Biotherapy 3: 1-8, 1991. © 1991 Kluwer Academic Publishers. Printed in the Netherlands.

Rationale for targeted drug delivery Peter W. Taylor & Colin Howes Advanced Drug Delivery Research, CIBA-Ge~gy Pharmaceuticals, Wimblehurst Road, Horsham, West Sussex RHI2 4AB, UK

The traditional approach to drug discovery has been largely based on a series of empirically derived procedures that rely on the demonstration of an appropriate pharmacological response in animal models of disease. Active compounds identified by such screening procedures commonly fail at some stage in the drug development process due to an inappropriate pharmacokinetic profile or to unacceptable side effects unrelated to the desired mechanism of action of the drug. Effective drug therapy, particularly in situations where there is a necessity for chronic drug administration, often requires the attainment of a desired pharmacological response at a selected site without undesirable drug interaction at other sites [1]. This can only be achieved if the correct amount of drug is absorbed into the body and transported to the site of action at the right time, and the subsequent rate of input of drug to the site controlled to produce therapeutically effective concentrations for as long as is necessary. At the same time, the distribution of the drug to tissues other than the site of action and organs of elimination must be severely restricted or eliminated [2]. Such a challenging profile is seldom, if ever, achieved, with the consequence that most drugs display a less than optimal benefit to risk ratio. The recent explosion in knowledge of the biochemical and molecular basis of a number of important disease states has led to significant changes in the way new drugs are discovered and developed, with more accent on establishing in vitro procedures that take into account the nature of the biochemical lesion

responsible for the disease. It is likely that such approaches will generate therapeutic agents with increased selectivity of drug action [1]. Concomitant with the introduction of these more sophisticated drug screening procedures has been the development of techniques to identify, clone, express and produce on a large scale many proteins and peptides of therapeutic interest. Such putative drugs, with regulatory or homeostatic functions, have a number of advantages for the correction of biochemical lesions responsible for much disease, and it is becoming increasingly likely that a number of such biotherapeutics, whether native or modified by a variety of molecular biological techniques, will find clinical use in the coming decades [3]. If such protein drugs can be produced in sufficient quantities and in a cost effective way, their mode of delivery to the site of action within the body is likely to become a issue which, if not adequately resolved, will limit their use as therapeutic entities. Unlike many conventional drugs, it is unlikely that they could be administered orally due to their susceptibility to degradation within the lumen of the gastrointestinal tract and to their inability to pass through the epithelial cell barrier [3]. If introduced parenterally into the body, they may fail to elicit an adequate pharmacodynamic response due to their susceptibility to enzymic degradation, their short circulating half life and a number of other factors related to the fact that many of these proteins have evolved to perform effector functions in very close anatomical proximity to their site of synthesis in vivo. Thus, the concepts of

2 targeted, or site-specific drug delivery, in which the drug can be protected from degradative processes within the body, delivered to the target tissue by exploitation of the body's own transport processes, and selectively released in an active form at that site, are likely to play an increasing role in translating recent promising developments into therapeutic reality.

Controlled release systems Traditional methods of drug administration such as oral dosing, ointments and eye drops present the body with a high initial systemic drug concentration, which then, either slowly or rapidly, decays through metabolic processing. This concentration profile is not ideal for most therapies, and relies on the condition that at some point between dosing and elimination, the drug passes through the optimum therapeutic concentration. However, during this period the body has been exposed to possibly toxic levels of drug, with potentially cumulative damage, in tissues which do not benefit from exposure to the drug, and for the remainder of the dosing cycle there is a therapeutically suboptimal concentration of drug present. Sustained release systems using, for example, slowly dissolving coatings, emulsions or resins, have been developed in an attempt to achieve stable drug levels in the body between doses. This technology has since lead to the development of controlled release systems which do not rely entirely upon the rate of dissolution of the drug, but introduce other rate controlling membranes. Therefore, to date the most successful drug delivery systems have been non-site-specific controlled release constructs which exploit physical, or occasionally physicochemical, mechanisms for modulation of the rate of drug liberation. These devices consist of either a polymer matrix, such as subdermal implants, ocular devices, or transdermal patches, and intra-uterine devices (IUD's), or a pump and reservoir system [4, 5]. For ex-

ample, applications of controlled release systems include hormonal treatments for diabetes and contraception. In these cases, the insulin treatment is administered from a reservoir by a pump, whereas contraceptive steroids have been administered by incorporation into intra-uterine devices. Controlled release systems are particularly useful in applications where the drug has a short biological half-life, but needs to be maintained at the therapeutically optimal concentration. The objective of this type of drug delivery is to allow the administration of a dose of drug which will have a long duration of efficacy by virtue of the fact that the drug is being continually released into the body compartment. This mode of administration avoids flooding the body with potentially toxic levels of drug which achieve only a proportionally low drug concentration at the desired site of action. Instead, controlled release only allows a limited concentration of free drug to become available in the body compartment. However, the usual release concentration profile from these devices consists of a high initial rate, followed by a prolonged, slowly decaying, release rate. This is because the release mechanism in devices other than the pump driven systems still relies upon diffusion of the drug through a controlled permeability membrane, and the solubility of the drug in the environment into which it is released. More advanced control of the release profile is being sought through the development of biodegradable cartier systems such as microparticles and polymers beating pendant drugs grafted onto the backbone [6-9]. The types of devices currently available are only a convenient form of administration if the principal means of drug consumption is through its action at the target site; this is very rarely the case. Thus, some degree of physical localisation of the carrier is desirable with these systems. This is the first hurdle to be overcome in any rationale for achieving site-specific release. Also, all current drug

delivery systems rely on the properties of the drug itself for the degree of accessibility, by diffusion or binding, to the target site. The carrier is not usually localised at the desired site of action although, as with the IUD, ocular, and transdermal patches, the device can be sited at the most therapeutically advantageous external locus on the body. With the exception of the pumped reservoir system, all of the controlled release systems on the market are used to deliver quite low molecular weight drugs. With the exception of pumps, none of the systems can be used for the administration of large peptides or proteins, because these molecules either cannot traverse the permeable polymer membrane of the delivery system, or they cannot cross the biological barriers encountered subsequent to release. Thus, oral, transdermal and ocular routes of administration must all be virtually excluded for the delivery of high molecular weight drugs; the oral route suffers from the additional drawback that digestive enzymes can quickly degrade the released peptidic drug. However, there has recently been increasing interest in nasal delivery for the administration of both high and low molecular weight drugs [ 10, 11]. The accepted dogma is that alternative routes of administration (intramuscular, subcutaneous, intravenous), are only acceptable in acute situations, and are not generally considered viable for use in chronic therapies. An objective therefore of controlled delivery is to reduce the frequency of administration by slow, long term drug release from biocompatible carriers. This is an area in which there is now a rapidly increasing amount of research being undertaken.

Current approaches to site-specific delivery Many drugs show little, if any, inherent specificity for their site of action; opportunities exist, therefore, for selective site delivery in order to improve the therapeutic effectiveness

of these molecules and limit side effects. One way in which the normal disposition of an active therapeutic agent can be altered to increase selectivity is through the design and use of prodrugs; these are pharmacologically inactive agents that are biotransformed to active drug at the site of action [ 12]. Activity in this area has met with some success; although attempts to exploit high concentrations of phosphatases in tumour cells to transform phosphate esters into active cytotoxic agents have proved disappointing [13], Wilk and coworkers [14] have demonstrated that v-glutamyl derivatives of L-dopa and dopamine are cleaved to form active drug in the kidney. ~,-glutamyl transpeptidase transfers the L-y-glutamyl group from the terminus of one peptide to another peptide or amino acid; the enzyme is concentrated in the brush borders of cells lining the kidney proximal tubules. Dopamine, a potent renal vasodilator, is generated slowly in the kidney, where it acts before being metabolised and excreted from the body, without causing systemic side effects. Other examples of this approach are cited by Stella and Himmelstein [ 13] and pharmacokinetic aspects of prodrugs are discussed by McMartin in this issue [ 15]. The idea of site-selective activation of drug can be taken a stage further by conjugating the drug to a soluble macromolecular carrier. Conventional drugs and some prodrugs frequently have a large volume of distribution in vivo as a result of their capacity to diffuse through physiological barriers in the body and deposit in non-target tissues in either a free or protein-bound state. This, and a degree of biotransformation between administration of the drug and its excretion from the body, mean that in most forms of conventional drug administration the dose must greatly exceed the therapeutic concentration in order to be effective. Linking a drug to a macromolecular carrier often alters dramatically the tissue localisation of the conjugated drug because the distribition in vivo of the complex is largely dictated by the properties

4

of the carrier molecule [ 16]. Linking the drug to a polymer in such a way that it will be specifically released at the site of action may result in improvements in drug stability, duration of action and a more desirable local concentration as well as the obvious advantage of increased site specificity [ 17]. Stable, biocompatible synthetic materials, such as those based on N-(2-hydroxyprophyl)methacrylamide copolymers, have been studied in combination with selectively cleaved peptidic drug linkers and proposed for cancer chemotherapy [18]. Similarly, soluble synthetic biodegradable polymeric systems are currently being investigated for a number of clinical applications [ 19]. A number of other carriers have been considered in order to achieve a degree of site selectivity and these have been reviewed in detail by Sezaki and Hashida [20] and Friend and Pangburn [17], the former in relation to cancer chemotherapy. Briefly, they include host proteins such as antibodies, albumin, various glycoproteins and lectins as well as polysaccharides such as dextrans, synthetic polypepfides such as polylysine, polyaspartic acid and polyglutamic acid, and polynucleic acids such as DNA. These carriers are biodegradable and will not, therefore, accumulate in the body. In some cases, the carrier is used because it recognises an antigen, receptor or some other marker at the target site, and such actively targeted systems may further increase the capacity of drug to localise and be retained at the desired site. In recent years, much progress has been made with efforts to target cytotoxic agents to tumor cells using monoclonal antibodies recognising tumour cell-associated antigenic markers [21]; immunotoxins, chimaeric constructs in which monoclonal antibodies are covalently linked to plant or bacterial toxins in order to elicit death of target cancer cells, have been particularly intensively studied and are addressed in this issue by Press [22]. As murine antibodies may themselves evoke a humoral immune response in species other

than the mouse, attention has also focused on chimaeric antibodies containing murine variable region sequences and human constant region sequences and is reviewed by Shin [23]. It may well be possible to direct drugs to specific intracellular locations by taking advantage of receptor-mediated pathways of trafficking within and through cells. A number of ligands of importance to metabolism are brought into the cell by receptor mediated endocytosis and include nutrients such as diferric iron (as an iron-transferrin complex) [24], low density lipoprotein (LDL)-cholesterol [25] and vitamin B~2 (as a B~2transcobalamin II complex) [26], growth factors such as insulin and epidermal growth factor [27], and asialoglycoproteins. The asialoglycoprotein receptor, exclusive to hepatocytes and responsible for the clearance of galactose terminated glycoproteins from the circulation [28], has been particularly well studied and attempts have been made to exploit this receptor for drug delivery purposes. Following binding of the ligand, most of the protein-receptor complex is transported via an endosomal compartment to the lysosomes for degradation [29]; systems have been devised to exploit this pathway for the delivery of antiviral agents [30], for the treatment of malaria with primaquine [31] and for redirecting plasma lipoproteins to the parenchymal cells [32]. In addition, a number of other potential applications have been discussed recently by Fallon and Schwartz [33]. In this issue, Wu and Wu [34] discuss strategies for the introduction of foreign genes into hepatocytes via the asialoglycoprotein receptor. Soluble systems remain attractive candidates for selective delivery in a variety of clinical situations, and these are reviewed by Eichler [35] in the current issue. Theoretically, however, greater loading of drug can be achieved using a number of particulate dehvcry systems, of which liposomes have been the most intensively studied. These artificial

vesicles composed of one or more concentric lipid bilayers, and ranging in size from 0.02 #m to 100 #m, have been the subject of a number of recent reviews to which the reader is referred [17,36-40]. Loading of drug into the aqueous compartment protects the drug from the bioenvironment. In certain disease states liposomes and other particulate drug carriers may extravasate into the diseased site [41] or may gain access to the target site by physical entrapment [40]. However, a number of problems that mitigate against their general use have been described [17], and these include leakage of contents prior to arrival at the target site, problems in controlling the release of drug at the target and instability of dosage forms. Although much effort has been expended on attempts to produce liposomes that evade uptake by cells of the reticuloendothelial system, it has been convincingly argued that liposomes would be best employed as carriers of drugs to the cells of the monocyte-macrophage lineage [42]. The article by Pak and Fidler [43] describes macrophage-directed liposome systems for the delivery of biological response modifiers and Crommelin [44] reviews liposomes as drug delivery vehicles for biotherapeutic agents.

Approaches to the targeting of peptide and protein drugs The latter part of this century is witnessing the development and increasing availability of both natural and synthetic peptide and protein drugs arising from the rapid progress being made in gene engineering, chemical synthesis, and fermentation technology. Several polypeptide drugs are now either in development or clinical use, including r-human growth hormone (hGH), r-human insulin, rinterferon-~, calcitonin, and luteinizing hormone/gonadotropinreleasing hormone (LHRH/GnRH). However, the emergence of these new kinds of drugs is accompanied by a

new set of challenges which are mostly unfamiliar in the sphere of conventional low molecular weight drug therapy. The questions arising include the risk of adverse effects from immunogenicity, and species specificity, as well as either short, or long term, harmful effects from interference in normal peptide mediated positive and negative feedback control mechanisms. In analogy to low molecular weight drugs, the dosing regimen of polypeptide drugs is also important, since administration in a pulsitile fashion or by continuous infusion have been shown to produce different efficacies owing to the diurnal and circadian cyclical regulation of mammalian physiology. Thus a great deal needs to be understood about the pharmacokinetics, and distribution of these peptides and proteins in-vivo to optimise the dosing regimen [45-47]. The various physicochemical problems associated with peptide and protein drugs are well known. In contrast to low molecular weight drugs, these large molecules are susceptible to inactivation by either peptidases, or simply an adverse local pH environment. In addition to some of the physicochemical disadvantages common with many small drug molecules, such as poor solubility in aqueous media, the permeation of biological membranes and subsequent disposition is influenced by the size, shape, and surface morphology, of the polypeptide [46]. Even if the large drug molecules can penetrate into the bloodstream, they may be rapidly removed by the action of the reticuloendothelial system or degraded prior to excretion. Current forms of administration of polypeptide drugs are limited to conventional direct intramuscular, intravenous or subcutaneous injection, but (as mentioned previously), there is significant interest in other routes using controlled release devices for nasal [10, 11,46], transdermal [48,49], and rectal [10, 49]administration. Unfortunately there are apparently poor prospects for oral [46, 49], and ocular [47], delivery of polypep-

tides. Nasal administration can also suffer from the particular problems of tolerance arising, or unique immunological responses. All of these methods also use absorption enhancers which may have damaging effects in chronic treatments. Another significant challenge facing the potential success of these drugs is the high production costs. The efficient use of the drug therefore becomes essential if the treatment is to be generally affordable and widely applicable. Thus the therapeutic utilisation of peptide and protein drugs provides the science and technology of targeted drug delivery with perhaps its greatest challenge. Approaches to the improved delivery and targeting of polypeptides have so far exploited a number of different strategies. Deletion mutagenesis employs a technique which excises sites on the protein which impart characteristics unfavourable to drug targeting [46]. This approach has been demonstrated with tissue-type human plasminogen activator (tPA) [50]. This is a 59,050 molecular weight macromolecule consisting of 567 amino-acid residues which is produced by the body as a thrombolytic agent, and has been used in the treatment of heart attacks and strokes [49]. The removal of functional regions of the protein such as glycosylation sites, fibronectin-type, and epidermal growth factor type regions, brought about a favourable alteration in the pharmacokinetic and thrombolytic characteristics of the protein. This example also provides a good insight into how the converse strategy of protein conjugation could work. Hybrid proteins may be built up from several protein fragments to give a product with favourable delivery and targeting features. Protein hybridisation is an approach which researchers are only just beginning to explore, and is being employed, for example, to elucidate the intracellular trafficking of proteins [27, 41, 51, 52]. This raises the long term possibility of achieving intracellular targeting. Thirdly, the conjugation of peptides or

proteins to synthetic polymers, to either improve solubility, stability, circulating half-life or reduce immunogenicity, has been a popular and successful strategy [41, 53, 55]. However, true in-vivo site-specific targeting has yet to be achieved. In this context, the importance of polysaccharides in the process of site-specific receptor recognition has also received some attention. Modifications to glycoproteins or derivatisation of polypeptides with polysaccharides appears to have some attractive possibilities [46]. These strategies overlap with the use of polypeptides and polysaccharides as carriers for drug targeting and to provide protection of low molecular weight drugs against rapid metabolic degradation. This area has so far been more extensively investigated than the topic of targeting polypeptides as drugs, and has been extensively reviewed elsewhere [17, 40, 41].

References I. Gardner CR. Potential and limitations of drug targeting: An overview. Biomaterials 1985; 6: 153-60. 2. Prescott LF. The need for improved drug delivery in clinical practice. In: Prescott LF, Nisso WS, eds. Novel drug delivery. Chichester: John Wiley & Sons, 1989; I ll. 3. Tomlinson E. Selective delivery and targeting of therapeutic proteins. In: Harris TJR, Hentschel CC, eds. Protein Production. Amsterdam: Elsevier, 1990; in press. 4. Beck LR, Pope VZ. Controlled release delivery systems for hormones. Drugs 1984; 27: 528-47. 5. Langer R. Implantable controlled release systems. Pharm Ther 1983; 21: 35-51. 6. Heller J. Controlled drug release from poly(ortho-esters). Ann New York Acad SO 1985; 446: 51-55. 7. Anderson JM. In-vivo and in-vitro studies of drug releasing poly(amino-acids). Ann New York Acad Sci 1985; 446: 67-75. 8. de Nijs H, Bowman TRM, Eeninik MJD. Controlled peptide delivery using biodegradable microcapsule formulations. Pharm Weekbl Sci Edn 1988; 10: 49. 9. Petersen R. Biodegradable drug delivery systems based on polypeptides. In: Gebelein GC, Carraher CE Jnr, eds. Bioactive polymeric systems 1985: 151-78. 10. Eppstein DA, Longenecker JP. Alternative delivery systems for peptides and proteins as drugs. CRC Crit Rev Ther Drug Carrier Syst 1988; 5: 99-139. ! 1. Su KSE. Intranasal delivery of peptides and proteins. Pharm Int 1986; 8-11.

12. Bundgaard H. Design of prodrugs: Bioreversible derivatives for various functional groups and chemical entities. In: Bundgaard H, ed. Design of prodrugs. Amsterdam: Elsevier, 1985; 1-92. 13. Stella VJ, Himmelstein KJ. Site-specific drug delivery via prodrugs. In: Bundgaard H, ed. Design of prodrugs. Amsterdam: Elsevier, 1985: 177-98. 14. Wilk S, Mizoguchi H, Orlowski M. ),-glutamyl dopa: A kidney-specific dopamin precursor. J Pharmacol Exp Ther 1978; 206: 227-32. 15. McMartin C. Pharmacokinetic requirements for successful site-directed targeting of drugs. Biotherapy 1991; 3: 9-23. 16. Ringsdorf H. Synthetic polymeric drugs. In: Kostelnik RJ, ed. Polymer Delivery Systems. New York: Gordon & Breach, 1978; 197-225. 17. Friend DR, Pangburn S. Site-specific drug delivery. Med Res Rev 1987; 7: 53-106. 18. Kopecek J. Development of tailor-made polymeric prodrugs for systemic and oral delivery. J Bioact Compat Polymers 1988; 3: 16-26. 19. Heller J. Biodegradable polymers in controlled drug delivery. CRC Crit Rev Ther Drug Carrier Syst 1984; 1: 39-90. 20. Sezaki H, Hashida M. Macromolecule-drug conjugates in targeted cancer chemotherapy. CRC Crit Rev Ther Drug Carrier Syst 1984; 1: 1-38. 21. Poste G, Kirsch R. Site-specific (targeted) drug deliver3," in cancer therapy. Biotechnology I983; I: 869-78. 22. Press OW. Immunotoxins. Biotherapy 1991; 3: 65-76. 23. Shin S-U. Chimeric antibody: Potential applications for drug delivery and immunotherapy. Biotherapy 1991; 3: 43 -53. 24. Dautry-Varsat A, Ciechanover A, Lodish HF. pH and the recycling of transferin during receptor mediated endocytosis. Proc Natl Acad Sci USA 1983; 80: 2258-62. 25. Brown MS, Goldstein JL. Lipoprotein receptors in the liver: Control signals for plasma cholesterol trattic. J Clin Invest 1983; 72: 743-7. 26. Seetharam B, Alpers DH, Allen RH. Isolation and characterization of the ileal receptor for intrinsic factor cobalamin. J Biol Chem 1981; 256: 3785-90. 27. Hopkins CR. Site-specific drug delivery: Cellular opportunities and challenges. In: Tomlinson E, Davis SS, eds. Site-specific drug delivery. Chichester: John Wiley & Sons, 1986; 27-48. 28. Ashwell G, MoreU A. The role of surface carbohydrates in the hepatic recognition and transport of circulating glycoproteins. Adv Enzymol 1974; 41: 99-128. 29. Geuze H J, Slot JW, Strous G JAM, Schwartz AL. The pathway of the asialoglycoprotein-ligand during receptormediated endocytosis: A morphological study with colloidal gold/ligand in the human hepatoma cell line, HepG2. Eur J Cell Biol 1983; 32: 38-44. 30. Fiume L, Mattioli A. Busi C, Balboni PG, BarbantiBroadano G, De Vries J, Altmann R, Wieland T. Selective inhibition of Ectromelia virus DNA synthesis in hepatocytes by ara-A and ara-AMP conjugated to asialofetuin. FEBS Lett 1980; 116: 185-8.

31. Hofsteenge J, Capuano A, Altszuler R, Moore S. Carrierlinked primaquine in the chomotherapy of malaria. J Med Chem 1986; 29: 1765-9. 32. van Berkel TJC, Kruijt JK, Kempen H-JM. Specific targeting of high density lipoproteins to liver hepatocytes by incorporation of a tris-galactoside-terminated cholesterol derivative. J Biol Chem 1985; 260: 12203-7. 33. Fallon RJ, Schwartz AL. Receptor-mediated delivery of drugs to hepatocytes. Adv Drug Del Rev 1989; 4: 49-63. 34. Wu GY, Wu CH. Delivery systems for gene therapy. Biotherapy 1991; 3: 87-95. 35. Eichler H-G. Clinical experience of targeted therapy. Biotherapy 1991; 3: 77-85. 36. Swenson CE, Popescu MC, Ginsberg RS. Preparation and use of liposomes in the treatment of microbial infections. Crit Rev Microbiol 1988; 15: S1-$31. 37. Weinstein JN, Leserman LD. Liposomes as drug carriers in cancer chemotherapy. Pharmac Ther 1984; 24: 207-33. 38. Kirsh R, Poste G. Liposome targeting to macrophages: Opportunities for treatment of infectious diseases. Adv Exp Med Biol 1986; 202: 171-84. 39. Poste G. Liposome targeting in vivo: Problems and opportunities. Biol Cell 1983; 47: 19-38. 40. Tomlinson E. Microsphere delivery systems for drug targeting and controlled release. Int J Pharm Tech Prod Mfr 1983; 4: 49-57. 41. Tomlinson E. Theory and practice of rite-specific drug delivery. Adv Drug Del Rev 1987; 1: 87-198. 42. Schroit AJ, Hart IR, Madsen J, Fidler IJ. Selective delivery of drugs encapsulated in liposomes: Natural targeting to macrophages involved in various disease states. J Biol Resp MOd 1983; 2: 97-100. 43. Pak CC, Fidler IJ. Liposomal delivery of biological response modifiers to macrophages. Biotherapy 1991; 3: 55-64. 44. Storm G, Wilms HP, Crommelin DJA. Liposomes and biotherapeutics. Biotherapy 1991; 3: 25-42. 45. Sternson LA. Obstacles to polypeptide delivery in biological approaches to the controlled delivery of drugs. In: Juliano RL, ed. Ann NY Acad Sci 1987; 507: 19-21. 46. Tomlinson E. Considerations in the physiological delivery of therapeutic proteins. In: Prescott LF, Nimmo WS, eds. Novel drug delivery. 1989; 245-62. 47. Lee V. H. L. Peptide and protein drug delivery: Opportunities and challenges. Pharmacy International 1986; 7: 208-12. 48. Chien YW, Siddiqui O, Sun Y, Shi WM, Liu JC. Transdermal iontophoretic delivery of therapeutic peptides/ proteins. In: Juliano RL, ed. Biological approaches to the controlled delivery of drugs. Ann NY Acad SCi 1987; 507: 32-51. 49. Banga AK, Chien YW. Systemic delivery of therapeutic peptides and proteins. Int J Pharm 1988; 48: 15-50. 50. Collen D, Stassen JM, Larsen G. Pharmacokinetics and thrombotytic properties of deletion mutants of human tissue-type ptasminogen activator in rabbits. Blood 1988; 71: 216-9. 51. Goldfarb DS, Cariepy J. Schoolnik G, Kornberg RD. Synthetic peptides as nuclear localisation signals. Nature 1986; 322: 641-4.

52. Moore HH, Kelly RB. Re-routing of a secretory protein by fusion with human growth hormone sequences. Nature 1986; 321: 443-6. 53. Abuchowski A, Palczuk NC, Van ET, Davis FF. Alteration of immunological properties of bovine serum albumin by covalent attachment of polyethylene glycol. J Biol Chem 1977; 252: 3578-81.

54. Abuchowski A, Van ET, Palczuk NC, Savoca K, Chert RHL, Pyatak P. Soluble non.antigenic polyethylene glycol boundary enzymes. Polym /'reprints 1979; 20: 35760. 55. Davis FF, Van ET, Palczuk NC. Non-immanogenic polypeptides. US patent 4,179,337: 1979.

Rationale for targeted drug delivery.

Biotherapy 3: 1-8, 1991. © 1991 Kluwer Academic Publishers. Printed in the Netherlands. Rationale for targeted drug delivery Peter W. Taylor & Colin...
838KB Sizes 0 Downloads 0 Views