification do not mediate specific biological events or selective interactions. Anyhow, also, these polymers may possess tropism for organs * To whom correspondence should be addressed. Phone: 0498275695. Fax: 049-8275366. E-mail: [email protected].

and tissues that reflects in body disposition of their conjugates by passive mechanisms based on diffusion or by endocytosis. Studies reported in the literature point out the effect of charge and molecular mass of macromolecular carriers on organ distribution of the conjugates (15-18). A typical example is the conjugation of an antitumor agent bound to a low molecular mass cationic dextran that showed a rapid disappearance from circulation due to accumulation in liver and spleen. High molecular mass cationic and anionic dextrans also promoted the localization of the conjugates in solid tumors (17, 18). In addition proteins, peptides and antitumor agents covalently modified with negatively charged divinyl ether-maleic anhydride copolymer (DIVEMA) or styrene-maleic anhydride copolymer (SMA) showed tumoritropism, lymphotropism and captation by phagocytic cells (6, 19). Also, uncharged amphiphilic olygomers can passively interact with biological structures stimulating endocytosis (20-24). On the basis of these properties, several polymeric carriers have been developed for lysosomotropic delivery of antitumoral drugs and successful results have been obtained, for example, with poly(hydroxypropyl)methacrylamide and polyaspartamide-poly(oxyethylene) (25-27). Furthermore, poly(ethylene glycol)derivatized proteins have been found to enter endothelial cells as well as blood cells (28, 29), while conjugates of poly(vinyl alcohol) have been found to accumulate in small amounts in various organs (30). All of these evidences indicate that biopharmaceutical properties of the conjugates must be carefully investigated since also slight differences in polymer structure may be reflected on different therapeutic performance of the conjugates (31-33).

10.1021/bc980155k CCC: $18.00 © 1999 American Chemical Society Published on Web 06/02/1999

Biopharmaceutical Properties of Polymer Conjugates

To give a further contribution to understand the influence of the physicochemical and structural properties of neutral macromolecules on the biopharmaceutical properties of the conjugates, four different monofunctional soluble polymers have been considered: poly(N-vinylpyrrolidone) (PVP), poly(N-acryloilmorpholine) (PAcM), linear monomethoxypoly(ethylene glycol) (PEG), and branched monomethoxypoly(ethylene glycol) (PEG2). Uricase, an enzyme of potential therapeutic value in urea clearance, has been used as protein model (34, 35) and a pharmacokinetic and biodistribution investigation of the native enzyme and its polymer conjugates was carried out in mice following intravenous administration. The concentration profiles in plasma, liver, kidney, spleen, heart, and lungs were evaluated and the main pharmacokinetic parameters have been calculated. MATERIALS AND METHODS

Uricase from Candida utilis was obtained from Toyobo (Osaka, Japan). [3H]succinimidyl propionate was from Amersham International (Amersham, U.K.) and Soluene 350, Instagel and Ionic Fluor were from Canberra Packard (Groninghen, The Netherlands). The Superose column and FPLC system were furnished by Pharmacia Biotech (Uppsala, Sweden), and the ultrafiltration system was from Amicon Inc. (Beverly, MA). All other reagents, of analytical grade, were purchased by Fluka (Buchs, Switzerland). Male Balb/c mice, weighing 30 ( 2 g and fed ad libitum, obtained from Charles River (Calco, Italy), were used for “in vivo” experiments. Polymer Preparation and Activation. Linear monomethoxypoly(ethylene glycol), Mw 5000 Da, obtained from Shearwater (Huntsville, AL), was end-carboxylated by introduction of a nor-leucine residue, and the free carboxylic group was activated as succinimidyl ester (36). Branched monomethoxypoly(ethylene glycol), Mw 10 000 Da, was prepared and activated as described in the literature (37). Carboxy-terminating poly(N-vinylpyrrolidone), Mw 6000 Da, and carboxy-terminating poly(Nacryloilmorpholine), Mw 6000 Da, were synthesized, fractionated, and activated according to the methods developed in our laboratory (38, 39). Preparation of Protein-Polymer Conjugates. Polymer activated as succinimidyl ester [15.4 µmol in the case of linear monomethoxypoly(ethylene glycol); 26.95 µmol of branched monomethoxypoly(ethylene glycol); 19.25 µmol of poly(N-vinylpyrrolidone); 23.1 µmol of poly(Nacryloilmorpholine)] was added under stirring to uricase (77 nmol) dissolved in 1 mL of 0.2 M borate buffer and 77 mM uric acid, pH 8.0. The reaction mixture was maintained under stirring at room temperature for 1 h, added to 9 mL of 0.1 M borate buffer, pH 8.0, and ultrafiltered using a membrane with 10 000 Da cut off to reach a volume of 1 mL. The remaining solution was diluted with 5 mL of the buffer reported above, and the ultrafiltration procedure was repeated three times. The ultrafiltered product was further purified by gel filtration chromatography using a Superose 12 preparative column eluted with 10 mM of phosphate buffer and 0.15 M NaCl, pH 7.2. The elution fractions were analyzed by OD at 280 nm for protein evaluation, by iodine assay for polymer determination (40) and by enzyme activity (41). The protein-polymer conjugate peak was concentrated by ultrafiltration. The protein concentration was finally determined by biuret method (42), the degree of modification by a trinitrobenzensulfonic acid colorimetric method (43) and by amino acid analysis on the basis of the nor-

Bioconjugate Chem., Vol. 10, No. 4, 1999 639

leucine content (36). The residual activity was assessed by enzyme assay. Preparation of Tritium-Labeled Proteins. Native or polymer conjugated uricase (77 nmol) in 1 mL of borate buffer, pH 8.0, was added under vigorous stirring to 20 µL of toluene containing 1 nmol of succinimidyl [3H]propionate. After 30 min of reaction, the solution was chromatographied using a Superose 12 preparative column eluted with 10 mM of phosphate buffer and 0.15 M NaCl, pH 7.2. The protein peak was collected and radioactivity was assessed by β-counter and expressed as disintegrations per minute per protein milligram (DPM/protein mg). Mouse serum albumin (144 nmol) was tritium-labeled following the procedure reported above for uricase. The radioactivity of the 3H-labeled derivatives was in the range of 0.6 × 106-0.8 × 106 DPM/mg. Pharmacokinetic and Biodistribution Studies. Animal treatments for the “in vivo” studies were performed according to institutional European guidelines. Five groups of 10 Balb/c male mice (30 ( 2 g) each were separately treated by injection in the tail vein of the tritium-labeled compounds: native uricase, linear monomethoxypoly(ethylene glycol)-uricase, branched monomethoxypoly(ethylene glycol)-uricase, poly(N-vinylpyrrolidone)-uricase, and poly(N-acryloilmorpholine)-uricase (450 µg in 0.1 mL of 20 mM phosphate buffer, 0.15 M NaCl, pH 7.2). The animals were sacrificed at scheduled times, and blood samples, liver, spleen, lungs, heart, and kidneys were taken. The blood samples were put in heparinized vials, centrifuged and the enzyme content in plasma was estimated by enzyme activity and by radioactivity evaluation after addition of 4 mL of Instagel cocktail to 50 µL of plasma. The organs were carefully washed in saline solution, dried on paper, and weighted. Soluene 350 was added to the organs (10 mL/g tissue) and steeped at 100 °C for 2 h. Two hundred and fifty microliters of samples was added to 2.75 mL of Ionic Fluor, and the radioactivity was evaluated by β-counter. Tritium-labeled mouse serum albumin (450 µg in 0.2 mL of sterile saline solution) was injected in the tail vein of 10 mice. The animals were sacrificed and blood samples and organs were taken and processed as described above for the estimation of the residual plasma in the organs. Data Elaboration. The plasma concentration means and the standard deviations ((SD) of the native and conjugated uricase as well as the mouse serum albumin were estimated on the basis of the enzyme activity and the radioactivity obtained from the plasma samples. The pharmacokinetic parameters were determined by computer elaboration of the enzyme concentrations in plasma obtained at the scheduled times according to the equation

C(t) ) Ae-Rt + Be-βt The concentration means and the standard deviations ((SD) of the native and conjugated uricase were determined on the basis of the values of the radioactivity obtained from the organ samples. The residual plasma volume in each organ was estimated by the [3H]MSA levels, and the obtained data were used to correct the disposition levels of the various uricase forms in the organs (22). The organ uptake indexes (CLin) were calculated, on the basis of the standard method reported in the litera-

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Table 1. Protein/Polymer Molar Ratios Used in Conjugate Preparation and Main Properties of Native Uricase, PAcM-Uricase, PVP-Uricase, PEG2-Uricase, and PEG-Uricase protein NH2/polymer molar ratio native uricase PacM-uricase PVP-uricase PEG-uricase PEG2-uricase

extent of modification (% derivatized molecular amino groups) mass (Da)

1:3 1:2.5 1:3 1:3.5

47 43 44 40

130 000 400 000 380 000 350 000 530 000

ture, according to the following expression:

CLin )

T(t)

∫C(t) dt

)

T(t1) AUC0ft1

where T(t1) is the percentage of dose per gram at 15 min and AUC0-t1 is the area under the curve in the range 0-15 min (23). RESULTS

Polymer-Protein Conjugate Preparation. To carry out a comparative study, conjugates at similar molecular mass or degree of modification were prepared with the various polymers. This could be realized using polymers with close molecular masses (in the range 5000-6000 Da) and by attachment of a similar number of polymer molecules to the protein. To overcome the different reactivity of the activated polymers, proper protein NH2/ polymer molar ratios in the conjugation reaction were used. Table 1 reports the conjugate preparation conditions, degree of modification, and molecular mass of unmodified uricase and of the four polymer-uricase derivatives. The table shows that, in the case of PAcM, PVP, and PEG, molecular mass in the range 350000400000 Da were obtained. The higher molecular mass of the PEG2 derivative (530 000 Da) is due to the fact that the reaching of the same degree of modification as the other conjugates was preferred to obtain a close overall mass.

The conjugation reaction was carried out in the presence of uric acid in order to prevent the dramatic loss in enzyme activity that the polymer binding can provoke in the absence of any active-site protection. Following this procedure, the conjugates could maintain enzyme activity in the range 20-80% of the unmodified uricase (44). Pharmacokinetic Studies. The pharmacokinetic behaviors obtained by intravenous injection to mice of native and polymer conjugated uricases are depicted in Figure 1. The time course profiles were obtained by radioactivity determination of the enzyme concentration in plasma. For the radioactivity evaluation few amino groups of native and conjugated proteins were covalently labeled with [3H]propionate, a method that prevents the “in vivo” release of the radioactive probe. Of note to report that in few samples the concentrations were also evaluated by enzyme activity and the values have been found in agreement with those obtained by radioactivity. The patterns in plasma were found to fit a biexponential curve, indicating that the various enzyme forms are cleared from circulation following a bicompartimental pharmacokinetic model. Furthermore the figure shows that, although the performance of the conjugates deeply depends on the polymer, in any case they are eliminated from blood much more slowly with respect to the native enzyme. The pharmacokinetic data reported in Table 2 point out that PVP-UC is, among the various uricase forms, the longest lasting in blood. The mean residence time (MRT) of PVP-UC is in fact about 10-fold of the native uricase, 3-fold of PAcM-UC, and 1.3-fold that of PEGuricase and PEG2-uricase. The β phase half time (t1/2β) and the elimination rate constants (ke) also reflect the slow PVP-UC elimination from the body, followed in the order by PEG-UC and PEG2-UC, PAcM-UC and native uricase. With regard to the distribution phase, the native uricase and the PAcM derivative show comparable R phase half times (t1/2R). These values that are about 10fold shorter than the ones obtained with the other enzyme forms indicate that a rapid distribution process takes place. The high distribution rate constants (k12 and k21) of native uricase and the PAcM derivative confirm

Figure 1. Time course profiles in plasma of radioactivity after intravenous administration to of native uricase (b), PAcM-uricase (O), PVP-uricase (9), PEG-uricase (0), and PEG2-uricase (2); (SD values are reported.

Biopharmaceutical Properties of Polymer Conjugates

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Table 2. Main Pharmacokinetic Parameters Obtained by Intravenous Injection in Mice of Native Uricase, PAcM-Uricase, PVP-Uricase, PEG2-Uricase, and PEG-Uricase A B R β t1/2R (min) t1/2β (min) AUC0- ∞ (µg/mL/min) Cl (mL/min-1) Vc (mL) MRT (min) Vss (mL) k12 k21 ke

native uricase

PAcM-uricase

PVP-uricase

PEG-uricase

PEG2-uricase

190.5 112.9 0.0118 0.0018 58 390 8 × 104 1.9 × 10-4 1.48 466 2.6 1.2 × 10-2 0.34 3.8 × 10-3

163.3 119.6 0.0088 0.0006 79 1190 2.2 × 105 7 × 10-5 1.59 1585 3.18 5.3 × 10-3 0.98 × 10-1 1.3 × 10-3

160.5 88.6 0.001 0.000 17 685 4075 6.8 × 105 2 × 10-5 1.8 4738 3.1 4.7 × 10-6 2.7 × 10-2 3.7 × 10-4

163.8 94.6 0.0011 0.0002 617 3384 6.1 × 105 2 × 10-5 1.74 3926 2.9 1 × 10-5 3.4 × 10-2 4.3 × 10-4

169.3 169.9 0.0033 0.000 27 206 2564 6.8 × 105 2 × 10-5 1.32 3448 2.3 0.5 × 10-3 4.7 × 10-2 5 × 10-4

Figure 2. Disposition profiles in liver of native uricase, PAcM-uricase, PVP-uricase, PEG-uricase, and PEG2-uricase after intravenous administration to mice; (SD values are reported.

the rapid equilibration of these enzyme forms with organs and peripheral tissues. On the other side PVP-UC, PEGUC and PEG2-UC display high t1/2R values and low k12 and k21 supporting that they slowly distributed in the peripheral tissues and slowly reach the equilibrium between blood and other tissues. In consideration of the t1/2R and k12 and k21 values it is therefore possible to state that the peripheral distribution process follows the order native uricase > PAcM-UC > PEG2-UC > PEG-UC > PVP-UC. Finally PVP-UC, PEG-UC, and PEG2-UC show similar high area under the curve (AUC), values indicating their significantly higher systemic availability, with respect to PAcM-UC and native enzyme. Organ Disposition. The uricase accumulation levels in liver, kidneys, spleen, heart, and lungs, obtained after intravenous administration to mice of the native and polymer conjugated enzyme and expressed as a percentage of the dose per gram of tissue up to 48 h from the administration are depicted in Figures 2-6. After this time, low levels of enzyme accumulation have been observed in the tissues with exception of kidneys that displayed a prolonged retention of the various enzyme forms, in particular of PAcM-UC and PEG2-UC. Liver. The distribution profiles reported in Figure 2 show an impressive amount of PAcM-UC in liver. The PAcM-UC disposition is practically instantaneous, and its concentration gradually increases by time to reach the

maximal level of approximately 26% within 5 h. After 24 h from injection, the PAcM-UC concentration drastically drops to 4%. Native uricase, PVP-UC, and PEG2UC also rapidly dispose in this organ and their distribution behaviors are very similar. Soon after administration these enzyme forms exhibit concentration levels ranging between 12 and 16%, but the clearance from this organ takes also place rapidly indicating a low affinity for the hepatic tissue. A different behaviour has instead been observed for PEG-UC that does not show any significant accumulation in liver in comparison to the other uricase forms. Levels below 4% of the dose were in fact found during the experiment. Kidneys. PAcM-UC is the uricase species with the highest affinity for kidneys. Figure 3 shows that the disposition process in this organ is practically instantaneous and the concentration increases with the time reaching the level of about 15% in 5 h, a value constantly retained up to 48 h. The kidney distribution time courses during the first 24 h of native uricase and the PAcM derivative are very similar, but after this time, the native form is rapidly cleared. PVP-UC, PEG-UC, and PEG2UC exhibit lower concentration values with respect to the native enzyme and the PAcM conjugate. Their accumulation is at the limit of detection after 15 min from injection and gradually rises to reach values of 3.3 and 4.8% after 24 h for the PVP and PEG2 derivatives, respectively, and of 5.6 after 48 h for the PEG one.

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Figure 3. Disposition profiles in kidneys of native uricase, PAcM-uricase, PVP-uricase, PEG-uricase, and PEG2-uricase after intravenous administration to mice; (SD values are reported.

Figure 4. Disposition profiles in spleen of native uricase, PAcM-uricase, PVP-uricase, PEG-uricase, and PEG2-uricase after intravenous administration to mice; (SD values are reported.

Spleen. The time courses of distribution in spleen depicted in Figure 4 indicate that PEG2-UC and PAcMUC undergo a rapid accumulation in spleen that reaches the maximal concentration level after 1 h from administration: 4 and 5%, respectively. After this time, the PEG2 derivative concentration is reduced by 2/3, while the PAcM conjugate is cleared much more slowly. Native uricase and PEG derivative dispose in spleen at a lower extent with respect to the PEG2 and PAcM counterparts but with a similar trend, while PVP-UC accumulation is practically negligible. Heart. Figure 5 describes the rapid and remarkable localization in heart of native uricase and PAcM-UC. In particular, the PAcM conjugate maintains constant concentration levels of about 3.5% for up to 5 h from the administration time, whereas the unmodified uricase is quickly cleared from this organ. Only a reduced localization in heart is instead found for PEG-UC and PEG2UC. These conjugates, that present a similar behavior, have concentration levels constantly maintained below 1%. PVP-UC failed to accumulate in heart and its concentration levels are practically undetectable.

Lungs. The distribution patterns in lungs depicted in Figure 6 show that native uricase, PAcM-UC, and PVPUC are rapidly localized in lungs and rapidly cleared from it. A high level of native enzyme is found after 15 min (13%). PEG-UC and PEG2-UC show, instead, a different disposition trend in comparison to the other uricase forms. Their concentrations gradually increase with time reaching a level of about 3% within 24-48 h. Organ Uptake Index. Table 3 reports the organ uptake rate indexes that quantitatively define the differences in organ translocation of the various uricase forms. Native uricase and PAcM-UC display, in the examined organs, the higher uptake rate values according to their consistent tissue disposition already observed in Figures 2-6. Interestingly the liver uptake of PAcM-UC is 1.3fold of the native uricase one, while the latter presents higher lungs and heart uptake indexes. PVP-UC is rapidly taken up only by liver, which is the organ involved in polymer elimination (45). The data of the table point out a rapid translocation of the PEG2-UC in liver and spleen, two organs rich of reticulo-endothelial

Biopharmaceutical Properties of Polymer Conjugates

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Figure 5. Disposition profiles in heart of native uricase, PAcM-uricase, PVP-uricase, PEG-uricase, and PEG2-uricase after intravenous administration to mice; (SD values are reported.

Figure 6. Disposition profiles in lungs of native uricase, PAcM-uricase, PVP-uricase, PEG-uricase, and PEG2-uricase after intravenous administration to mice; (SD values are reported. Table 3. Organ Uptake Rate Indexes Obtained with Native Uricase, PAcM-Uricase, PVP-Uricase, PEG2-Uricase, and PEG-Uricase CLin (µL/g/h)

liver kidneys spleen heart lungs

native uricase

uricasePAcM

uricasePVP

uricasePEG

uricasePEG2

816 624 128.4 441 958

1056 762 241.6 255 493

749 45 19.8 2.79 43.9

83.4 9.6 108 46.7 9.7

532 0.9 120.2 0 41.7

cells, while the linear PEG does not present significant index values in any studied organ. These results are in agreement with the enhanced uptake by Kuppfer cells observed with high molecular mass poly(ethylene glycol)s (23). DISCUSSION

The present study was aimed at evaluating the influence of different neutral amphiphilic polymers on biopharmaceutical properties of polymer-protein conju-

gates. This is of primary importance in the choice of the polymer for protein modification because polymers with apparently similar physicochemical properties can affect in different way the biopharmaceutical properties of the conjugates. Uricase was chosen for this investigation since its therapeutic value in the treatment of hyperuricemiaassociated diseases such as nephropathologies and haematological malignancies and poly(ethylene glycol) and dextran conjugates have been recently prepared in order to overcome its considerable immunological and pharmacokinetic character (15, 34, 35). The four polymers used in this study were selected because of their relevance in the preparation of protein conjugates for medical use. Indeed they have the prerequisites for medical use and possess similar properties such as good solubility in both aqueous and organic solvents, absence of charge, molecular shape, mass, and monofunctionality in reaction. Furthermore, previous studies carried out in our laboratories already demonstrated that monofunctional PVP, PAcM, and PEG2 provide for an excellent alternative to the most used linear PEG in protein modification (37-39, 46-48).

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The biopharmaceutical investigation reported here clearly indicates that the protein conjugation with these polymers yields derivatives with prolonged permanence in blood, although different clearance from circulation and organ distribution behaviors have been obtained. With regard to the permanence in blood, we can say that the increased residence time of the conjugates, with respect to the native counterpart, can only partially be ascribed to reduction in renal ultrafiltration due to their high molecular volume, as often reported with other proteins. Native uricase “per se” has in fact a high molecular mass (128 000 Da) and Stokes’ radius (42.7 Å) that overcome the renal filtration limit and therefore its main elimination route is by hepatic route (15). The organ disposition behaviors and uptake indexes of native uricase suggest that its rapid disappearance is by localization in organs, a behavior that is certainly followed by the uricase conjugates also. In particular, the high disposition of native uricase in reticulo-endotelial cell rich organs such as liver and lungs seems to suggest that its elimination takes place by phagocytic processes, followed by biliary excretion and/or degradation to low molecular mass peptides that can be cleared with urine. It is therefore worth noting that the changes in protein organ disposition determined by the polymer binding are accompanied by enhancing the uricase permanence in blood. In addition, the resistance of the conjugates to proteolytic digestion and to recognition by the immunosystem already observed in our laboratories (44) also contribute to prevent the uricase conjugate disappearance from circulation. In this regard it may be of interest to remember the agreement between the enzyme activity and radioactivity levels in blood of the conjugated uricase a behavior that is in favor of the stability in blood of the conjugates despite their long permanence in it. This behavior points out that the protein degradation to inactive long circulating fragments does not take place. If we now examine the “in vivo” performance of the four polymer conjugates, we can observe that PAcM promotes significantly the organ accumulation of the derivative in the main organs, in particular in liver, spleen, and kidneys. The high uptake rate indexes of these organs, and especially of liver, explains its rapid disappearance during the distribution phase (R phase). Nevertheless, the conjugate has a considerable higher permanence in blood (β phase) compared to the native enzyme probably due to a reduced degradation and a slow release from the tissues that guarantee sustained levels in blood. For what the PVP derivative is concerned its long permanence in plasma reflects the low levels of tissue translocation. The conjugate in fact disposes significantly only in liver, in agreement with the data reported in the literature that indicate the hepatic uptake by endocytosis of this polymer as the main PVP elimination route (24). Interesting results have been obtained with the PEG and PEG2 derivatives that show very different accumulation profiles in the examined organs. According to the literature data, the linear PEG derivative does not show specific localization in peripheral districts, and low amounts of its conjugate are found in all the tissues (23). On the other hand, the PEG2 adduct accumulates at significant extent in liver and spleen, two organs rich of reticulo-endothelial cells. These results suggest that the branched shape of this polymer can affect the cell/ conjugate interaction process and the higher accumulation of the PEG2 derivative is probably related to the higher molecular volume of this conjugate that, with respect to the linear one, can more efficiently stimulate the cell phagocytic process.

Caliceti et al.

The reasons that are at the basis of the differences in organ disposition among the conjugates are difficult to be understood. Differences in stability to proteolytic enzymes of the various enzyme forms, observed in “in vitro” experiments, seem not to be relevant in determining the disposition profiles of the various enzyme forms since all of them were found do not degrade in the experiment time. We can instead suggest that peculiar lipohilic/hydrophilic properties of the polymers can play a significant role in the interaction with the organ cell membranes indicating the importance of their amphiphilic character. CONCLUSIONS

A careful choice of the polymer used for protein modification allows for controlling the “in vivo” fate of the conjugates. However, charge and molecular mass are not the only parameters playing a role in dictating the biopharmaceutical properties of the conjugates as often reported in the literature. The data reported here indicate that neutral polymers, with apparently similar physicochemical properties but different chemical composition and architecture, can confer to the conjugates peculiar biopharmaceutical properties. Therefore, the assumption that the covalent conjugation of uncharged and relatively low molecular mass polymers to proteins avoids the organ disposition of the conjugates, based on the low disposition properties of the most studied PEG derivatives, must be carefully reconsidered. This suggests the possibility to use uncharged amphiphilic polymers for preparation of tailored conjugates that can preferentially accumulate in organs where their therapeutic effect is expected or from where they can be slowly released in the active form in order to ensure sustained concentrations in plasma. Furthermore, in addition to the therapeutic value of the conjugates, also their toxicological aspect must be considered. Accumulation of the conjugates in organs where they can house for a long time can in fact represent a serious limit of their application, also in consideration of the usual high stability to degradation processes of the conjugates that prevents their elimination. However, it is important to observe that the non biodegradable polymers studied here do not induce any irreversible accumulation of the conjugates in organs that could cause toxicity problems. LITERATURE CITED (1) Delgado, C., Francis, G. E., and Fisher, D. (1992) The uses and properties of PEG-linked proteins. Crit. Rev. Ther. Drugs Carrier Syst. 9, 249-304. (2) Veronese, F. M., Caliceti, P., Schiavon, O., and Sartore, L. (1991) In Poly(ethylene glycol): chemistry, biochemical and biomedical applications (J. M. Harris, Ed.) Plenum Publication Corp., New York. (3) Kartre, K. (1993) The conjugation of proteins with poly(ethylene glycol) and other polymers. Adv. Drug Deliv. Rev. 10, 91-114. (4) Caliceti, P., Schiavon, O., Hirano, T., Ohashi, S., and Veronese, F. M. (1996) Modification of physicochemical and biopharmaceutical properties of superoxide dismutase by conjugation to the polymer of divinyl ether and maleic anhydride. J. Controlled Release 39, 27-34. (5) Ito, Y., Kotoura, M., Chung, D. J., and Imanishi, Y. (1993) Bioconjugate Chem. Trypsin modification by vinyl polymers with variable solubilities that responses to external signals, 4, 358-361. (6) In Neocarzinostatin: the past, present and future of an anticancer drug (1997) (H. Maeda, K. Edo, and N. Ishida, Eds.) Spinger-Verlag, Tokyo.

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