Aerosolization of Superoxide Dismutase

Aerosolization of Superoxide Dismutase

Aerosolization of Superoxide Dismutase* Augmentation of Respiratory Epithelial Lining Fluid Antioxidant SCreen by Aerosolization of Recombinant Human ...

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Aerosolization of Superoxide Dismutase* Augmentation of Respiratory Epithelial Lining Fluid Antioxidant SCreen by Aerosolization of Recombinant Human Cu++lZn++ Superoxlde Dismutase Adrian Gillissen, M.D.; james H. Roum, M.D ., Ph.D. ; Robert F. Hoyt, D.V.M.; and Ronald G. Crystal, M.D . Various human pulmonary diseases are characterized by an increased oxidant burden on the respiratory epithelial surface. As a step toward developing a therapy to augment the antioxidant defenses of respiratory epithelial lining 8uid (ELF) of the human lung, we have evaluated the feasibility of aerosolizing a human protein antioxidant to the respiratory epithelial surface of an experimental animal sufficiently large to permit repetitive sampling of ELF. To accomplish this, recombinant human Cu++fZn++ superoxide dismutase (rSOD) was aerosolized to sheep, and the levels of human superoxide dismutase (SOD) and antisuperoxide anion (Ol~) capacity were quantified in ELF over time. In vitro aerosolization did not alter the speciSc activity of rSOD (p>O.5). In vivo aerosolization of rSOD (100 mg) to sheep (n 7) resulted in peak amounts of human Cu+ +/ Zn++ SOD in ELF of 3.1±O.6 ....mollL, with a parallel increase in the anti-O; capacity of ELF. For the duration of the study (5 h), levels of SOD and anti-O," in ELF

remained elevated, with a value 50 percent of the peak at 5 h. Aerosolization of phosphate-buffered saline (n 5) had DO effect on SOD or anti-Ol~ levels in ELF. In animals receiving rSOD, there was DO change in the speci6c activity of SOD recovered in ELF compared to the starting material (p>O.4). We conclude that rSOD can be delivered by aerosol to the ELF of a large animal with preservation of speci6c activity and that a substantial increase in both the amount of SOD and the anti-O; capacity can be achieved for a period of time applicable to human therapy, supporting the rationale for evaluation of rSOD aerosol as an antioxidant in human pulmonary disease. (Chat 1993; 104:811-15)

progressive damage to the respiratory epithelium is a prominent feature of a variety of acute and chronic inflammatory pulmonary diseases,"? In many of these disorders, particularly those characterized by large numbers of activated alveolar macrophages, neutrophils, or eosinophils in respiratory epithelial lining fluid (ELF), the epithelial surface is barraged by an increased burden of oxidants released by the inflammatory cells .v'? Although ELF and the epithelial cells contain antioxidants to contend with such oxidants, when the inflammation is overwhelming, the antioxidant screen is inadequate, resulting in oxidantmediated epithelial injury.3.4.11-13 In the context of these concepts, approaches to therapy for such disorders might be to augment the antioxidant screen of the epithelial cells, thus making the epithelium more resistant to external oxidant attack. Alternatively, and perhaps more easily, the antioxidant screen of ELF might be augmented, thus elevating the antioxidant barrier, reducing the burden of oxidants that reach the epithelial cells.4.11.12.14-23

Human recombinant Cu + +!Zn + + superoxide dismutase (rSOD) represents one example of an antioxidant that might serve this purpose. This recombinant molecule is similar to the naturally occurring form of Cu + + IZn + + superoxide dismutase (SOD) (EC 1.15.1.1), a 33-kd cytoplasmic enzyme that normally catalyzes the metabolism of superoxide anion (0/), the initial oxidant produced by inflammatory cells.13.24 In the context of these considerations, the purpose of the present study is to evaluate whether it is possible to aerosolize rSOD to augment the antioxidant screen of the respiratory epithelial surface in a fashion applicable to human therapy. Using sheep as the experimental model, the data demonstrate that it is feasible, suggesting a possible strategy of using a recombinant human antioxidant to augment the antioxidant protective screen of respiratory ELF that is directly applicable to humans.

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·From the Pulmonary Branch (Drs. Gillissen, Room, and Crystal), and the Section of Laboratory Animal Medicine and Surgery (Dr. Hoyt), National Heart, Lung, and Blood Institute, NationaI Institutes of Health. Bethesda. Md . Manuscript received May 29, 1992; revision accepted February 11, 1993. Reprint requests: Dr. Crystal, NIH, 9000 Rockoille Pike, Bldg 10, Rm 6D03 , Bethesda 20892

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ELF = resPu.:8tory epithelial Os" = superoxide anion; PBS phosphate-bufl'ered • ; RIA radIoimmunoassay; rSOD = human recombinant Cu + + fZ.o + + superoxide dismutase; SOD = superoxide dismutase

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MATERIALS AND METHODS

Human Recombinant Cu + + IZn+ + Superoxide DUmutase The rSOD (Bio-Technology General; Rehovot, Israel) was produced by Escherichia coli under the direction ofa plasmid containing the entire human SOD coding sequence and was subsequently purified." Quantification of rSOD was performed by radioimmunoassay (RIA) using an anti-rSOD mouse monoclonal antibody, "'I-labeled rSOD, and a standard curve of rSOD . The enzymatic activity of the rSOD standard or ELF samples (10 ..,1 a1iquots) was evaluated as "anti-O," capacity:' This was carried out by spectroCHEST I 104 I 3 I SEPTEMBER. 1993

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pbotomebic analysis by measuring the inhibition offerricytocbrome C reduction caused by 0; generation by xanthine/xanthine 00due.- Anti-Q.· capacity was expressed as units per milliliter, wbere a unit was defined as the quantity of SOD needed for a SOpercent inhibition of the rate of reduction of ferricytocbrome C using xanthine (SO j.LIIIoVL), xanthine oxidase (0.01 U), and ferricytochrome C (10 j.LIIIoVL) (Sigma Cbemical Co) in a total volume of 3m!.

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To determine whether aerosolization of rSOD would alter its enzymatic activity, the amount of rSOD and antt-O," capacity were quantified tn otIro before and after aerosolization using a standard aerosol system.11." Briefly, rSOD (100 mg in 4 mI of phosphatebuft'ered saline [PBS)) was aerosolized via a compressed-air nebulizer, and the aerosolized droplets were coUected in a reservoir containing ISO mI of PBS (pH 1). The specific activity of SOD (in units per microgram), defined as the ratio of the anti-O," capacity (in units per milliliter) to the amount determined by RIA (in micrograms per milliliter), was quantified for the starting solution and the resulting post-aerosol solution, and the values were com-

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ErperimenttJl Model To evaluate the feasibility of aerosolizing rSOD to a large experimental animal, rSOD was aerosolized to sheep, and bronchoalveolar lavage used to recover ELF before and after aerosolization. II Briefly, female mixed-breed sheep (total n = 12; mean weight, 29 ± 2 kg) were anesthetized with intravenous thiopental sodium and were intubated with a cuffed endotracheal tube attached to a positivepressure ventilator. (All data are presented as means±SEM; all statistical comparisons were made using the two-tailed Students' t test). The animals were ventilated with SOpercent O. and SOpercent nibous onde with 1.0 to 1.5 percent halothane (F1uothane). at a tidal volume of 12 mIIkg of body weight with a positive endexpiratory pressure of 5 em H.O and a respiratory rate of Hi/min throughout the study. \;lnous access was achieved by placing a catheter in a jugular vein. Of the 12 sheep, 7 received an aerosol of rSOD in PBS (100 m'{/ 4 m1). Bronchoalveolar lavage was used to retrieve ELF before and at I , 2, 3, and 5 h after rSOD aerosolization. As a control, 5 sheep received an aerosol of PBS alone (4 ml) and had ELF evaluated before and at 1 and 6 h after aerosolization. FOr all animals, at each time point, the lavage procedure was carried out at a new, randomly selected site in the lung. Lavage was performed via a fiberoptic bronchoscope (length, 1 m; external diameter, 5.2 mm ; Machida) using a single 5O-ml aliquot of PBS . Simultaneously, blood samples were obtained from the jugular vein catheter. The amount and activity of SOD in the lavage samples and SOD in plasma samples were quantified as described previously. The volume of ELF was determined by the urea method,- and the amount and activity of SOD were expressed per milliliter of ELF. The specific activity of SOD, as defined previously, was determined for each lavage sample. RESULTS

Evaluation of the rSOD preparation in vitro demonstrated it could be placed in aerosol droplets without loss of activity (Fig 1). In this regard the starting material had a specific activity of 4.9 ± 0.7 UltLg. After aerosolization and collection in vitro, analysis of the collected droplets demonstrated a similar specific activity (5.4 ± 0.5 UltLg; >0.5 compared to the starting material). Aerosolization of rSOD to sheep was associated with no adverse reactions in regard to vital signs and local 812

FIGURE

1. Maintenance of activity of rSOD foUowingaerosolization

in otIro . Activity and amount of SOD were measured before and after aerosolization tn otIro of rSOD via compressed-air nebulizer. Specific activity of SOD, defined as ratio of activity to amount, is

shown before and after aerosolization.

effects. There were no changes observed in the airways at bronchoscopy. Analysis of lavage fluid showed no

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2. Levels of SOD in ELF of sheep following aerosolization

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difference in the amount of ELF recovered from the animals before therapy compared to after therapy, or therapy with rSOD compared to PBS (p>O.3 for all comparisons). Following aerosolization of 100 mg of rSOD, the levels of human SOD in ELF increased to approximately 3 J.LmoVL over the first 2 h and then declined (Fig 2). Five hours after aerosol administration, the ELF levels were still SO percent of the peak levels. As a control, aerosolization of PBS caused no change in the amount of human SOD detected in ELF; human SOD remained undetectable throughout the study. Likewise, SOD levels in plasma were unchanged throughout the study (p>O.1 for all comparisons). As was observed in vitro, aerosolization in vivo was associated with the maintenance of activity of the SOD that impacted on the epithelial surface (Fig 3). The pattern of anti-O," capacity in ELF increased and declined in a parallel fashion with the antigenic levels of SOD (Fig 2), suggesting that rSOD is not inactivated in vivo following aerosolization but most likely decreases by a combination of absorption and possibly adsorption. Quantification of the average specific activity of the rSOD before aerosolization showed no difference from that of the SOD recovered from ELF (p>OA). Aerosolization of PBS had no effect on the anti-O," capacity of ELF. DISCUSSION

These observations demonstrate that it is feasible to aerosolize rSOD to the respiratory epithelial surface of a large animal with marked augmentation of fune-

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FICURE 3. Augmentation of antisuperoxide anion (0.-'-) capacity of ELF of sheep following aerosolization of rSOD. Anti-O.'" capacity was measured by inhibition of reduction of ferricytochrome C caused by 0.-'- generated by xanthine and xanthine oxidase. Values are shown for measurements before and after aerosolization of rSOD (circles; 100 mg) or, as control, before and after aerosolization of PBS (squares ~

tional SOD levels in ELF for a period of time feasible for human applications. In regard to applying this therapy to human pulmonary disease, several considerations are relevant to safety and efficacy. In regard to safety, several reasons suggest that aerosolization ofrSOD will not cause problems per se. First, no adverse effects were noted in the sheep receiving rSOD. Secondly, SOD is a normal constituent of ELF,211 and a variety of human proteins have been aerosolized to humans with no adverse effects, including plasma ai-antitrypsin, lET recombinant a l antitrypsin,ZlI recombinant interferon gamma,211 interferon alpha, and recombinant interferon alpha,30 and recombinant deoxyribonuclease.31 Thirdly, the specific activity of the rSOD did not change following aerosolization in vitro or in vivo, suggesting that the molecule is not altered by the aerosolization process. Finally, rSOD has been administered by the intravenous route to humans with no adverse effects. 32 •33 In regard to the possible efficacy of this form of therapy for human disease, the issues include the disease target and the relevance of SOD as a therapeutic agent. The presumed need for an increased anti-O/ capacity in ELF to combat an increased oxidant burden on the respiratory epithelial surface is situational, ie, different oxidant burdens for different diseases. Current concepts are that this burden is a function, in large part, of the number of inflammatory cells present on the epithelial surface and the respective inflammatory cell "activation state" for release of oxidants.3-8·13,14 Evidence for an increased ELF 0/ burden compared to normal individuals has been shown in several human lung disorders, including cystic fibrosis,9 idiopathic pulmonary fibrosis,5 interstitial pulmonary diseases associated with the collagen vascular disorders," inorganic dust diseases," cigarette smoking," human immunodeficiency virus mfection," sarcoidosis, 35 hypersensitivity pneumonitis,36 tropical pulmonary eosfnophilia," and the adult respiratory distress syndrome.!" Superoxide dismutase has not been administered directly to the lung as a therapeutic agent for human pulmonary disease, but it has been used in a variety of animal studies, including a number of animal models of lung inflammation, where it has been shown to protect the airway epithelium from oxidant-induced damage.17.18,23 In these models, SOD has been delivered by aerosolizatton'" and intratracheal instillation,18as well as by subcutaneous, ~ intravascular,23.38.4O and intraperitoneal administration.F The therapeutic efficacy of these studies vary, likely secondary to the type of oxidant injury and to the proportion of the administered SOD reaching the targeted physiologic compartment.v'" In an attempt to augment the therapeutic effect of SOD, altered forms of the SOD molecule have been used in hopes of either increasing its active half-life or improving its CHEST I 104 I 3 I SEPTEMBER, 1993

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targeting to selected compartments. Some of these approaches include liposomal encapsulated SODISand SOD conjugated with molecules such as polyethylene glycolllll•22 •39 and vinyl ether-maleic acid copolymer." Cu ++/Zn ++ SOD is one of the three forms of human SOD known.4. 11•12 At this time, there is no a priori reason to choose the Cu++fZn++ SOD form over Mn++ SOD (the mitochondrial form of SOD) or extracellular SOD for aerosol administration, other than the fact that recombinant Cu ++fZn ++ SOD has been administered to humans. 32.33 The relevance of any form of SOD as a potential therapeutic agent for human pulmonary disease relates to the oxidant target it suppresses. Superoxide dismutase acts by reducing the parent of all oxidant molecules released by inflammatory cells.UJ · 13 While SOD may prevent OH· formation via the Haber-Weiss reaction by removing SOD converts to H 20 2 , the parent oxidant of OH· and HOCI. Thus, it may be that a combination of SOD with an anti-H 20 2 antioxidant (eg, catalase) would provide maximally effective antioxidant protection to the respiratory epithelium in inflammatory lung diseases. 17,21 .3lI In this regard, catalase, by converting H 20 2 to oxygen and water, would prevent both its oxidant effect and its inhibitory effect on SOD.41 If this combined approach is advantageous, the results of the present study support the feasibility of this approach from the SOD standpoint; that is, biologically active SOD can be delivered to ELF by aerosol.

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ACKNOWLEDGMENTS: ~ thank J. R. DeLeonardis and M. Waters, Section of Laboratory Animal Medicine and Surgery, National Heart , Lung, and BloOd Institute, lOr help in this sfudy: A. Gonenne, Bio-TechnoJosly General, for providing the recombinant Cu++/Ln++ SOD anaanti-SOD antibody, and A. Avent for editorial assistance. REFERENCES

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C, et al, Alveolar macrophages in the asymptomatic HIVseropositive state: evidence for in 0100 activation not directed towards host defense [abstract]. Clin Res 1990; 38:485A Room JH, 8uhl R, McElvaney N, Borak Z, Chernick M, Caplan 0 , et al. In oitro evaluation of the ability of glutathione to suppress the inflammatory cell derived oxidant burden in respiratory epithelial lining fluid of individuals with cystic fibrosis {abstract] . Clin Res 1990: 38:440A Baldwin SR, Simon RH , Grum CM, Ketal LH, Boxer LA , Devall LJ. Oxidant activity in expired breath of patients with adult respiratory distress syndrome. Lancet 1986: 1:11-3 Davis W8, Pacht ER. Extracellular oxidant defenses. In : Crystal RG, West JB , eds, The lung : scientific foundations. New York: Raven Press, 1991; 1821-28 Heffner JE , Repine JE. Antioxidants and the lung . In : Crystal RG, ~st J8, eds, The lung: scientific foundations. New York: Raven Press, 1991; 1811-20 Schraufstatter IU, Cochrane CG . Oxidants: types, sources, and mechanisms of injury. In: Crystal RG, West J8, eels. The lung: scientific foundations. New York: Raven Press, 1991: 1803-10 Cantin A, Crystal RG. Oxidants, antioxidants and the pathogenesis of emphysema. Eur J Respir Dis 1985: 66(suppll39):7-17 Borok Z, 8uhl R, Grimes G , Bokser A, Hubbard RC , Holroyd KJ, et al, Glutathione aerosol therapy to suppress the burden of oxidants on the alveolar epithelial surface in idiopathic pulmonary Bbrosis, Lancet 1991; 388:215-16 Buhl R, Vogelmeier C , Crittenden M, Hubbard RC, Hoyt RF, Wilson EM , et al , Augmentation of glutathione in the fluid lining of the epithelium of the lower respiratory tract by directly administering glutathione aerosol. Proc Nat! Acad Sci USA 1990; 87:~

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