- Email: [email protected]
[40] Medical applications of immobilized proteins, enzymes, and cells
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[40] M e d i c a l A p p l i c a t i o n s o f I m m o b i l i z e d P r o t e i n s , E n z y m e s , a n d Cells By T. M. S. CHANG
Introduction Discussion of the medical applications of immobilized proteins, enzymes, and cells 1-9 usually emphasizes methods of enzyme immobilization and areas of possible application. Since extensive in oivo evaluations are being carried out in this area of research, methodologies for in vivo applications will also be covered in this chapter. About 20 years ago immobilized enzymes in the form of artificial cells containing catalase, asparaginase, or urease were shown to be effective for replacing hereditary enzyme deficiency in acatalasemic miceJ°; for suppressing the growth of lymphosarcoma in mice~; or for decreasing system urea levels in animals. ~2,13 Since that time, despite extensive efforts by many researchers using all available immobilization technologies, progress toward the actual large-scale clinical application of immobilized enzymes and cells has been very slow.l-9 This does not negate the earlier promise of the potential medical applications of immobilized enzymes and cells but shows the complexities and problems associated with this type of approach. Although proteins, enzymes, and cells are potentially much more specific, powerful, and useful, there are also major problems related to immunogenicity, toxicity, and needs for targeting. Furthermore, there T. M. S. Chang, "Artificial Cells." Thomas, Springfield, lllinois, 1972. 2 T. M. S. Chang (ed.), "Biomedical Applications of Immobilized Enzymes and Proteins," Vols. 1 and 2. Plenum, New York, 1977. 3 T. M. S. Chang (ed.), "Microencapsulation Including Artificial Cells." Humana, New York, 1984. 4 I. Chibata, "Immobilized Enzymes." Wiley (Interscience), New York, 1978. 5 j. S. Hoicenberg and J. Roberts (eds.), "Enzymes as Drugs." Wiley (Interscience), New York, 1981. 6 j. S. Holcenberg, Annu. Rev. Biochem. 51, 795 (1982). 7 K. J. Widder and R. Green, eds., this series, Vol. 112, p. 1. s T. M. S. Chang, Int. J. Biomater., Artif. Cells Artif. Organs 15, 1 (1987). 9 T. M. S. Chang, Appl. Biochem. Biotechnol. 10, 5 (1984). l0 T. M. S. Chang and M. J. Poznansky, Nature (London) 218, 242 (1968). 11 T. M. S. Chang, Nature (London) 229, 117 (1971). ~2 T. M. S. Chang, Science 146, 524 (1964). 13 T. M. S. Chang, Trans. Am. Soc. Artif. Intern. Organs 12, 13 (1966).
METHODS IN ENZYMOLOGY, VOL. 137
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
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are also problems related to the availability of suitable enzyme systems, the need for multienzyme system with cofactor requirements, and the viability of cell cultures. Recent progress in biotechnology is beginning to solve some of these problems. As a result an increasing number of researchers are seriously investigating the medical applications of immobilized proteins, enzymes, and cells. Since many of the applications are described elsewhere in this volume, we will discuss other examples not presented herein.
lnborn Error~ of Metabolism A number of years ago we reported the successful use of microencapsulated catalase for enzyme replacement in acatalasemic mice with congenital deficiency of the enzyme catalase.l° Whereas repeated injections of heterogenous catalase resulted in anaphylactic shocks, microencapsulated catalase did not result in immunological reactions and continued to carry out its functions. 1,1°,14However, this demonstration 15 years ago did not result in routine clinical application in this and other types of inborn errors of metabolism. There are many problems to be solved before immobilized enzymes and cells can be used routinely. Three of the most important problems are (1) the availability of enzymes, (2) the requirement for multienzyme systems in many conditions, and (3) the need for targeting to specific organs or intracellular locations in many types of congenital enzyme defects. Extensive effort has been carried out to solve these problems. One of the most intensively studied areas involves the use of liposomes for the microencapsulation of enzymes. 15Thus it has been reported that liposomes containing sulfatide, phosphatidylcholine, and cholesterol may be incorporated into the central nervous system. 16 Surface incorporation of antibodies onto liposomes has also been used in targeting. 15 Other modifications include surface attachment of lectins, glycoproteins, ligands, and others. Liposomes have the advantage of being biodegradable; however, they appear to enhance immune response to the entrapped protein. This area has been reviewed in detail elsewhere. 17 Red blood cells have also been used to microencapsulate enzymes by 14 M. J. Poznansky and T. M. S. Chang, Biochim. Biophys. Acta 334, 103 (1974). ~5 G. Gregoriadis, in "Enzyme Replacement Therapy of Lysosomal Storage Diseases" (J. M. Tager, J. M. Hooghwinkel, and W. T. Daoms, eds.), p. 131. North-Holland, Amsterdam, 1974. 16 M. Naoi and K. Yagi, Biochem. Int. 1, 591 (1980). 17 G. Gregoriadis, "Drug Carders in Biology and Medicine." Academic Press, New York, 1979.
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hemolysis and resealing. ~8Rh antibody-coated human red blood cells containing fl-glucosidase have been used for targeting in Gaucher's disease. 17 Other immobilization techniques have also been extensively investigated, 8 including the cross-linkage of enzymes with proteins 19,2° or polymers. 2~,22 Other approaches involve the modification of the enzyme by selective removal of carbohydrates, coupling of the recognition marker, and selection of a specific isoenzyme. 23,24 Multienzyme systems enclosed within artificial cells have been used to convert ammonia or urea into amino acids. 25-29 In our recent studies, artificial cells can convert urea or ammonia into essential amino acids, such as leucine, isoleucine, and valine. 3° Enzymes in the urea cycle have also been immobilized to carry out reactions in the urea cycle. 3~ By using artificial cells containing bacterial phenylalanine ammonialyase, we have solved the problem of the availability of the human enzyme system and the requirement for cofactor recycling. 32 By administering these systems orally we have also solved the problem of in vivo accumulation, resulting in ease of administration. 33'34We have carried out randomized control studies in rats with phenylketonuria33,34 and found that oral administration in the phenylketonuria rat model for 7 days resulted in the lowering of system phenylalanine levels from the control group 0f331.4 - 26.4 mg/dl to the treated group of 82.7 -+ 7.0 mg/dl (p < 0 . 0 0 1 ) . 33,34 The level in the treated group is not significantly different from that of normal rats (33.6 ± 29.3 mg/dl).
is G. Ihler and R. Glew, in "Biomedical Applications of Immobilized Enzymes and Proteins" (T. M. S. Chang, ed.), p. 219. Plenum, New York, 1977. 19 T. M. S. Chang, Biochem. Biophys. Res. Commun. 44, 1531 (19~1). 20 M. J. Poznansky, J. Appl. Biochem. Biotechnol. 2, 41 (1984). 21 A. Abuchowski and F. F. Davis, in "Enzymes as Drugs" (J. S. Holcenberg and J. Roberts, eds.), p. 367. Wiley (Interscience), New York, 1981. 22 R. L. Foster and T. Wileman, J. Pharm. Pharmacol. 31 (Suppl.), 37P (1979). G. A. Grabowski and R. J. Desnick, in "Enzymes as Drugs" (J. S. Holcenberg and J. Roberts, eds.), p. 167. Wiley (Interscience), New York, 1981. 24 G. Gregoriadis and M. F. Dean, Nature (London) 278, 603 (1981). 25 T. M. S. Chang, Enzyme Eng. 5, 225 (1980). 26 y. T. Yu and T. M. S. Chang, Enzyme Microb. Technol. 4, 327 (1982). 27 T. M. S. Chang, this series, Vol. 136, p. 67. E. Ilan and T. M. S. Chang, Appl. Biochem. Biotechnol. 13, 221 (1986). 29 H. P. Wahl and T. M. S. Chang, J. Mol. Catalysis 39, 147 (1986). 3o K. F. Gu and T. M. S. Chang, Int. J. Biomater., Artif Cells Artif. Organs 15, 297 (1987). 31 N. Siegbahn and K. Mosbach, FEBS Lett. 137, 6 (1982). 32 L. Bourget and T. M. S. Chang, Appl. Biochem. Biotechnol. 10, 57 (1984). 33 L. Bourget and T. M. S. Chang, FEBS Lett., in press (1985). L. Bourget and T. M. S. Chang, Biochim. Biophys. Acta 883, 432 (1986).
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Asparaginase and Other Enzymes in Chemotherapy Since the first publication by Broome, 35 extensive research has been carried out using asparaginase and other enzymes which can degrade essential amino acids required by tumor cells. 5,6 The use of these enzymes is associated with problems related to toxicity, immunogenicity, and duration of action. Initial attempts at immobilization involved the successful use of microencapsulated asparaginase to suppress the growth of lymphosarcoma in mice. 1~Because of the importance of cancer treatment, extensive study has been carried out since then by many groups using all available immobilization approaches, j-8,21,22,36-41 Details of the different immobilization approaches have been discussed under the section "Inborn Errors of Metabolism" in this chapter. Asparaginase itself can also be modified: For example, it can survive longer in circulation by deamination, acylation, and carbodiimide reactions with free amino groups. Certain poly(amino acids) have also been incorporated into the enzyme to prolong half-life and decrease immunogenicity. 6 Poly(N-vinylpyrrolidine) conjugated to fl-o-N-acetylhexosaminidase A increased survival and decreased immunogenicity. 6
Detoxification Many of the detoxifying functions in the body are carried out by enzymatic reactions; as a result it is not too surprising that research has been carried out to investigate the use of immobilized enzymes in detoxification. It is now clearly established that the earlier high expectations of immobilized enzymes in detoxifying applications have been overshadowed by the much more rapid advances in the use of artificial cell-immobilized adsorbents for detoxification.l,8,42-48 Microencapsulated activated 35 j. D. Broome, J. Exp. Med. 118, 99 (1963). 36 T. M. S. Chang, Enzyme 14, 95 (1973). 37 E. D. SiuChong and T. M. S. Chang, Enzyme 18, 218 (1974). 38 T. Mori, T. Tosa, and I. Chibata, Biochim. Biophys. Acta 321, 653 (1973). 39 L. D. S. Hudson, M. B. Fiddler, and R. J. Desnick, J. Pharmacol. Exp. Ther. 208, 507 (1979). 40 G. Schmer and J. S. Holcenberg, in "Enzymes as Drugs" (J. S. Holcenberg and J. Roberts, eds.), p. 385. Wiley (Interscience), New York, 1981. 4i D. A. Cooney, H. H. Weetall, and E. Long, Biochem. Pharmacol. 24, 503 (1975). 42 T. M. S. Chang, J. F. Coffey, P. Barre, A. Gonda, J. H. Dirks, M. Levy, and C. Lister, Can. Med. Assoc. J. 108, 429 (1973). 43 T. M. S. Chang, J. F. Coffey, C. Lister, E. Taroy, and A. Stark, Trans. Am. Soc. Artif. Intern. Organs 19, 87 (1973). 44 T. M. S. Chang, Clin. Toxicol. 17, 529 (1980). 45 M. C. Gelfand, J. F. Winchester, J. H. Knepshield, K. M. Hansen, S. L. Cohan, B. S. Stranch, K. L. Geoly, A. C. Kennedy, and G. E. Schreiner, Trans. Am. Soc. Artif. Intern. Organs 23, 599 (1977).
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charcoal when used in extracorporeal circulation in hemoperfusion is much more effective than standard hemodialysis or any presently available immobilized enzyme system for the removal of many drugs. This is particularly so in the case of poisoning. Many of the commonly used medications in suicidal overdose can be very effectively removed by hemoperfusion at a level close to the blood flow rate. 8,42-48 As a result, hemoperfusion using artificial cells containing activated charcoal has now replaced standard hemodialysis as the treatment of choice for many types of poisoning. However, this approach is not as specific as enzyme systems. Where it is important to have specificity there is still an important place for immobilized enzyme systems.
Chronic Renal Failure Renal failure results in the inability of the body to excrete waste metabolites, electrolytes, and water. The standard treatment is hemodialysis. Studies on patients demonstrated that 100 g of artificial cells containing activated charcoal can remove uremic metabolites and toxins much more efficiently than the hemodialysis machine.l,8,46-51 Hemoperfusion in series with dialysis49-55 has been successfully used to cut down the time required for hemodialysis and also for the treatment of uremic complications. 46-55 A recent crossover control clinical trial shows that 8 hours/ week hemoperfusion-hemodialysis is as effective as 12 hours/week hemodialysis alone. 55 A second-generation artificial kidney has been developed consisting of 100 g of artificial cells containing activated char-
46 V. Bonomini and T. M. S. Chang (eds.), "Hemoperfusion" (Contributions to Nephrology Series). Karger, Basel, Switzerland, 1982. 47 S. Sideman and T. M. S. Chang (eds.), "Hemoperfusion: I. Artificial Kidney and Liver Support and Detoxification." Hemisphere, Washington, 1980. 48 T. M. S. Chang and N. Nicolaev, eds., Int. J. Biornater., Artif. Cells Artif. Ogans 15, 1 (1987). 49 T. M. S. Chang, E. Chirito, P. Barre, C. Cole, and M. Hewish, Trans. Am. Soc. Artif. Intern. Organs 21, 502 (1975). 50 T. M. S. Chang, Kidney Int. 10, $305 (1976). 51 T. M. S. Chang, Clin. Nephrol. 11, 111 (1979). 52 j. F. Winchester, M. T. Apiliga, J. M. MacKay, and A. C. Kennedy, Kidney Int. 10, 315 (1976). 53 S. Stefoni, L. Coli, G, Feliciangeli, L. Beldrati, and V. Bonomini, Int. J. Artif. Organs 3, 348 (1980). 54 A. M. Martin, T. K. Gibbins, T. Kimmit, and F. Rennie, Dial. Transplant. 8, 135 (1979). 55 T. M. S. Chang, P. Barre, and S. Kuruvilla, Trans. Am. Soc. Artif. Intern. Organs 31, 572 (1985).
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coal in series with a small ultrafiltrator.49,56This way uremic metabolites, water, and sodium chloride can all be controlled. Oral adsorbents can be used to remove potassium and phosphate. The only additional step required before this second-generation artificial kidney is completed is the need to remove urea. Unfortunately, adsorbents available at present do not have sufficient capacity for urea. Artificial cells containing urease can be used in extracorporeal blood circulation to rapidly convert urea into ammonia? 3 However the amount of ammonium adsorbent required is too large for extracorporeal blood recirculation in patients. This principle has been successfully adopted for use in dialyzate regeneration where a large amount of adsorbent can be u s e d . 57 The use of microencapsulated urease together with ammonium adsorbent for oral administration ~,58,59resulted in a significant lowering of systemic urea levels in the rat. This has been developed further6° and tested clinically in patients. 6~Initial clinical trials in patients demonstrated that this approach is effective in lowering the urea level in chronic renal failure patients; however the volume required for ingestion needs to be decreased. 6~ We have recently carried out further study and analysis and demonstrated that with proper design of the immobilized system it should be possible to decrease the volume required. 62 This promising approach using immobilized urease may become the one additional step required to complete the second-generation artificial kidney based on hemoperfusion and ultrafiltration. Instead of using ammonia adsorbent, we have prepared artificial cells containing a multienzyme system of urease, glutamate dehydrogenase, glucose, dehydrogenase, and a transaminase. 25--'9 This way each artificial cell can convert urea to ammonia which is then converted to glutamic acid and other amino acids. The cofactor required is recycled by glucose dehydrogenase. More recently, by using artificial cells with another multienzyme system and cofactor recycling, we can convert urea into essential amino acids (leucine, isoleucine, and valine.) 3°
56 T. M. S. Chang, E. Chirito, P. Barre, C. Cole, C. Lister, and E. Resurreccion, Artif. Organs 3, 127 (1979). ~7 A. Gordon, A. J. Lewin, M. H. Maxwell, and R. Martin, in "Artificial Kidney, Artificial Liver and Artificial Cells" (T. M. S. Chang, ed.), p. 23. Plenum, New York, 1978. 58 T. M. S. Chang and S. K. Loa, Physiologist 13, 70 (1970). 59 Z. M. S. Chang, Kidney Int. 10, $218 (1976). 6o D. L. Gardner, R. D. Falb, B. C. Kim, and D. C. Emmerling, Trans. Am. Soc. Artif. Intern. Organs 17, 239 (1971), 61 C. Kjellstrand, H. Borges, C. Pru, D. Gardner, and D. Fink, Trans. Am. Soc. Artif. Intern. Organs 27, 24 (1981). 62 E. A. Wolfe and T. M. S. Chang, Int. J. Artif. Organs 10, 43 (1987).
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Liver Failure
Hemoperfusion using artificial cells containing activated charcoal can result in the temporary recovery of consciousness of grade IV coma patients. 63-65 When treated in the earlier grades of acute fulminant hepatic failure there is a 70% long-term recovery rate as compared to 30% in control groups. 66-68 However this is only useful in acute liver failure. In order to complete the artificial liver support system for use in chronic hepatic failure like cirrhosis, many other metabolic functions of the liver in addition to detoxification have to be supplemented. Metabolic disturbances in amino acids is very marked in liver failure, with elevations of aromatic acids like tyrosine and phenylalanine. We have microencapsulated tyrosinase in artificial cells. These cells are then used in an extracorporeal system for hemoperfusion in galactosamineinduced fulminant hepatic failure r a t s . 69,7° Hemoperfusion results in significant lowering of the tyrosine level. We have also prepared artificial cells containing phenylalanine ammonia-lyase to effectively remove phenylalanine in vitro and also in v i o o . 32-34 Another waste metabolite to be removed is ammonia. We have carried out studies where artificial cells containing multienzyme systems of glutamate dehydrogenase, glucose dehydrogenase, and a transaminase have been used to convert ammonia to amino acid with the required cofactor recycled by glucose dehydrogenase. 25-z9Further research has led to the use of artificial cells to convert ammonia into essential amino acids of leucine, isoleucine, and valine. 3° Other groups have used other methods of immobilizing multienzyme systems. An exciting approach is the immobilization of the enzymes required for the urea cycle. 31 Microencapsulation within artificial cells with hepatic organelles to carry out the required enzymatic functions has also been studied. 71Attempts to supplement the detoxifying function have also been 63 T. M. S. Chang, Lancet 2, 1371 (1972). 64 R. Williams and I. M. Murray-Lyon, "Artificial Liver Support." Pitman, London, 1975. 65 M. C. Gelfand, J. F. Winchester, J. H. Knepshield, S. L. Cohan, and G. E. Schreiner, Trans. Am. Soc. Artif. Intern. Organs 24, 239 (1978). 66 T. M. S. Chang, C. Lister, E. Chirito, P. O'Keefe, and E. Resurreccion, Trans. Am. Soc. Artif. Intern. Organs 24, 243 (1978). 67 T. M. S. Chang, in "Artificial Liver Support" (G. Brunner and F. W. Schmidt, eds.), p. 126. Springer-Verlag, Berlin, 1981. 6s A. E. S. Gimson, S. Brande, P. J. Mellon, J. Canalese, and R. Williams, Lancet 2, 681 (1982). 69 C. D. Shu and T. M. S. Chang, Int. J. Artif. Organs 4, 82 (1981). 7o Z. Q. Shi and T. M. S. Chang, Trans. Am. Soc. Artif. lntern. Organs 28, 205 (1982). 71 Z. Y. Yuan and T. M. S. Chang, Int. J. Artif. Organs 9(1), 63 (1986).
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analyzed using enzymes extracted from liver cells and immobilized in Sepharose. 72 A recent review is available. 73 Microencapsulated Cell Cultures in Diabetes Mellitus and Other Applications Artificial cells can be used to microencapsulate biological cells, and we have proposed the use of this approach for cells like islet cells, hepatocytes, and other cells for in vivo replacement to avoid immunological rejection. 1,74,75 This has now been supported by Sun et al. who have microencapsulated islet cells. 76'77 They have shown that intraperitoneal injection can successfully maintain diabetic rats for over 12 months. The microencapsulated islet cells respond to glucose and secrete the required amount of insulin to maintain a suitable glucose level in diabetic rats. Microencapsulation of hybridoma cell cultures have also been used for the large-scale production of monoclonal antibodies and interferon. TM Our recent studies show that microencapsulated hepatocyte culture can increase the survival of acute liver failure rats. 79 lmmunoadsorbents We have complexed albumin to collodion-coated activated charcoal to improve blood compatibility and to allow albumin to facilitate the transport of loosely bound protein molecules.l,59 Terman found that this albumin-collodion-charcoal system could also be used to remove albumin antibodies in a perfusion system. 8° He later replaced albumin with other types of antigens or antibodies for immunosorbents in perfusion. 8° He found in preliminary studies that plasma perfusion with protein A-collodion-activated charcoal decreased the size of breast carcinoma in pa72 G. Brunner and F. W. Schmidt (eds.), "Artificial Liver Support." Springer-Verlag, Berlin, 1981. 73 T. M. S. Chang, Sem. Liver Dis. Ser. 6, 148 (1986). 74 T. M. S. Chang, Ph.D. thesis. McGill University, Montreal, Quebec, 1965. 75 T. M. S. Chang, F. C. Macintosh, and S. G. Mason, Can. J. Physiol. Pharmacol. 44, 115 (1966). 76 F. Lira and A. M. Sun, Science 210, 908 (1980). 77 A. M. Sun, G. M. O'Shea, and M. F. A. Goosen, J. Appl. Biochem. Biotechnol. 10, 87 (1984). 78 Damon Corp., "Bulletin on Tissue Microencapsulation." Damon Corp., Needham Heights, Massachusetts, 1981. 79 H. Wong and T. M. S. Chang, Int. J. Artif. Organs 9, 335 (1986). 8o D. S. Terman, in "Sorbents and Their Clinical Applications" (C. Giordano, ed.), p. 470. Academic Press, New York, 1980.
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tients, s~ However, we have pointed out that this way the immobilized protein A may be released into the circulation acting as a slow release system. 82 Protein A has also been immobilized by covalent linkage to Sepharose for hemoperfusion in patients by groups. 83 A synthetic immunoadsorbent has been prepared for the removal of blood group antibodies anti-A and anti-B. However, the problems of blood compatibility and release of particulates has limited its application. We found that by using an ultrathin coating of collodion and albumin, a synthetic immunoadsorbent could be made that was blood compatible and would not release particles so that it could be used for hemoperfusion. 84 Clinical trials have been conducted on patients to remove anti-A and anti-B blood group antibodies before bone marrow transplantation. 85 Blood Substitutes
Immobilization technology has also been used in the search for a red blood cell substitute. Outside the red blood cell, hemoglobin is converted in the circulation to a dimer and removed rapidly. We have earlier demonstrated that hemoglobin could be cross-linked by bifunctional agents to polyhemoglobin. 1.12 This resulted, however, in an increase in oxygen affinity so that oxygen is not readily released for use when required. Benesch et al. 86 demonstrated that pyridoxalation of hemoglobin decreases oxygen affinity. There is a renewed interest at present in the use of polyhemoglobin.87 Work being carried o u t 87-91 has shown that peridoxylated hemoglobin can be cross-linked to soluble polyhemoglobin which can reversibly carry oxygen. In this form the polyhemoglobin can survive in the circulation so that 3 hours after intravenous injection 77% still remains in the circulation as compared to 25% for free hemoglobin. ss-9° It has also been shown that this type of polyhemoglobin is effective in resuscitating rats with hemorrhagic s h o c k . 92 Unlike fluorocars~ D. S. Terman, J. B. Young, W. T. Shearer, and Y. Daskal, N. Engl. J. Med. 305, 1195 (1981). 82 T. M. S. Chang, N. Engl. J. Med. 306, 936 (1982). 83 I. M. Nilsson, S. Jonsson, S. B. Sundquist, A. Ahlberg, and S. E. Bergentz, Immunology 14, 38 (1981). T. M. S. Chang, Trans. Am. Soc, Artif. Intern. Organs 26, 546 (1980). 85 W. I. Bensinger, D. A. Baker, C. D. Buckner, R. A. Clift, and E. D. Thomas, N. Engl. J. Med. 304, 160 (1981). 86 R. E. Benesch, R. Benesch, R. D. Renthal, and N. Maeda, Biochemistry 11, 3576 (1972). 87 T. M. S. Chang, Trans. Am. Soc. Artif. Intern. Organs 26, 354 (1980). a8 p. Keipert, J. Minkowitz, and T. M. S. Chang, Int. J. Artif. Organs 5, 383 (1982). 89 p. Keipert and T. M. S. Chang, Trans. Am. Soc. Artif. Intern. Organs 29, 329 (1983). 9o p. Keipert and T. M. S. Chang, Appl. Biochem. Biotechnol. 10, 133 (1984). 9J R. B. Bolin, R. P. Geyer, and G. J. Nemo (eds.), Adv. Blood Substitute Res. (1983).
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b o n s , 91 cross-linked hemoglobin is biodegradable in the body after use.
Repeated injection of homologous polyhemoglobin is not immunogenic. 93,94 Extensive research is being carried out using pyridoxalated polyhemoglobin or microencapsulated hemoglobin as blood substitute .93-% In Vivo Evaluations As shown in the above discussions, extensive studies have already been carried out on the use of different types of immobilization techniques. Perhaps one of the next major steps should be to study the different possible routes of in vivo actions of the immobilized systems. For eventual clinical application, the routes used in experimental animal studies may have to be greatly modified. The following factors are extremely important in considering any system for actual clinical application. Mode o f Action If the mode of action of the immobilized enzymes and cells requires rapid interactions with body fluids, then one of the best routes is to use extracorporeal blood circulation. Intraperitoneal administration is another alternative although this is usually used in experimental investigations in animals rather than for actual clinical applications. If the metabolites can diffuse freely across the intestinal tract, another possible approach is to use oral ingestion of immobilized enzymes and cells. In controlled release, intramuscular or subcutaneous injection could be carried out. The greatest problem is encountered when the immobilized enzyme or cells require localization in specific organs, cells, or intraceUular organelles. Biocompatibility Depending on the mode of action and therefore the route of administration, the requirements for biocompatibility are different. In extracorporeal blood circulation the immobilized system has to be blood compatible, nontoxic, and should not release any harmful material into the circulation. In systems involving intraperitoneal injection, intramuscular injection, or subcutaneous injection, the material also has to be biocompatible, sterile, 92 p. 93 C. 94 T. 9~ T. T.
Keipert and T. M. S. Chang, J. Biomater., Artif. Organs Med. Devices 13, 1 (1985). M. Hertzman, P. E. Keipert, T. M. S. Chang, Int. J. Artif. Organs 9, 179 (1986). M. S. Chang and R. Varma, Int. J. Biomater., Artif. Cells Artif. Organs 15,443 (1987). M. S. Chang, Int. J. Biomater., Artif. Cells Artif. Organs 15, 323 (1987). M. S. Chang and R. Geyer, eds., "Blood Substitutes." Dekker, New York, 1988.
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and nontoxic, in addition to being noncarcinogenic and nonimmunogenic. Oral administration involves the least requirements for biocompatibility. Accumulation
Immobilized enzymes and cells implanted by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection are retained for some time in the body. Since the duration of action of immobilized enzymes and cells is not indefinite, it is necessary to repeat the injections at various intervals. This may not be a problem in feasibility studies in animals. However, accumulation of immobilized material in the body could be a major problem in actual clinical applications if the treatments have to be repeated over a long period of time. Biodegradable polymers like poly(lactic acid), poly(glycolic acid), or cross-linked protein could solve the problem. However, proteins, enzymes, and cells which have been immobilized would be exposed and left behind when the biodegradable materials are removed. It is important to assure that this does not give rise to allergic and immunological reactions. To solve the problem of introduction of material into the body one could use the extracorporeal route, for instance, extracorporeal blood circulation, in those cases where this is applicable. The immobilized materials, having carried out their functions extracorporeally, can be disconnected and removed with no problems of accumulation. Another approach to solve the problem of in vivo accumulation is the use of oral administration in those few cases where this route is applicable. On the basis of the above factors, one of the following approaches could be selected. Methodology Intraperitoneal Administration
The intraperitoneal route of administration is a convenient way for investigating the in vivo action of immobilized enzymes and cells on substances present in body fluids. While this approach is very convenient and useful for initial feasibility studies in animals, the peritoneal cavity is an extremely sensitive area. Intraperitoneal introduction of foreign materials in humans may result in peritonitis due to reaction to foreign materials or infections. Furthermore, chronic fibrotic reaction to the long-term intraperitoneal introduction of foreign material could result in intestinal obstruction and other major problems. Thus in most cases, the peritoneal route should be used only as a convenient initial feasibility study in animals, to be followed by other approaches for actual clinical applications.
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Although large animals such as dogs and primates can be used, this approach is most convenient in introducing large amounts of immobilized proteins, enzymes, and cells into smaller animals like mice and rats. The procedure in small animals is very simple and can be summarized as follows. When the particles are about 50/~m in diameter, they can be injected as a suspension through an 18- or 20-gauge needle. Care should be taken to make sure that the injection is not made into the subcutaneous tissue or into the lumen of the gastrointestinal tract. Two millimeters of suspension can be introduced into each rat without causing any problems. With particles much larger than 50/zm diameter, one has to use larger needles. In these cases, it may be necessary to suture the hole made by the larger bore needle in smaller animals like rats and mice. Small flexible particles smaller than 8 p.m are removed by the lymphatic system from the peritoneal cavity. Larger particles are retained in the peritoneal cavity to act on metabolites equilibrating across the peritoneal membrane. However, unless the material is very biocompatible, it will be encased by a fibrous capsule because of foreign body reactions. This is a convenient approach which has been used in the initial investigation of many of the earlier studies employing immobilized enzymes and cells. These involved the use of microencapsulated urease as a model system,l,12,74,75 microencapsulated catalase for replacement of enzyme deficiency or acatalsemia, 1,1°,~4 microencapsulated asparaginase for tumor suppression separation, 11,36-38 microencapsulated islet cells for diabetes m e l l i t u s , 76'77 and microencapsulated hepatocytes for acute liver failure. 79
Extracorporeal Peritoneal Recirculation Extracorporeal peritoneal recirculation attempts to make use of the intraperitoneal approach without introducing any foreign materials into the peritoneal cavity. Immobilized enzymes or cells are retained in a small column; 2 ml sterilized peritoneal dialysis fluid is injected into the peritoneal cavity of each anesthetized mouse. The peritoneal fluid is continuously recirculated through the extracorporeal column and returned to the peritoneal cavity. At the completion of the recirculation the extracorporeal immobilized enzymes and cells are discarded or stored for later use. This approach has been used successfully in acatalasemic mice for the enzymatic removal of peroxides by artificial cells containing catalase. 1,1°
Extracorporeal Blood Recirculation Immobilized proteins, enzymes, and cells are retained in an extracorporeal column. 13 Blood from the animal is recirculated through the column by means of a pump. This technique can be used conveniently in
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animals as small as rats and in acute studies can be carried out in anesthetized animals with femoral artery and vein cannulation. In cases where repeated extracorporeal circulation is required, one can use chronic cannulation so that repeated extracorporeal circulation can be carried out in fully conscious animals. A number of techniques can be used depending on the size of the animal. When using rats two approaches are possible. Using the jugular vein and carotid artery to form an arteriovenous shunt, the cannula is exteriorized near the top of the head, and the tubings are attached permanently to the top of the cage. This results in restrictions in the mobility of the animal. Another approach is to use the femoral artery and vein and exteriorize the cannulae through the tail veins. 97 This is much more convenient since the animal, when not being treated, can be freely mobile. In the case of extracorporeal blood circulation one does not introduce foreign material into the body. However the immobilized enzymes and cells have to be blood compatible, sterile, and not release any harmful material. Artificial cell-immobilized adsorbents have already become a routine procedure in clinical practice in patients for the treatment of poisoning, 42-48 chronic renal failure, 46-56 and removal of aluminum. 98 With the large amount of clinical experience already available for immobilized adsorbents, clinical applications of immobilized enzymes and cells could be easily developed. Experimental investigation of immobilized enzymes and cells includes the use of immobilized urease, 13asparaginase, 4°,41 tyros i n a s e , 8'69'7° heparinase, 99 and other enzymes.~-8
Oral Administration In some cases, the metabolites to be acted on can equilibrate readily from the bloodstream into the lumen of the gastrointestinal tract. In these cases oral administration of immobilized enzymes is possible. This is one of the most convenient approaches. A gastric tubing can be easily introduced into the stomach of an unanesthetized rat and materials inserted by means of a syringe. This approach has been used for the removal of urea using artificial cell-immobilized urease and ammonia adsorbent in r a t s 1,58-62 and the recent use of artificial cell-immobilized phenylalanine ammonia-lyase in phenylketouria r a t s . 32-34 In humans, oral route of administration is the most convenient and least problematic, since it will be equivalent to drinking a suspension or taking a few pills.
97 y . Tabata and T. M. S. Chang, J. Artif. Organs 6, 213 (1982). 98 T. M. S. Chang and P. Barre, Lancet 2, 1051 (1983). 99 R. Langer, P. J. Blackshear, T. M. S. Chang, M. D. Klein, and J. S. Schultz, Trans. Am. Soc. Artif. Intern. Organs 32, 639 (1986).
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Intravascular Injection
Immobilized enzymes and cells can be injected intravenously into the systemic circulation, e.g., through foreleg veins in dog, ear veins in rabbits, and tail veins in rats and mice. Chronic cannulations could also be used for long-term intermittent administration. Most intravenously injected particulate matters larger than 2 t~m in diameter are filtered out in the pulmonary circulation. Most of the smaller particles which pass through the pulmonary circulation are removed by the reticuloendothelial system particularly the liver and spleen. Immobilized enzymes which have been injected intravenously localize in the reticuloendothial system. 1,2,5-8A5-~8 Immobilized enzymes or proteins in the soluble form such as cross-linked hemoglobin, 87-96 enzymes cross-linked to albumin] ° or enzymes cross-linked to soluble polymer2~,22 can survive much longer in the circulation. The use of magnetic microcapsules ~3 allows the material to be localized by externally applied magnetic forces. Intraarterial injections have been used to localize material in specific organs. Intramuscular or Subcutaneous Injection
Intramuscular or subcutaneous injection routes are more suitable in cases where the immobilized system is used as a depot for slow release of immobilized materials like hormones and drugs. Discussion The first in vivo studies carried out about 20 years ago demonstrated the possible medical application of immobilized enzymes and cells 1,t°-13 ; however, many different approaches are now available. 1-8 Further progress toward actual routine clinical applications will depend on careful design of in vivo experiments which can be adapted for use in clinical applications. The clinical uses of artificial cells containing adsorbents have developed rapidly so that, although this technique started much later than immobilized enzymes, it is already in routine clinical use. 4L42Experience gained can be applied to future large-scale clinical applications of immobilized proteins, enzymes, and cells. With the recent rapid progress in biotechnology, the required proteins, enzymes, and cells will be more readily available. Many factors will contribute to the future possibilities of clinical application of immobilized proteins, enzymes, and cells. Artificial cells or microencapsulated enzyme in the intestine can act effectively on plasma substrates which can enter the intestine. ~°° This may lead to an easy route for routine applications of immobilized enzymes. 100 L. Bourget and T. M. S. Chang, J. Biomat. Artif. Cells Artif. Organs (in press).
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[40] M e d i c a l A p p l i c a t i o n s o f I m m o b i l i z e d P r o t e i n s , E n z y m e s , a n d Cells By T. M. S. CHANG
Introduction Discussion of the medical applications of immobilized proteins, enzymes, and cells 1-9 usually emphasizes methods of enzyme immobilization and areas of possible application. Since extensive in oivo evaluations are being carried out in this area of research, methodologies for in vivo applications will also be covered in this chapter. About 20 years ago immobilized enzymes in the form of artificial cells containing catalase, asparaginase, or urease were shown to be effective for replacing hereditary enzyme deficiency in acatalasemic miceJ°; for suppressing the growth of lymphosarcoma in mice~; or for decreasing system urea levels in animals. ~2,13 Since that time, despite extensive efforts by many researchers using all available immobilization technologies, progress toward the actual large-scale clinical application of immobilized enzymes and cells has been very slow.l-9 This does not negate the earlier promise of the potential medical applications of immobilized enzymes and cells but shows the complexities and problems associated with this type of approach. Although proteins, enzymes, and cells are potentially much more specific, powerful, and useful, there are also major problems related to immunogenicity, toxicity, and needs for targeting. Furthermore, there T. M. S. Chang, "Artificial Cells." Thomas, Springfield, lllinois, 1972. 2 T. M. S. Chang (ed.), "Biomedical Applications of Immobilized Enzymes and Proteins," Vols. 1 and 2. Plenum, New York, 1977. 3 T. M. S. Chang (ed.), "Microencapsulation Including Artificial Cells." Humana, New York, 1984. 4 I. Chibata, "Immobilized Enzymes." Wiley (Interscience), New York, 1978. 5 j. S. Hoicenberg and J. Roberts (eds.), "Enzymes as Drugs." Wiley (Interscience), New York, 1981. 6 j. S. Holcenberg, Annu. Rev. Biochem. 51, 795 (1982). 7 K. J. Widder and R. Green, eds., this series, Vol. 112, p. 1. s T. M. S. Chang, Int. J. Biomater., Artif. Cells Artif. Organs 15, 1 (1987). 9 T. M. S. Chang, Appl. Biochem. Biotechnol. 10, 5 (1984). l0 T. M. S. Chang and M. J. Poznansky, Nature (London) 218, 242 (1968). 11 T. M. S. Chang, Nature (London) 229, 117 (1971). ~2 T. M. S. Chang, Science 146, 524 (1964). 13 T. M. S. Chang, Trans. Am. Soc. Artif. Intern. Organs 12, 13 (1966).
METHODS IN ENZYMOLOGY, VOL. 137
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
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are also problems related to the availability of suitable enzyme systems, the need for multienzyme system with cofactor requirements, and the viability of cell cultures. Recent progress in biotechnology is beginning to solve some of these problems. As a result an increasing number of researchers are seriously investigating the medical applications of immobilized proteins, enzymes, and cells. Since many of the applications are described elsewhere in this volume, we will discuss other examples not presented herein.
lnborn Error~ of Metabolism A number of years ago we reported the successful use of microencapsulated catalase for enzyme replacement in acatalasemic mice with congenital deficiency of the enzyme catalase.l° Whereas repeated injections of heterogenous catalase resulted in anaphylactic shocks, microencapsulated catalase did not result in immunological reactions and continued to carry out its functions. 1,1°,14However, this demonstration 15 years ago did not result in routine clinical application in this and other types of inborn errors of metabolism. There are many problems to be solved before immobilized enzymes and cells can be used routinely. Three of the most important problems are (1) the availability of enzymes, (2) the requirement for multienzyme systems in many conditions, and (3) the need for targeting to specific organs or intracellular locations in many types of congenital enzyme defects. Extensive effort has been carried out to solve these problems. One of the most intensively studied areas involves the use of liposomes for the microencapsulation of enzymes. 15Thus it has been reported that liposomes containing sulfatide, phosphatidylcholine, and cholesterol may be incorporated into the central nervous system. 16 Surface incorporation of antibodies onto liposomes has also been used in targeting. 15 Other modifications include surface attachment of lectins, glycoproteins, ligands, and others. Liposomes have the advantage of being biodegradable; however, they appear to enhance immune response to the entrapped protein. This area has been reviewed in detail elsewhere. 17 Red blood cells have also been used to microencapsulate enzymes by 14 M. J. Poznansky and T. M. S. Chang, Biochim. Biophys. Acta 334, 103 (1974). ~5 G. Gregoriadis, in "Enzyme Replacement Therapy of Lysosomal Storage Diseases" (J. M. Tager, J. M. Hooghwinkel, and W. T. Daoms, eds.), p. 131. North-Holland, Amsterdam, 1974. 16 M. Naoi and K. Yagi, Biochem. Int. 1, 591 (1980). 17 G. Gregoriadis, "Drug Carders in Biology and Medicine." Academic Press, New York, 1979.
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hemolysis and resealing. ~8Rh antibody-coated human red blood cells containing fl-glucosidase have been used for targeting in Gaucher's disease. 17 Other immobilization techniques have also been extensively investigated, 8 including the cross-linkage of enzymes with proteins 19,2° or polymers. 2~,22 Other approaches involve the modification of the enzyme by selective removal of carbohydrates, coupling of the recognition marker, and selection of a specific isoenzyme. 23,24 Multienzyme systems enclosed within artificial cells have been used to convert ammonia or urea into amino acids. 25-29 In our recent studies, artificial cells can convert urea or ammonia into essential amino acids, such as leucine, isoleucine, and valine. 3° Enzymes in the urea cycle have also been immobilized to carry out reactions in the urea cycle. 3~ By using artificial cells containing bacterial phenylalanine ammonialyase, we have solved the problem of the availability of the human enzyme system and the requirement for cofactor recycling. 32 By administering these systems orally we have also solved the problem of in vivo accumulation, resulting in ease of administration. 33'34We have carried out randomized control studies in rats with phenylketonuria33,34 and found that oral administration in the phenylketonuria rat model for 7 days resulted in the lowering of system phenylalanine levels from the control group 0f331.4 - 26.4 mg/dl to the treated group of 82.7 -+ 7.0 mg/dl (p < 0 . 0 0 1 ) . 33,34 The level in the treated group is not significantly different from that of normal rats (33.6 ± 29.3 mg/dl).
is G. Ihler and R. Glew, in "Biomedical Applications of Immobilized Enzymes and Proteins" (T. M. S. Chang, ed.), p. 219. Plenum, New York, 1977. 19 T. M. S. Chang, Biochem. Biophys. Res. Commun. 44, 1531 (19~1). 20 M. J. Poznansky, J. Appl. Biochem. Biotechnol. 2, 41 (1984). 21 A. Abuchowski and F. F. Davis, in "Enzymes as Drugs" (J. S. Holcenberg and J. Roberts, eds.), p. 367. Wiley (Interscience), New York, 1981. 22 R. L. Foster and T. Wileman, J. Pharm. Pharmacol. 31 (Suppl.), 37P (1979). G. A. Grabowski and R. J. Desnick, in "Enzymes as Drugs" (J. S. Holcenberg and J. Roberts, eds.), p. 167. Wiley (Interscience), New York, 1981. 24 G. Gregoriadis and M. F. Dean, Nature (London) 278, 603 (1981). 25 T. M. S. Chang, Enzyme Eng. 5, 225 (1980). 26 y. T. Yu and T. M. S. Chang, Enzyme Microb. Technol. 4, 327 (1982). 27 T. M. S. Chang, this series, Vol. 136, p. 67. E. Ilan and T. M. S. Chang, Appl. Biochem. Biotechnol. 13, 221 (1986). 29 H. P. Wahl and T. M. S. Chang, J. Mol. Catalysis 39, 147 (1986). 3o K. F. Gu and T. M. S. Chang, Int. J. Biomater., Artif Cells Artif. Organs 15, 297 (1987). 31 N. Siegbahn and K. Mosbach, FEBS Lett. 137, 6 (1982). 32 L. Bourget and T. M. S. Chang, Appl. Biochem. Biotechnol. 10, 57 (1984). 33 L. Bourget and T. M. S. Chang, FEBS Lett., in press (1985). L. Bourget and T. M. S. Chang, Biochim. Biophys. Acta 883, 432 (1986).
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Asparaginase and Other Enzymes in Chemotherapy Since the first publication by Broome, 35 extensive research has been carried out using asparaginase and other enzymes which can degrade essential amino acids required by tumor cells. 5,6 The use of these enzymes is associated with problems related to toxicity, immunogenicity, and duration of action. Initial attempts at immobilization involved the successful use of microencapsulated asparaginase to suppress the growth of lymphosarcoma in mice. 1~Because of the importance of cancer treatment, extensive study has been carried out since then by many groups using all available immobilization approaches, j-8,21,22,36-41 Details of the different immobilization approaches have been discussed under the section "Inborn Errors of Metabolism" in this chapter. Asparaginase itself can also be modified: For example, it can survive longer in circulation by deamination, acylation, and carbodiimide reactions with free amino groups. Certain poly(amino acids) have also been incorporated into the enzyme to prolong half-life and decrease immunogenicity. 6 Poly(N-vinylpyrrolidine) conjugated to fl-o-N-acetylhexosaminidase A increased survival and decreased immunogenicity. 6
Detoxification Many of the detoxifying functions in the body are carried out by enzymatic reactions; as a result it is not too surprising that research has been carried out to investigate the use of immobilized enzymes in detoxification. It is now clearly established that the earlier high expectations of immobilized enzymes in detoxifying applications have been overshadowed by the much more rapid advances in the use of artificial cell-immobilized adsorbents for detoxification.l,8,42-48 Microencapsulated activated 35 j. D. Broome, J. Exp. Med. 118, 99 (1963). 36 T. M. S. Chang, Enzyme 14, 95 (1973). 37 E. D. SiuChong and T. M. S. Chang, Enzyme 18, 218 (1974). 38 T. Mori, T. Tosa, and I. Chibata, Biochim. Biophys. Acta 321, 653 (1973). 39 L. D. S. Hudson, M. B. Fiddler, and R. J. Desnick, J. Pharmacol. Exp. Ther. 208, 507 (1979). 40 G. Schmer and J. S. Holcenberg, in "Enzymes as Drugs" (J. S. Holcenberg and J. Roberts, eds.), p. 385. Wiley (Interscience), New York, 1981. 4i D. A. Cooney, H. H. Weetall, and E. Long, Biochem. Pharmacol. 24, 503 (1975). 42 T. M. S. Chang, J. F. Coffey, P. Barre, A. Gonda, J. H. Dirks, M. Levy, and C. Lister, Can. Med. Assoc. J. 108, 429 (1973). 43 T. M. S. Chang, J. F. Coffey, C. Lister, E. Taroy, and A. Stark, Trans. Am. Soc. Artif. Intern. Organs 19, 87 (1973). 44 T. M. S. Chang, Clin. Toxicol. 17, 529 (1980). 45 M. C. Gelfand, J. F. Winchester, J. H. Knepshield, K. M. Hansen, S. L. Cohan, B. S. Stranch, K. L. Geoly, A. C. Kennedy, and G. E. Schreiner, Trans. Am. Soc. Artif. Intern. Organs 23, 599 (1977).
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charcoal when used in extracorporeal circulation in hemoperfusion is much more effective than standard hemodialysis or any presently available immobilized enzyme system for the removal of many drugs. This is particularly so in the case of poisoning. Many of the commonly used medications in suicidal overdose can be very effectively removed by hemoperfusion at a level close to the blood flow rate. 8,42-48 As a result, hemoperfusion using artificial cells containing activated charcoal has now replaced standard hemodialysis as the treatment of choice for many types of poisoning. However, this approach is not as specific as enzyme systems. Where it is important to have specificity there is still an important place for immobilized enzyme systems.
Chronic Renal Failure Renal failure results in the inability of the body to excrete waste metabolites, electrolytes, and water. The standard treatment is hemodialysis. Studies on patients demonstrated that 100 g of artificial cells containing activated charcoal can remove uremic metabolites and toxins much more efficiently than the hemodialysis machine.l,8,46-51 Hemoperfusion in series with dialysis49-55 has been successfully used to cut down the time required for hemodialysis and also for the treatment of uremic complications. 46-55 A recent crossover control clinical trial shows that 8 hours/ week hemoperfusion-hemodialysis is as effective as 12 hours/week hemodialysis alone. 55 A second-generation artificial kidney has been developed consisting of 100 g of artificial cells containing activated char-
46 V. Bonomini and T. M. S. Chang (eds.), "Hemoperfusion" (Contributions to Nephrology Series). Karger, Basel, Switzerland, 1982. 47 S. Sideman and T. M. S. Chang (eds.), "Hemoperfusion: I. Artificial Kidney and Liver Support and Detoxification." Hemisphere, Washington, 1980. 48 T. M. S. Chang and N. Nicolaev, eds., Int. J. Biornater., Artif. Cells Artif. Ogans 15, 1 (1987). 49 T. M. S. Chang, E. Chirito, P. Barre, C. Cole, and M. Hewish, Trans. Am. Soc. Artif. Intern. Organs 21, 502 (1975). 50 T. M. S. Chang, Kidney Int. 10, $305 (1976). 51 T. M. S. Chang, Clin. Nephrol. 11, 111 (1979). 52 j. F. Winchester, M. T. Apiliga, J. M. MacKay, and A. C. Kennedy, Kidney Int. 10, 315 (1976). 53 S. Stefoni, L. Coli, G, Feliciangeli, L. Beldrati, and V. Bonomini, Int. J. Artif. Organs 3, 348 (1980). 54 A. M. Martin, T. K. Gibbins, T. Kimmit, and F. Rennie, Dial. Transplant. 8, 135 (1979). 55 T. M. S. Chang, P. Barre, and S. Kuruvilla, Trans. Am. Soc. Artif. Intern. Organs 31, 572 (1985).
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coal in series with a small ultrafiltrator.49,56This way uremic metabolites, water, and sodium chloride can all be controlled. Oral adsorbents can be used to remove potassium and phosphate. The only additional step required before this second-generation artificial kidney is completed is the need to remove urea. Unfortunately, adsorbents available at present do not have sufficient capacity for urea. Artificial cells containing urease can be used in extracorporeal blood circulation to rapidly convert urea into ammonia? 3 However the amount of ammonium adsorbent required is too large for extracorporeal blood recirculation in patients. This principle has been successfully adopted for use in dialyzate regeneration where a large amount of adsorbent can be u s e d . 57 The use of microencapsulated urease together with ammonium adsorbent for oral administration ~,58,59resulted in a significant lowering of systemic urea levels in the rat. This has been developed further6° and tested clinically in patients. 6~Initial clinical trials in patients demonstrated that this approach is effective in lowering the urea level in chronic renal failure patients; however the volume required for ingestion needs to be decreased. 6~ We have recently carried out further study and analysis and demonstrated that with proper design of the immobilized system it should be possible to decrease the volume required. 62 This promising approach using immobilized urease may become the one additional step required to complete the second-generation artificial kidney based on hemoperfusion and ultrafiltration. Instead of using ammonia adsorbent, we have prepared artificial cells containing a multienzyme system of urease, glutamate dehydrogenase, glucose, dehydrogenase, and a transaminase. 25--'9 This way each artificial cell can convert urea to ammonia which is then converted to glutamic acid and other amino acids. The cofactor required is recycled by glucose dehydrogenase. More recently, by using artificial cells with another multienzyme system and cofactor recycling, we can convert urea into essential amino acids (leucine, isoleucine, and valine.) 3°
56 T. M. S. Chang, E. Chirito, P. Barre, C. Cole, C. Lister, and E. Resurreccion, Artif. Organs 3, 127 (1979). ~7 A. Gordon, A. J. Lewin, M. H. Maxwell, and R. Martin, in "Artificial Kidney, Artificial Liver and Artificial Cells" (T. M. S. Chang, ed.), p. 23. Plenum, New York, 1978. 58 T. M. S. Chang and S. K. Loa, Physiologist 13, 70 (1970). 59 Z. M. S. Chang, Kidney Int. 10, $218 (1976). 6o D. L. Gardner, R. D. Falb, B. C. Kim, and D. C. Emmerling, Trans. Am. Soc. Artif. Intern. Organs 17, 239 (1971), 61 C. Kjellstrand, H. Borges, C. Pru, D. Gardner, and D. Fink, Trans. Am. Soc. Artif. Intern. Organs 27, 24 (1981). 62 E. A. Wolfe and T. M. S. Chang, Int. J. Artif. Organs 10, 43 (1987).
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Liver Failure
Hemoperfusion using artificial cells containing activated charcoal can result in the temporary recovery of consciousness of grade IV coma patients. 63-65 When treated in the earlier grades of acute fulminant hepatic failure there is a 70% long-term recovery rate as compared to 30% in control groups. 66-68 However this is only useful in acute liver failure. In order to complete the artificial liver support system for use in chronic hepatic failure like cirrhosis, many other metabolic functions of the liver in addition to detoxification have to be supplemented. Metabolic disturbances in amino acids is very marked in liver failure, with elevations of aromatic acids like tyrosine and phenylalanine. We have microencapsulated tyrosinase in artificial cells. These cells are then used in an extracorporeal system for hemoperfusion in galactosamineinduced fulminant hepatic failure r a t s . 69,7° Hemoperfusion results in significant lowering of the tyrosine level. We have also prepared artificial cells containing phenylalanine ammonia-lyase to effectively remove phenylalanine in vitro and also in v i o o . 32-34 Another waste metabolite to be removed is ammonia. We have carried out studies where artificial cells containing multienzyme systems of glutamate dehydrogenase, glucose dehydrogenase, and a transaminase have been used to convert ammonia to amino acid with the required cofactor recycled by glucose dehydrogenase. 25-z9Further research has led to the use of artificial cells to convert ammonia into essential amino acids of leucine, isoleucine, and valine. 3° Other groups have used other methods of immobilizing multienzyme systems. An exciting approach is the immobilization of the enzymes required for the urea cycle. 31 Microencapsulation within artificial cells with hepatic organelles to carry out the required enzymatic functions has also been studied. 71Attempts to supplement the detoxifying function have also been 63 T. M. S. Chang, Lancet 2, 1371 (1972). 64 R. Williams and I. M. Murray-Lyon, "Artificial Liver Support." Pitman, London, 1975. 65 M. C. Gelfand, J. F. Winchester, J. H. Knepshield, S. L. Cohan, and G. E. Schreiner, Trans. Am. Soc. Artif. Intern. Organs 24, 239 (1978). 66 T. M. S. Chang, C. Lister, E. Chirito, P. O'Keefe, and E. Resurreccion, Trans. Am. Soc. Artif. Intern. Organs 24, 243 (1978). 67 T. M. S. Chang, in "Artificial Liver Support" (G. Brunner and F. W. Schmidt, eds.), p. 126. Springer-Verlag, Berlin, 1981. 6s A. E. S. Gimson, S. Brande, P. J. Mellon, J. Canalese, and R. Williams, Lancet 2, 681 (1982). 69 C. D. Shu and T. M. S. Chang, Int. J. Artif. Organs 4, 82 (1981). 7o Z. Q. Shi and T. M. S. Chang, Trans. Am. Soc. Artif. lntern. Organs 28, 205 (1982). 71 Z. Y. Yuan and T. M. S. Chang, Int. J. Artif. Organs 9(1), 63 (1986).
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analyzed using enzymes extracted from liver cells and immobilized in Sepharose. 72 A recent review is available. 73 Microencapsulated Cell Cultures in Diabetes Mellitus and Other Applications Artificial cells can be used to microencapsulate biological cells, and we have proposed the use of this approach for cells like islet cells, hepatocytes, and other cells for in vivo replacement to avoid immunological rejection. 1,74,75 This has now been supported by Sun et al. who have microencapsulated islet cells. 76'77 They have shown that intraperitoneal injection can successfully maintain diabetic rats for over 12 months. The microencapsulated islet cells respond to glucose and secrete the required amount of insulin to maintain a suitable glucose level in diabetic rats. Microencapsulation of hybridoma cell cultures have also been used for the large-scale production of monoclonal antibodies and interferon. TM Our recent studies show that microencapsulated hepatocyte culture can increase the survival of acute liver failure rats. 79 lmmunoadsorbents We have complexed albumin to collodion-coated activated charcoal to improve blood compatibility and to allow albumin to facilitate the transport of loosely bound protein molecules.l,59 Terman found that this albumin-collodion-charcoal system could also be used to remove albumin antibodies in a perfusion system. 8° He later replaced albumin with other types of antigens or antibodies for immunosorbents in perfusion. 8° He found in preliminary studies that plasma perfusion with protein A-collodion-activated charcoal decreased the size of breast carcinoma in pa72 G. Brunner and F. W. Schmidt (eds.), "Artificial Liver Support." Springer-Verlag, Berlin, 1981. 73 T. M. S. Chang, Sem. Liver Dis. Ser. 6, 148 (1986). 74 T. M. S. Chang, Ph.D. thesis. McGill University, Montreal, Quebec, 1965. 75 T. M. S. Chang, F. C. Macintosh, and S. G. Mason, Can. J. Physiol. Pharmacol. 44, 115 (1966). 76 F. Lira and A. M. Sun, Science 210, 908 (1980). 77 A. M. Sun, G. M. O'Shea, and M. F. A. Goosen, J. Appl. Biochem. Biotechnol. 10, 87 (1984). 78 Damon Corp., "Bulletin on Tissue Microencapsulation." Damon Corp., Needham Heights, Massachusetts, 1981. 79 H. Wong and T. M. S. Chang, Int. J. Artif. Organs 9, 335 (1986). 8o D. S. Terman, in "Sorbents and Their Clinical Applications" (C. Giordano, ed.), p. 470. Academic Press, New York, 1980.
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tients, s~ However, we have pointed out that this way the immobilized protein A may be released into the circulation acting as a slow release system. 82 Protein A has also been immobilized by covalent linkage to Sepharose for hemoperfusion in patients by groups. 83 A synthetic immunoadsorbent has been prepared for the removal of blood group antibodies anti-A and anti-B. However, the problems of blood compatibility and release of particulates has limited its application. We found that by using an ultrathin coating of collodion and albumin, a synthetic immunoadsorbent could be made that was blood compatible and would not release particles so that it could be used for hemoperfusion. 84 Clinical trials have been conducted on patients to remove anti-A and anti-B blood group antibodies before bone marrow transplantation. 85 Blood Substitutes
Immobilization technology has also been used in the search for a red blood cell substitute. Outside the red blood cell, hemoglobin is converted in the circulation to a dimer and removed rapidly. We have earlier demonstrated that hemoglobin could be cross-linked by bifunctional agents to polyhemoglobin. 1.12 This resulted, however, in an increase in oxygen affinity so that oxygen is not readily released for use when required. Benesch et al. 86 demonstrated that pyridoxalation of hemoglobin decreases oxygen affinity. There is a renewed interest at present in the use of polyhemoglobin.87 Work being carried o u t 87-91 has shown that peridoxylated hemoglobin can be cross-linked to soluble polyhemoglobin which can reversibly carry oxygen. In this form the polyhemoglobin can survive in the circulation so that 3 hours after intravenous injection 77% still remains in the circulation as compared to 25% for free hemoglobin. ss-9° It has also been shown that this type of polyhemoglobin is effective in resuscitating rats with hemorrhagic s h o c k . 92 Unlike fluorocars~ D. S. Terman, J. B. Young, W. T. Shearer, and Y. Daskal, N. Engl. J. Med. 305, 1195 (1981). 82 T. M. S. Chang, N. Engl. J. Med. 306, 936 (1982). 83 I. M. Nilsson, S. Jonsson, S. B. Sundquist, A. Ahlberg, and S. E. Bergentz, Immunology 14, 38 (1981). T. M. S. Chang, Trans. Am. Soc, Artif. Intern. Organs 26, 546 (1980). 85 W. I. Bensinger, D. A. Baker, C. D. Buckner, R. A. Clift, and E. D. Thomas, N. Engl. J. Med. 304, 160 (1981). 86 R. E. Benesch, R. Benesch, R. D. Renthal, and N. Maeda, Biochemistry 11, 3576 (1972). 87 T. M. S. Chang, Trans. Am. Soc. Artif. Intern. Organs 26, 354 (1980). a8 p. Keipert, J. Minkowitz, and T. M. S. Chang, Int. J. Artif. Organs 5, 383 (1982). 89 p. Keipert and T. M. S. Chang, Trans. Am. Soc. Artif. Intern. Organs 29, 329 (1983). 9o p. Keipert and T. M. S. Chang, Appl. Biochem. Biotechnol. 10, 133 (1984). 9J R. B. Bolin, R. P. Geyer, and G. J. Nemo (eds.), Adv. Blood Substitute Res. (1983).
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b o n s , 91 cross-linked hemoglobin is biodegradable in the body after use.
Repeated injection of homologous polyhemoglobin is not immunogenic. 93,94 Extensive research is being carried out using pyridoxalated polyhemoglobin or microencapsulated hemoglobin as blood substitute .93-% In Vivo Evaluations As shown in the above discussions, extensive studies have already been carried out on the use of different types of immobilization techniques. Perhaps one of the next major steps should be to study the different possible routes of in vivo actions of the immobilized systems. For eventual clinical application, the routes used in experimental animal studies may have to be greatly modified. The following factors are extremely important in considering any system for actual clinical application. Mode o f Action If the mode of action of the immobilized enzymes and cells requires rapid interactions with body fluids, then one of the best routes is to use extracorporeal blood circulation. Intraperitoneal administration is another alternative although this is usually used in experimental investigations in animals rather than for actual clinical applications. If the metabolites can diffuse freely across the intestinal tract, another possible approach is to use oral ingestion of immobilized enzymes and cells. In controlled release, intramuscular or subcutaneous injection could be carried out. The greatest problem is encountered when the immobilized enzyme or cells require localization in specific organs, cells, or intraceUular organelles. Biocompatibility Depending on the mode of action and therefore the route of administration, the requirements for biocompatibility are different. In extracorporeal blood circulation the immobilized system has to be blood compatible, nontoxic, and should not release any harmful material into the circulation. In systems involving intraperitoneal injection, intramuscular injection, or subcutaneous injection, the material also has to be biocompatible, sterile, 92 p. 93 C. 94 T. 9~ T. T.
Keipert and T. M. S. Chang, J. Biomater., Artif. Organs Med. Devices 13, 1 (1985). M. Hertzman, P. E. Keipert, T. M. S. Chang, Int. J. Artif. Organs 9, 179 (1986). M. S. Chang and R. Varma, Int. J. Biomater., Artif. Cells Artif. Organs 15,443 (1987). M. S. Chang, Int. J. Biomater., Artif. Cells Artif. Organs 15, 323 (1987). M. S. Chang and R. Geyer, eds., "Blood Substitutes." Dekker, New York, 1988.
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and nontoxic, in addition to being noncarcinogenic and nonimmunogenic. Oral administration involves the least requirements for biocompatibility. Accumulation
Immobilized enzymes and cells implanted by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection are retained for some time in the body. Since the duration of action of immobilized enzymes and cells is not indefinite, it is necessary to repeat the injections at various intervals. This may not be a problem in feasibility studies in animals. However, accumulation of immobilized material in the body could be a major problem in actual clinical applications if the treatments have to be repeated over a long period of time. Biodegradable polymers like poly(lactic acid), poly(glycolic acid), or cross-linked protein could solve the problem. However, proteins, enzymes, and cells which have been immobilized would be exposed and left behind when the biodegradable materials are removed. It is important to assure that this does not give rise to allergic and immunological reactions. To solve the problem of introduction of material into the body one could use the extracorporeal route, for instance, extracorporeal blood circulation, in those cases where this is applicable. The immobilized materials, having carried out their functions extracorporeally, can be disconnected and removed with no problems of accumulation. Another approach to solve the problem of in vivo accumulation is the use of oral administration in those few cases where this route is applicable. On the basis of the above factors, one of the following approaches could be selected. Methodology Intraperitoneal Administration
The intraperitoneal route of administration is a convenient way for investigating the in vivo action of immobilized enzymes and cells on substances present in body fluids. While this approach is very convenient and useful for initial feasibility studies in animals, the peritoneal cavity is an extremely sensitive area. Intraperitoneal introduction of foreign materials in humans may result in peritonitis due to reaction to foreign materials or infections. Furthermore, chronic fibrotic reaction to the long-term intraperitoneal introduction of foreign material could result in intestinal obstruction and other major problems. Thus in most cases, the peritoneal route should be used only as a convenient initial feasibility study in animals, to be followed by other approaches for actual clinical applications.
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Although large animals such as dogs and primates can be used, this approach is most convenient in introducing large amounts of immobilized proteins, enzymes, and cells into smaller animals like mice and rats. The procedure in small animals is very simple and can be summarized as follows. When the particles are about 50/~m in diameter, they can be injected as a suspension through an 18- or 20-gauge needle. Care should be taken to make sure that the injection is not made into the subcutaneous tissue or into the lumen of the gastrointestinal tract. Two millimeters of suspension can be introduced into each rat without causing any problems. With particles much larger than 50/zm diameter, one has to use larger needles. In these cases, it may be necessary to suture the hole made by the larger bore needle in smaller animals like rats and mice. Small flexible particles smaller than 8 p.m are removed by the lymphatic system from the peritoneal cavity. Larger particles are retained in the peritoneal cavity to act on metabolites equilibrating across the peritoneal membrane. However, unless the material is very biocompatible, it will be encased by a fibrous capsule because of foreign body reactions. This is a convenient approach which has been used in the initial investigation of many of the earlier studies employing immobilized enzymes and cells. These involved the use of microencapsulated urease as a model system,l,12,74,75 microencapsulated catalase for replacement of enzyme deficiency or acatalsemia, 1,1°,~4 microencapsulated asparaginase for tumor suppression separation, 11,36-38 microencapsulated islet cells for diabetes m e l l i t u s , 76'77 and microencapsulated hepatocytes for acute liver failure. 79
Extracorporeal Peritoneal Recirculation Extracorporeal peritoneal recirculation attempts to make use of the intraperitoneal approach without introducing any foreign materials into the peritoneal cavity. Immobilized enzymes or cells are retained in a small column; 2 ml sterilized peritoneal dialysis fluid is injected into the peritoneal cavity of each anesthetized mouse. The peritoneal fluid is continuously recirculated through the extracorporeal column and returned to the peritoneal cavity. At the completion of the recirculation the extracorporeal immobilized enzymes and cells are discarded or stored for later use. This approach has been used successfully in acatalasemic mice for the enzymatic removal of peroxides by artificial cells containing catalase. 1,1°
Extracorporeal Blood Recirculation Immobilized proteins, enzymes, and cells are retained in an extracorporeal column. 13 Blood from the animal is recirculated through the column by means of a pump. This technique can be used conveniently in
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animals as small as rats and in acute studies can be carried out in anesthetized animals with femoral artery and vein cannulation. In cases where repeated extracorporeal circulation is required, one can use chronic cannulation so that repeated extracorporeal circulation can be carried out in fully conscious animals. A number of techniques can be used depending on the size of the animal. When using rats two approaches are possible. Using the jugular vein and carotid artery to form an arteriovenous shunt, the cannula is exteriorized near the top of the head, and the tubings are attached permanently to the top of the cage. This results in restrictions in the mobility of the animal. Another approach is to use the femoral artery and vein and exteriorize the cannulae through the tail veins. 97 This is much more convenient since the animal, when not being treated, can be freely mobile. In the case of extracorporeal blood circulation one does not introduce foreign material into the body. However the immobilized enzymes and cells have to be blood compatible, sterile, and not release any harmful material. Artificial cell-immobilized adsorbents have already become a routine procedure in clinical practice in patients for the treatment of poisoning, 42-48 chronic renal failure, 46-56 and removal of aluminum. 98 With the large amount of clinical experience already available for immobilized adsorbents, clinical applications of immobilized enzymes and cells could be easily developed. Experimental investigation of immobilized enzymes and cells includes the use of immobilized urease, 13asparaginase, 4°,41 tyros i n a s e , 8'69'7° heparinase, 99 and other enzymes.~-8
Oral Administration In some cases, the metabolites to be acted on can equilibrate readily from the bloodstream into the lumen of the gastrointestinal tract. In these cases oral administration of immobilized enzymes is possible. This is one of the most convenient approaches. A gastric tubing can be easily introduced into the stomach of an unanesthetized rat and materials inserted by means of a syringe. This approach has been used for the removal of urea using artificial cell-immobilized urease and ammonia adsorbent in r a t s 1,58-62 and the recent use of artificial cell-immobilized phenylalanine ammonia-lyase in phenylketouria r a t s . 32-34 In humans, oral route of administration is the most convenient and least problematic, since it will be equivalent to drinking a suspension or taking a few pills.
97 y . Tabata and T. M. S. Chang, J. Artif. Organs 6, 213 (1982). 98 T. M. S. Chang and P. Barre, Lancet 2, 1051 (1983). 99 R. Langer, P. J. Blackshear, T. M. S. Chang, M. D. Klein, and J. S. Schultz, Trans. Am. Soc. Artif. Intern. Organs 32, 639 (1986).
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Intravascular Injection
Immobilized enzymes and cells can be injected intravenously into the systemic circulation, e.g., through foreleg veins in dog, ear veins in rabbits, and tail veins in rats and mice. Chronic cannulations could also be used for long-term intermittent administration. Most intravenously injected particulate matters larger than 2 t~m in diameter are filtered out in the pulmonary circulation. Most of the smaller particles which pass through the pulmonary circulation are removed by the reticuloendothelial system particularly the liver and spleen. Immobilized enzymes which have been injected intravenously localize in the reticuloendothial system. 1,2,5-8A5-~8 Immobilized enzymes or proteins in the soluble form such as cross-linked hemoglobin, 87-96 enzymes cross-linked to albumin] ° or enzymes cross-linked to soluble polymer2~,22 can survive much longer in the circulation. The use of magnetic microcapsules ~3 allows the material to be localized by externally applied magnetic forces. Intraarterial injections have been used to localize material in specific organs. Intramuscular or Subcutaneous Injection
Intramuscular or subcutaneous injection routes are more suitable in cases where the immobilized system is used as a depot for slow release of immobilized materials like hormones and drugs. Discussion The first in vivo studies carried out about 20 years ago demonstrated the possible medical application of immobilized enzymes and cells 1,t°-13 ; however, many different approaches are now available. 1-8 Further progress toward actual routine clinical applications will depend on careful design of in vivo experiments which can be adapted for use in clinical applications. The clinical uses of artificial cells containing adsorbents have developed rapidly so that, although this technique started much later than immobilized enzymes, it is already in routine clinical use. 4L42Experience gained can be applied to future large-scale clinical applications of immobilized proteins, enzymes, and cells. With the recent rapid progress in biotechnology, the required proteins, enzymes, and cells will be more readily available. Many factors will contribute to the future possibilities of clinical application of immobilized proteins, enzymes, and cells. Artificial cells or microencapsulated enzyme in the intestine can act effectively on plasma substrates which can enter the intestine. ~°° This may lead to an easy route for routine applications of immobilized enzymes. 100 L. Bourget and T. M. S. Chang, J. Biomat. Artif. Cells Artif. Organs (in press).