Procesamiento de imágenes

The attachment of poly(DL-alanine), a non-immunogenic (Davis et al., 1991) synthetic amino acid polymer, to asparaginase (from Erwinia carotovora and

Escherichia coli) leads to a conjugate with increased heat stability and resistance to proteolysis (Uren & Ragin, 1979). Modified asparaginase of either of the two sources was completely resistant to tryptic digestion and about five-fold more resistant to chymotrypsin. Both enzymes suffered considerable loss of activity upon modification (between 35 and 80%) but the Km remained unchanged.

Native E. coli and E. carotovora asparaginase exhibited in intraperitoneally injected mice, half-lives of 3 and 5 h respectively compared to those of 20 and 36 h observed for the poly-(DL-alanine) derivatives. In intravenously injected rats, modified asparaginase showed a biphasic clearance (ti/2a= 4 h; ti/2P=13 h) compared to a monophasic one for the native (ti/2=1.5 h). The improved residence of poly-(DL- alanine)-asparaginase in the blood circulation was accompanied by a longer depletion of the plasma asparagine. However, only the preparations with a half-life greater than 24 h showed superior therapeutic activity (as compared to that of native asparaginase) against L5178 murine leukaemia. In addition, the immunogenicity and antigenicity of asparaginase in mice was reduced by conjugation to poly-(DL-alanine). In mice with low antibody titers to native E. coli asparaginase, the modified enzyme circulated for extended periods (although its half-life was shorter than observed in the intact animal) but was rapidly cleared from the plasma of highly immune animals. Native

asparaginase in immune mice was short-lived, independently of the antibody titers (Uren & Ragin, 1979).

1.5.3 Dextran

Dextran is routinely used as a plasma expander in man and its low toxicity is well established (Wileman et al., 1983; Wileman et al., 1986). Table 1.3 shows some of the proteins that have been modified with dextran and the respective circulating half- lives as compared to the native counterparts. In every case (except for uricase), the dextran-enzyme’s blood residence was markedly improved by a factor of between 5 and 71. The half-life of the adducts usually increased with increasing size of the dextran. However, dextran of extremely high molecular weight may not be as efficient as one with a lower molecular weight (Wileman et al., 1986). The circulation times of asparaginase-dextran conjugates compared particularly well with the native enzyme in pre-immune mice (Table 1.3). Catalase-dextran conjugates attained maximum plasma activity levels 4.5 h after intraperitoneal injection, as compared to the 2 h shown by the native enzyme (Marshall et al., 1977). This probably reflects the decreased ability of the conjugate to extravasate from the peritoneal cavity, as anticipated from the increased size.

Natural glycoproteins are usually more stable than non-glycosylated proteins (Davis et al., 1991; Marshall, 1978) and the enzyme-dextran conjugates also show improved stability. The neoglycoproteins were more resistant to heat, exogenous proteases (e.g. trypsin and chymotrypsin) and protein dénaturants (e.g. urea) (Marshall, 1978). Asparaginase-dextran conjugates showed considerable resistance to trypsin and stability increased with the molecular weight of the dextran (Wileman et al., 1986).

Table 1.3 Circulating half-lives o f proteins modified with dextran o f different molecular weights. When half-lives were not reported the % o f protein in circulation at a certain time is given instead. Results were obtained on first injection; * indicates half-lives obtained after repeated dosing and (a) and (p) denote that half-lives were respectively calculated from the a and P phases o f the pharmacokinetic profiles. Subscripts denote the molecular weight o f the dextran: 10000, 40000, 70000 and 250000 Da.

w

Os

Protein Source Route Animal

Native

Circulating half-life

Modified Fold increase

Reference

a-Amylase Bacillus IV Rat 2 h: 16% 2 h: 75% 5 Marshall et al., 1977

amylol iquefaciens

Asparaginase Erwinia carotovora IV Man 12 h l i d ® 2 2 Wileman et al., 1983

Erwinia carotovora IV Rabbit I l h 190 h 17 Benbough et al., 1979

Erwinia carotovora IV New Zealand 8 h 46 h ® 6 Wileman et al., 1986

white rabbits 56 h ® 7

36 h ® 5

<0 .1 h* 1 .6 h ® * 16

1.7 h ® * 17 7.1 h ® * 71

Catalase Bovine liver IV Acatalasémie 17 min (a) 140 min (a) 8 Marshall et al., 1977 mice

Uricase ? IV ddY mice 25 min (a) 3 1 min (a) 1.2 Fujita et al., 1990 3.5 h(P ) 2 . 3 h ( p f < I

Activation of dextran by periodate oxidation produces dialdehyde-dextrans that are responsible for intra and intermolecular cross-linking between enzyme and polysaccharide. Dextran multipoint attachment thus results in a rigid conformation that, together with the change in the protein’s degree of hydration, is believed to account for the increased stability of the dextran conjugates (Marshall, 1978).

Table 1.4 Immunogenicity and antigenicity o f dextran conjugated enzymes

Dextran-protein Test system /Animal (route)

Reference Asparaginase Precipitin

Antigenicity

Reduced Wileman et al., 1986 Superoxide

dismutase

Immunodiffusion Reduced Miyata et al., 1988 Asparaginase New Zealand

white rabbits (IM)

Immunogenicity

Abohshed Wileman et al., 1986 Superoxide

dismutase

1

Mice (IP) ^ 1 Increased Miyata et al., 1988

^^^o antibodies were detected either against the enzyme or the dextran moieties.

The immunological properties of dextran conjugates have not been extensively investigated but seem to vary with the protein. Asparaginase modified with dextran (70000 Da), for example, was non-immunogenic in rabbits (Table 1.4). After repeated injection with the dextran conjugate, native enzyme circulated as in intact animals (Wileman et al., 1986). No hypersensitivity reactions were observed when the conjugate was given as a single injection to patients suffering fi-om lymphoblastic

Table 1.5 Effect o f dextran modification on protein activity retention.

Protein Dextran

molecular weight

Degree of modification (% lysine residues)

% Remaining activity Reference

a-Amyiase 60000-90000 1 0:1'^' 43 Marshall et ah, 1977

Asparaginase 40000 ? 50 Wileman et ah, 1986

70000 ? 50

250000 ? 50

1 1 0 0 0 0 17 34 Benbough et ah, 1979

2 0 0 0 0 0 0 17 36

70000 ? 50 Wileman et ah, 1983

Catalase 60000-90000 1 0:l(^) 71 Marshall et ah, 1977

17000 ? 49-67 Davis et ah, 1991

40000 ? 77-82

Superoxide dismutase 80000 34 67 Miyata et ah, 1988

Relative amount o f carbohydrate to protein on a weight basis.

UJ

leukaemia (Wileman et al., 1983). On the other hand, dextran modification of superoxide dismutase increased the enzyme’s immunogenicity (Table 1.4). Other enzymes such as catalase and a-amylase showed reduced but not abrogated immunogenicity (Davis et al., 1991). Antigenicity was usually reduced (Table 1.4 and Marshall, 1978). In the case of dextran-asparaginase, antigenicity decreased in vivo

with increasing size of the dextran (Wileman et al., 1986). Concerns about the use of dextran as a protein modifier derive from its reported immunogenicity in humans (Torii et al., 1976) and non-biodegradability in the lysosomes.

The therapeutic efficacy of catalase-dextran constructs was superior to the native enzyme (Marshall, 1978): the majority of acatalasémie mice challenged with hydrogen peroxide survived if the animals were simultaneously injected with the catalase construct; all the mice given native catalase and untreated animals died.

Loss of enzymatic activity is usually concomitant with dextran attachment, the degree of which can be inversely proportional to the molecular weight of the dextran (Table 1.5). The activity retention of asparaginase-dextran conjugates did not however, seem dependent on the molecular weight of the carrier (Table 1.5). The relationship (if any) between molecular weight of the polymer and the degree of modification of the final construct has not been evaluated.