Figure 8 Commonly used coupling methods for the preparation of macromolecule-drug conjugates. Functional groups of the drug and macromolecule are interchangeable with each other.

activity of the drug. The preparation of ester-linked conjugates is typically achieved by activating the carboxylic acid group with a water-soluble or water-insoluble carbodiimide [e.g., l-ethyl-3-(dimethylaminopropyl) carbodiimide and N, A^'-dicyclohexylcarbodiimide, respectively], optionally in the presence of a base catalyst [e.g., 4-(dimethylamino) pyridine], followed by a reaction with an alcohol group-containing agent. The use of a mixture of carbodiimide and Af-hydroxysuccinimide has also been extensively used (277).

The fate of macromolecular drug conjugates in vivo depends on their distribution and elimination properties. In general, the plasma half-life of a macromolecule-drug conjugate depends on its molar mass, ionic nature, configuration, and interaction tendencies in the physiological milieu. It has been reported that macromolecules with molar mass less than 40,000 are cleared rapidly, while those with molar mass higher than 40,000 remain in the circulation for prolonged periods. Although the RES is the natural target, other organs in the body can be targeted as well. Because many tumors possess vasculature that is hyperpermeable to macromolecules, soluble macromolecules also serve as potential drug carriers in the treatment of cancers. Present evidence suggests that the greatest levels of tumor accumulation are achieved by using macromolecules that carry a negative charge. Several excellent reviews that describe the distribution of soluble macromolecules in biological systems have been published (37,56,58,67,278).

The choice of a macromolecular carrier depends on the intended clinical objectives and the nature of the therapeutic agents being used. In general, the properties of an ideal soluble carrier system include the following (276,279): (i) the carrier and its degradation products must be biodegradable (or, at least, should not show accumulation in the body), the carrier must be nontoxic and nonantigenic and must not alter the antigenicity of the therapeutic agents being transported; (ii) the carrier must have an adequate drug-loading capacity; that is, the carrier must have functional groups for chemical fixation of the therapeutic agent; (iii) the carrier must remain soluble in water when loaded with drug; (iv) the molar mass of the carrier should be large enough to permit glomerular filtration but small enough to reach all cell types; (v) the carrier-drug conjugate must retain the specificity of the original carrier and must maintain the original activity of the therapeutic agent until it reaches the targeted site(s); (vi) the carrier-drug conjugate must be stable in body fluids but should slowly degrade in extracellular compartments or in lysosomes; and (vii) for lysosomotropic drug delivery, the macromolecule should not interfere with pinosome formation at the cell surface and subsequent intracellular fusion events. Furthermore, the macromolecule-drug linkage must be sensitive to acid hydrolysis or degradation by specific lysosomal enzymes.

Various natural and synthetic soluble macromolecules that have been investigated together with their uses are listed in Table 9. Natural polymers are monodisperse (i.e., same chain lengths), have rigid structures, and are biodegradable. The rigid structures may facilitate interactions between the determinant groups on the natural polymer and the binding region of immunoglobulin. Synthetic polymers, by contrast, are less immunogenic and can be better tailor-made to predetermined specifications (i.e., molecular size, charge, hydrophobicity, and their capacity for drug loading can be optimized). They are also easier and cheaper to produce in large quantity and high purity, and the chemistry to load drugs is much less laborious. In addition, they are more robust and are thus more stable during manipulation and storage. Another interesting field that is developing is the design of polymers that mimic biopolymers (280). These synthetic polymers have properties similar to those of proteins and RNA and present new opportunities to design new novel structures with desirable functions, including drug therapy. A detailed discussion of some natural and synthetic soluble carrier systems follows.

Table 9 Suggested Soluble Macromolecular Drug Delivery Systems

Carrier Targets/disease/therapy


Antibodies, antibody fragments (e.g., collagen specific drug-toxin conjugates) Albumin-drug conjugates Glycoproteins

Lipoproteins Lectins

Hormones (toxin-drug-hormone conjugate) Dextrans (e.g., enzyme-drug-dextran conjugates) Deoxyribonucleic acid (drug-conjugate;

lysosomotropic carrier) Synthetic polymers

Poly(L-lysine)and polyglutamic acid Poly(L-aspartic acid) Polypeptide-mustard conjugates Af-(2-hydroxypropyl)methacrylamide copolymers Pyran copolymers

Source: From Ref. 37.

Injured sites of blood vessels' walls, tumor cells

Cancer cells (lysosomotropic) Hepatocyte-specific agents (infectious disease, especially viruses) Liver/cancers of ovaries and gonads General carrier/recognition ligands Tumors Tumors Cancer cells

Carrier for targeting cancer Hydrolyzable targeting carrier for cancer Lung targeting, tumor targeting Lysosomotropic carrier for cytotoxics

Lysosomotropic carrier for cytotoxics

Antibodies Antibodies are circulating plasma proteins of the globulin group. They are produced by plasma cells that arise as a result of differentiation and proliferation of B-lymphocytes. Antibodies interact specifically with antigenic determinants (molecular domains of the antigens) that elicit their formation. In humans, there are five main types of antibodies, commonly designated as IgG, IgA, lgM, IgE, and IgD. Of these, IgG is the most abundant. It constitutes 75% of the serum immunoglobulins and is the only immunoglobulin that crosses the placental barrier and is incorporated in the circulatory systems of the fetus, thereby protecting a newborn from infection. The basic structure of the immunoglobulin molecule consists of two identical heavy (long) chains, each with a molar mass of 50,000, and two light (short) chains with a molecular mass of 23,000 (Fig. 9). These chains are held together by noncovalent forces as well as by disulfide linkages. Each chain also contains interchain disulfide linkages. In addition, each chain contains a region of a constant amino acid sequence and a region of variable amino acid sequence. Antibodies belonging to the same class share the constant region in their heavy chains and may have k- or X,-type light chains. The ends of the constant region (carboxyl ends) of the heavy chains form the Fc region, which is responsible for binding to Fc receptors present on many cells. The specificity of a particular antibody is determined by amino acid sequences of the variable region that are similar in the light and heavy chains. The ends of the variable region, referred to as amino (NH2) terminals, serve as the binding sites for antigens.

The use of antibodies for active targeting of drugs to specific cell types in vivo has long been recognized (281-285). They have been extensively explored as carriers in cancer diagnosis and in targeting drugs, toxins, and other therapeutic agents to tumor cells. Monoclonal antibodies of defined class and antigen specificity can be obtained in a highly purified form and in virtually unlimited amounts by immunizing mice with human tumor cells, followed by hybridizing their spleen cells with myeloma cells and, subsequently, screening the hybridoma cells for the formation of antibodies that bind only

Constant region

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