Bioadhesives And Drug Targeting To The Lung

The use of bioadhesive targeting as a means for specific delivery of drugs has gained some impetus since the late 1980s [180]. The term bioadhesion refers to interactions involving multiple molecular and usually noncovalent bonds. However, a bioadhesive agent, to be effective as a drug carrier, must initially be trapped or sequestered by endothelial or epithelial cells, followed by multiligand binding of the surface material of the carrier particle to cell surface determinants, which then induces the rapid (10 to 15-minute) envelopment of the carrier by the cell either by transcytosis or migrational overgrowth mechanisms, followed by transfer to the proximal tissues. Bioadhesion targeting is, therefore, a combination of biophysical trapping and biochemical adherence. The carrier is usually a hydrophilic macromolecule or microparticulate, and, for systemic delivery, a particle size between 3 and 5 mm (nonembolizing) is preferable for pulmonary trapping, whereas larger particle sizes between 5 and 250 mm (embolizing) are also usable. Multivalent binding agents may comprise substance such as heparins, lectins, and antibodies to endothelial antigens, epithelial antigens, and glycosylated albumin.

Lectins are naturally occurring glycoproteins of nonimmunological origin. They have the unique ability to recognize and bind to exposed carbohydrate residues on glycoproteins, such as those exposed at the surface of epithelial cells, and therefore have been classified as second-generation bioadhesives. In a recent study [181], lectin-functionalized liposomes, in contrast to lectin-free liposomes, specifically bound to A 549, a tumor-derived cell line. This suggests that lectin-mediated bioadhesion and uptake of liposome-carriers may provide a useful technology for lung cancer treatment. The administration of liposome-encapsulated drugs by aerosols seems to be a feasible way of targeting these delivery systems to the lung. The tolerability and safety of liposome aerosols has been tested in animals as well as human volunteers, and no unwanted effects have been observed [182].

Both transendothelial and transepithelial migration of particles and molecular aggregates larger than about 2 nm in diameter have been shown to be accelerated by application of surface coatings that bind multiply to cell surface receptors or antigens [138]. Such particles conjugate at least two molecules of drug or diagnostic agent and a multivalent binding agent (bioadhesive) that is specific for cell surface determinants. Table 2 lists carrier particles, multivalent binding agents, and endothelial and epithelial cell surface determinants of potential usefulness in bioadhesive targeting to the lung. This list is by no means exhaustive. Carrier particles, preferably having a size between 1 nm and 250 mm, or coatings comprising carbohydrates, oligosaccharides, or monosaccharides, proteins or peptides, and lipids or other biocompatible synthetic polymers are usually used. The chemical structure may be a simple single-chain polymer, a molecular microaggregate (i.e., a molecular carrier or aggregate that acts as both the cell target binding moiety and the backbone for linking prodrug moieties), or a more complex structure incorporating multiple matrix material or serial coating that is able to interact with multiple cell surface determinants, resulting in rapid sequestration and transport of the carrier. Substances from a wide range are known that bind to native endothelium and epithelium and to basement membrane constituents. All are promising candidates with potential usefulness as bioadhesive agents for lung-targeting studies. Laminin is a noncollagenous high-molecular-weight (106) protein that interacts with glycosaminoglycans and promotes adhesion of various cell types. Laminin is located in the lamina lucida

Table 2 Composition of Bioadhesives

Carrier particles

Multivalent binding agents

Endothelial and epithelial surface determinants

Macromolecules Heparin and heparin derivatives

Receptors

Microaggregates Heparin fragments Microparticles Lectins Microspheres Antibodies

Enzyme active sites Antigens

Endothelial and epithelial

Nanospheres Dextrans Liposomes Peptides

Microemulsions Enzyme inhibitors tissue factors Subcellular tissue moieties Fibrin D-D dimers Glycoprotein complexes

Receptor agonists;

and antagonists Albumins and glycosylated albumins of a cell's basal surface and the supporting matrix of type IV (basement membrane) collagen [183-185]. Laminin receptors occur on cells that normally interact with basement membranes as well as on cells that extravasate, for example, metastasizing cancer cells, macrophages, and leukocytes [186]. In vitro studies have clearly shown that laminin can mediate the attachment of a wide variety of epithelial and endothelial cells to type IV collagen [183,184], and a cell surface receptor protein for laminin has been isolated from murine fibrosarcoma cells that may mediate the interaction of the cell with its extracellular matrix [187].

Fibronectin, another constituent of basement membrane, may also be an effective constituent for promoting extravascular migration of particulate matter [188,189], and glycosylated serum albumins appear to undergo greater vesicular endothelial micropinocytosis by rat microvessels as compared to unmodified albumin, which may indicate a useful role for nonenzymatic glycosylated serum albumin in drug targeting [190].

Heparin sulfates, the side-chain moieties of cell surface proteoglycans, are important factors in cell recognition phenomena. The proteoheparan sulfates are ubiquitous cell surface proteglycan components of the cell coat or glycocalyx. They undergo chain-chain self-association in a structure-specific manner. Studies show that such compounds are useful bioadhesives [191]. Lipoprotein lipase also attaches to endothelial cells through heparin sulfate interaction on the cell surface and is released by heparin through a detachment from this binding site [192]. Factor VIII antigen has been widely used as a marker for endothelial cells [193], and studies have shown that Ulex europaeus 1 agglutinin (UEA-1), a lectin that is specific for a-L-fucose-containing glycocompounds, is also a marker for vascular endothelium in human tissues [193]. UEA-1 appears to be a more sensitive marker than factor VIII antigen for the factor VIII binding site and has a particularly high affinity for alveolar capillary endothelium and bronchial epithelium [194]. In vivo studies indicate that intravenously injected fucose-blocked UEA-1-coated microspheres in CBA mice allowed approximately 90% of the injected dose to be concentrated in the lung after 20 minutes, with 80% of this amount being in extravascular locations [138]. The use of UEA-1 lectin as a diagnostic agent for tumors derived from human endothelial cells was recently described [195].

Anionic sites on the lumenal surface of pulmonary microvascular endothe-lium have been shown to bind cationic ferritin in isolated, perfused rat lung studies [196]. The cationic ferritin is taken up by vesicles and discharged into the capillary membrane. Similar anionic sites are also present on alveolar epithelial surfaces [25].

In some cases, cell surface expression of certain species can be induced; for example, interleukin-1 has been shown to induce the biosynthesis and cell surface expression of procoagulant activity in human vascular endothelial cells [197]. Such materials may also be exploitable as candidates for bioadhesion studies. Millions of lives of patients with diabetes have been saved since the introduction of insulin therapy. However, several daily injections of insulin are required to maximize glucose control in diabetic patients. Insulin is administered by subcutaneous injection, but this route of administration has a slow onset and subsequent prolonged duration of action. These limitations show up more when higher doses of insulin are injected, which results in a long duration of action and forces the patients to consume additional amounts of food to limit the risk of hypoglycemia [198].

This limitation has been reduced by the availability of newer, short-acting insulin analogues (Lipro and Aspart). However, this form of insulin must be injected subcutaneously. Technosphereâ„¢ insulin is a formulation of regular human and Technosphereâ„¢, a new drug-delivery system for pulmonary administration. The formulation is designed for efficient transport of insulin across the intact respiratory epithelium into the systemic circulation [199]; its duration of action is more than 3 hours, and maximal serum insulin levels can be reached within 13 minutes after inhalation [200], which is considerably shorter than those observed with rapid-acting insulin analogues administered subcutaneously or other insulin inhalations [201].

Other molecules have been suggested as being useful lung-specific bioadhesive agents [137,138], for example, insulin, transferrin, prostaglandins, hirudin-inhibited thrombin (which binds thrombomodulin), anionic polysacchar-ides, oligosaccharides (such as dextran sulfate, dermatan sulfate, chondroitin sulfate, hyaluronic acid), peptides (such as benzoyl-phe-ala-pro [BPAPI] that bind angiotensin-converting enzyme, 5'-nucleotidies that bind 50 nucleases, and inactive analogues of biogenic amines (such as 5-hydroxytryptamine) that interact with surface neuroreceptors. Such a list of compounds also includes antibodies directed against cell surface targets, such as factor VIII antigen and type IV collagen of the basement membrane, glycoproteins, and other antigens (see the later section on monoclonal antibody conjugates).

Bioadhesive agents have been hypothesized to interact with endothelial or epithelial cell surface determinants, inducing the cell to undergo transient separation or opening, thereby exposing subcellular determinants for which the agent may also have binding affinity. The interaction results in an acceleration of transport across at least one of the associated endothelial and epithelial structures or subcellular structures into a proximal tissue compartment. The basic premise is that this phenomenon will result in an improvement in the therapeutic index so that a reduced total dose of drug (or diagnostic agent) is required to obtain pharmacological effects comparable to significantly higher doses of free drug (or diagnostic agent).

Ranney [138] showed that intravenously administered heparin-amphoter-icin Pluronic B-F108 nanospheres and microspheres in adult male CBA/J mice are specifically targeted to lung, endothelium uptake being complete within 15 minutes after injection (i.e., zero blood levels) (Table 3). The results indicate that preferential and rapid uptake in the lung occurs with both subernbolizing

Table 3 Organ Localization of Heparin-Amphotericin B Formulations After Intravenous Injection into Adult Male CBA/J Mice

Organ concentration (mg amphotericin/g tissue) (%) 1 hr after injection

Table 3 Organ Localization of Heparin-Amphotericin B Formulations After Intravenous Injection into Adult Male CBA/J Mice

Organ concentration (mg amphotericin/g tissue) (%) 1 hr after injection

Tissue

Amphotericin Ba (unbound)

Heparin (nonembolizing) (nanospheresb)

Heparin (marginally embolizing) (microspheresc)

Lung

4.0 (14)d

15.0 (52)d

25.7 (94)d

Liver

6.7 (24)

8.3 (29)

5.0 (18d)

Kidney

2.5 (9.2)

1.1 (3.8)

0.4 (1.4)d

Spleen

11.0 (38.3)

6.2 (21)

2.2 (8.2)

Heart

0.3 (1.3)

0.2 (0.7)

0.3 (0.4)

Brain

0.0 (0)

1.4 (4.8)

0.2 (0.5)

aBiodistribution of fungizone (amphotericin (b-deoxycholate nanoemulsion). bTotal body drug recovered, 70%. cTotal body drug recovered, 55%.

d Percent of injected dose localized per gram of tissue (wet weight).

aBiodistribution of fungizone (amphotericin (b-deoxycholate nanoemulsion). bTotal body drug recovered, 70%. cTotal body drug recovered, 55%.

d Percent of injected dose localized per gram of tissue (wet weight).

and embolizing particle diameters. Such a rapid and efficient uptake was not observed for dextran and agarose placebo particles that lack the heparin surface coat.

Histochemical analysis of lung deposition showed the amphotericin B to be distributed in the alveoli, pulmonary interstitium, respiratory epithelium, and bronchial and tracheal lymph nodes, thus indicating extensive tissue percolation of the drug carrier; no significant kidney deposition was observed. (Note: This is a major site of amphotericin toxicity.) The results establish that it is possible to achieve high-efficiency endothelial bioadhesion, selective drug uptake, and retention in the lung using this approach.

In the same study, intravenous injection of Ulex europaeus 1 lectin microspheres were shown to be specifically taken up by lung endothelial cells and rapidly underwent migration into the extravascular tissues and into the airspace within 5-10 minutes after injection, with 90% of the injected dose being identified in the lung. Intratracheal administration of heparin nanospheres of 200 to 800-nm diameter containing iron oxide (Fe3O4) and ionic iron (Fe3+) to pentobarbital-anesthetized adult male CBA/J mice indicated a very similar deposition profile to that observed after intravenous injection of amphotericin B-containing nanospheres (Table 3) [138].

Liver and kidney deposition was negligible to very low, showing that stabilized heparin nanosphere carriers with heparin surfaces are taken up by epithelial transport and that a high proportion of the dose becomes localized in lungs relative to other organs when administered by the airways. This novel example of epithelial uptake may provide the rationale for administering drug -bioadhesive carrier conjugates by the inhalation route and may be particularly useful for the topical or systemic delivery of highly toxic drugs (e.g., antitumor drugs, antifungals, antivirals, antibiotics), drugs that are very labile (e.g., peptides, proteins, oligonucleotides), or drugs that for one reason or another exhibit poor access by conventional formulations to pulmonary tissues.

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