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Urease (E. coli cells engineered

Removal of urea in kidney failure

24,25

to produce urease)

to produce urease)

biodegradable, and should possess high purity and homogeneous molecular-weight (MW) distribution (2). Besides, one should bear in mind that covalent coupling of enzymes to polymers may result in conformational alterations and decrease significantly enzymatic activity (27).

One of the most popular polymers currently used for the modification of peptides and proteins with therapeutic potential is polyethylene glycol (PEG). PEG is inexpensive, biocompatible, nontoxic, and is already approved by drug regulatory agencies (28). Pegylation may be used to improve the properties of the enzymes without causing any loss in the activity of the enzymes, which could be later used as protein drugs or as catalysts in bioreactors (29). Currently, several enzyme/PEG conjugates are used in clinics; some of these are listed by Michel Vellard in a recent review (30) and comprise recombinant human tissue-plasminogen activator, adenosine deaminase, arginine deaminase, and asparaginase.

The immobilization by inclusion methods present some advantages over the methods used for the immobilization by binding: there is no need for derivatization of the enzyme (26), the systems provide a higher degree of protection against enzymatic degradation and other destructive factors, allow for much higher drug loads, and enzymes can be immobilized into the system in a more stabilized form, such as multienzymatic systems, enzyme mixtures, or even cells producing a certain enzyme (1).

Enzyme Entraped
Fig. 1. Structure of a liposome. Hydrophilic drugs are encapsulated in the inner aqueous space, whereas lipophilic drugs are entrapped in the phospholipid membrane.

However, inclusion methods present disadvantages related to the use of synthetic materials, which can be solved by the use of biodegradable polymers (e.g., polymers and copolymers derived from lactic and glycolic acid, alginate, chitosan) or by the use of more biocompatible immobilization carriers such as liposomes or red blood cells (1,2).

Among particulate drug carriers, liposomes are the most extensively studied and possess the most suitable characteristics for polypeptide encapsulation. Liposomes are microscopic vesicles composed of membrane-like phospholipid bilay-ers enclosing aqueous compartments (see Fig. 1). They are biologically inert, biocompatible, and cause very little toxic or antigenic reaction. By the encapsulation of enzymes into liposomes, the in vivo blood circulation of enzymes can be increased and, moreover, the liposomes can mask their antigen determinants, avoiding the adverse immunological reactions (31).

An alternative to liposomes are controlled-release systems made of biodegradable polymers, such as nanoparticles. Micro/nanoparticles are polymeric colloidal systems, ranging in size from 10 nm to micrometers, in which the drug is dissolved, entrapped, encapsulated, or adsorbed (see Fig. 2). Nanoparticles, because of their versatility for formulation, sustained release properties, subcellular size, and biocompatibility with tissue and cells appear to be a promising system for the immobilization of enzymes and protein drugs (32).

One of the enzymes that has been immobilized in different supports and has received great attention from researchers is asparaginase. L-Asparaginase (EC 3.5.1.1) catalyzes the hydrolysis of L-asparagine (an essential amino acid) to L-aspartic acid and ammonia (33). Leukemic cells lacking asparagine synthase are unable to

Enzyme Immobilization
Fig. 2. Enzyme immobilization into polymeric micro- or nanoparticles (E enzyme). The entrapped enzyme may be dispersed (A) or dissolved (B) in the micro- or nanoparticulate system.

synthesize asparagine and are dependent on an exogenous source of asparagine for their survival. Treatment with L-asparaginase causes a rapid depletion of extracellular asparagines, leading to selective killing of the leukemic cells. Normal cells, able to synthesize asparagine intracellularly, are less sensitive to L-asparaginase oncolytic activity. L-Asparaginase therapy reduces the plasma levels of the amino acid and thus interferes in protein synthesis (34). Figure 3 shows the mechanism of action of L-asparaginase.

L-asparaginase (usually from Escherichia coli or Erwinia crysanthemi) is often used in protocols for the treatment of leukemia in combination with vincristine, daunomycin, methotrexate, or cyclophosphamide (34). Morever, recent clinical trials have proven the efficacy of asparaginase for the treatment of other childhood lymphoid malignancies (i.e., lymphoblastic lymphoma) (35).

Although a wide experience has been obtained from the treatment of acute lym-phoblastic leukemia (ALL) in recent years, still there are some limitations to the administration of L-asparaginase as a result of its short in vivo half-life, which implies the need for multiple injections in order to reach therapeutic levels of the enzyme, and to the immunological side effects, ranging in severity from mild allergic reactions to anaphylactic shock because this enzyme is a protein foreign to humans, especially upon the repetitive administration needed to reach therapeutic levels of the enzyme.

Therefore, alternative therapy or substitutes for L-asparaginase has been investigated over the past years. One of the approaches studied in clinics that has given successful results, with less immunogenicity and longer half-life, is binding to polyethylene glycol, originating a new molecule called pegaspargase. This approach is already approved by the FDA under the trade name of Oncaspar®. This modification leads to a less frequent administration and results in less immunogenic reaction (33). Nevertheless, some authors do not recommend routine substitution of L-asparaginase for pegaspargase owing to a possible higher incidence of pancreatitis associated with pegaspargase (36).

In this chapter, we will focus on the immobilization of L-asparaginase by inclusion into two delivery systems, namely liposomes and nanoparticles. These methods can be adapted for the immobilization of other therapeutic enzymes.

Asparaginase Immobilization

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