Pharmacokinetics and Distribution of Recombinant Secretory Leukocyte Proteinase Inhibitor in Rats1 , 2
ALAIN GAST, WAYNE ANDERSON, ALESSANDRO PROBST, HANSPETER NICK, ROBERT C. THOMPSON, STEPHEN P. EISENBERG, and HANSPETER SCHNEBLI
Introduction
It is believed that an imbalance between proteases and their naturally occurring inhibitors results in an abnormal degradation of connective tissue, thus playing a major role in the development of lung diseases such as emphysema (1) and cystic fibrosis. The local concentration of leukocyte elastase exceeds that of active inhibitors, namely, at-proteinase inhibitor (u.Pl), secretory leukocyte proteinase inhibitor (SLPI), and some not-yet-characterized proteins (2, 3), leading to tissue damage. SLPI is synthesized in the central airways by serous glandular cells and in the lower respiratory tract by Clara cells and goblet cells (4, 5). This potent human leukocyte elastase inhibitor (6) is a single polypeptide chain with a molecular weight of 11,700 containing two highly homologous domains. Site-directedmutagenesis of the SLPI gene (in preparation) and X-ray crystallography of the SLPIchymotrypsin complex (7) indicate that the C-terminal domain inhibits trypsinlike enzymes, chymotrypsinlike enzymes, and leukocyte elastase (8). A striking feature of this molecule is its unusual resistance against denaturation by heat and acid, probably because of the presence of eight disulfide bridges. In order to develop SLPI as a therapeutic agent, the gene for this polypeptide was cloned and expressed in Escherichia coli (9). Here, we report the pharmacokinetics and distribution of SLPI after intravenous, intraperitoneal, and intratracheal administration in rats. Some preliminary results have been presented elsewhere (10). Methods Recombinant SLPI and Eglin c Recombinant SLPI was produced in E coliand purified by affinity chromatography on anhydrochymotrypsin AffigellO (11). [3SS]SLPI was produced by either of two methods. In the first method, a 17Q-ml culture of the strain containing the SLPI gene was grown at 37° C to a density of rv 2 x 108cells/ml in M9 medium containing 15 J.1g tetracycline/ml. The
SUMMARY secretory leukocyte proteinase inhibitor (SLPI) is a potent elastase, trypsin, and chymotrypsin inhibitor occurring in all mucous secretions. Its inhibitory potency and profile suggested that it may become a therapeutic adjuvant In diseases where protelnases play a pathogenetic role. In the course of developing recombinant SLPI for therapeutic purposes, we studied Its pharmacokinetics after intravenous, Intraperitoneal, and Intratracheal application to rats. In plasma, SLPI was determined with an ELISA or by following a radlotracer ((35S1SLPI). In bronchoalveolar lavage fluid (BALF), SLPI was determined additionally by a functiQl'I"j assay (elastase inhibitory capaclty).lntr. venously applied SLPI (2 mg/kg) was rapidly cleared, with half-times o! distribution of 6 min and half-times of elimination of 50 min. Very little « 5%) appeared in the urirJ~ even after 24 h. ApproXImately 80% of Intraperitoneally Inlected SLPI (12mg/kg) was absorbed and generated maximal plasma concentration of 6 to 10 I!g/ml 30 to 120 min after administration. When given Intratracheally (8.6 mg/kg), SLPI disappeared from the lungs, with a half-time of 4 to 5 h. This value was the same whether the remaining SLPlln BALF was determined radiometrically, by ELISA or by the functional assay, indicating minimal metabolism in the lung. As in the case of Intraperitoneal application, SLPI was absorbed systemically, resulting In a maximal plasma level of about 2 I!g/ml1 to 2 h after application. In contrast to the measurements in BALF, the ELISA and radlotracer measurements In plasma correlated only for the first 2 h after application and diverged progressively after that, suggesting breakdown of the molecule once it reaches the plasma. The data provided demonstrate that in the rat, intratracheally applied SLPI remains in the lung in active form for several hours and may thus provide effective protection against proteinase-mediated lung damage. AM REV RESPIR DIS 1990; 141:889-894
SLPI gene was induced with IPTG (l mM), and incubation was continued for 80 min. At this point, 5.6 mCi of [35S]methionine (rv 1,000 Ci/mmol) (New England Nuclear, Boston, MA) was added to the culture, and incubation was continued for an additional 10min. Cells wereharvested by centrifugation at 5,000 x g for 10 min at 4° C, washed with 0.18 M cold NaCl, repelleted, and suspended in 0.4 ml of 0.1 M TRIS-HCl at pH 7.2. Lysis of cells and purification of PSS]SLPIwas performed as previously described (9, ll). In the alternate method, the bacterial cells were grown in a medium similar to the one described above, except that the sulfate ion concentration was reduced to 0.2 mM. This culture was grown to a density of 3 x 108 cells/ml. The cells were pelleted by centrifugation at 5,000 x g for 10min at 25° C, and then resuspended in medium as described above with the sulfate ion concentration now at 0.05 mM. The SLPI gene was induced as above and onetwentieth volume of [35S]sulfuric acid (1,300 Ci/mmol) (New England Nuclear) was immediatelyadded. The culture wasincubated with aeration for 4 h, the cells were harvested, and the [3SS]SLPI was purified as above. The gene of eglin c was synthesized and expressed in E. coli (12). Apart from its acetylated N-terminus the purified recombinant
protein is indistinguishable from the nati...e eglin isolated from the leech as demonstrated by inhibitory activity, dodecyl sulfate-polyacrylamide gel electrophoresis, and amino acid analysis (12).
Immunologic Assays Recombinant SLPI was measured in rat plasma and lung lavage fluid using the sandwich ELISA technique described by Kramps and coworkers (13) with the following modifications. PVC micro titer plates (Nunc-Immune plate I; Roskilde, Copenhagen, Denmark) were coated for 1 h at 37° C and then overnight at 4° C with 100 ul of a 5-ltg/ml solution of rabbit immunoglobulin raised against recombinant SLPI. Samples and SLPI standards were applied in triplicate and allowed to bind to the antibody for 3 h at 37° C in a humid chamber. Rabbit anti-SLPI conjugated
(Received in original form April 10, 1989) 1 From the Pharmaceutical Division, Ciba-Geigy Ltd, Basel, Switzerland, and Synergen Inc., Boulder, Colorado. 2 Correspondence and requests for reprints should be addressed to Hanspeter Nick, Pharmaceutical Division, Ciba-Geigy Ltd, 4002 Basel, Switzerland.
889
890 to horseradish peroxidase (Boehringer, Mannheim, FRG) diluted 1:1,500 was added and allowed to complex for 3 h at room temperature. The antigen-antibody complex was visualized with the addition of o-phenylenediamine (4 mg/ml) in citrate phosphate buffer at pH 5 containing 0.012% H 202 • The plates were placed in the dark for 40 min, and the absorbance was read at 490 nm after the color reaction was terminated with 2.5 M H 2S04 , The standard curve was similar to that obtained by Kramps and coworkers (13) and could accurately measure SLPI at concentrations between 0.4 and 1.5 ng/ml, An internal standard (human EDTA plasma diluted 1:50) was run in triplicate on each plate, and the experiment was repeated if the concentration was not 50 ± 10 ng/ml (50 ng/ml being the mean value of SLPI in the plasma). At the dilutions of the samples used in the assay, rat plasma and rat lung lavage did not interfere with the assay. Eglin concentrations in lung lavage were quantified with a competitive ELISA similar to that described for aprotinin (14)except that the eglin-horseradish conjugate was diluted 1:20,000.
Elastase Inhibitory Capacity The elastase inhibitory activity of BALF was assessed by measuring its ability to inhibit the hydrolysis of methoxysuccinyl-(alanylj.-prolyl-valyl-p-nitroanilide by human leukocyte elastase. Leukocyteelastase was purified using the method described by Baugh and Travis (15). Increasing amounts of unconcentrated lavage fluids were incubated with a constant amount of active elastase (20 nM) at 37° C for 15 min in 50 mM TRIS, 100 mM NaCl, and 0.05070 Triton XlOO at pH 7.7 (total reaction volume: I ml). Then, 10 IIIof a lOO-mM substrate solution in dimethyl sulfoxide were added, and after 10min at 37° C the reaction was stopped by the addition of 100 11112.5% acetic acid, and the absorbance at 405 nm was immediately read. An SLPI solution of known anti elastase activity was used as a standard. Radioactivity Measurement The "S activityof the samples was measuredin a liquid scintillationcounter (Packard CD 460; Canberra Packard, Downers Grove, IL) or LKB counter (Wallach 1215; Wallach, Turku, Finland) according to published methods (16). Blood and Urine Collection After intravenous and intraperitoneal administration, the animals were anesthetized with ether, and blood samples of about 0.5 ml were withdrawn from the retro-orbital plexus into small, heparinized glass tubes using capillary pipettes at set time points after dosing. Plasma was obtained by centrifugation at 2,000 rpm for 15 min at 4° C and stored frozen. Urine and feces were collected in standard metabolic cages after intravenous injection of 35S-labeled SLPI. After intratracheal administration, blood
GAST, ANDERSON, PROBST, NICK, THOMPSON, EISENBERG, AND SCHNEBLI
wascollected bycardiac puncture into syringes containing heparin (20IE/ml blood). Animals in the 8-h treatment group wereplaced in standard metabolic cages with free access to water. Urine was collected during this period.
Intravenous and Intraperitoneal Administration SLPI (unlabeled or 35S-labeled) was diluted in PBS to a concentration of 0.93 mg/ml. Approximately 500 III of this was injected into healthy random-bred male rats (Tif: RAIF [SPFj raised on the premises of Ciba-Geigy, weighing 200 to 230 g) intravenously into a tail vein or intraperitoneally. The rats were housed singly in metabolic cages designed to separate urine and feces. For distribution studies the animals were exsanguinated under ether anesthesia, and organs and tissues were obtained by dissection. Intratracheal Administration Intratracheal instillation was performed as described by Brain and coworkers (17). Male rats (weighing 300 to 350 g) were lightly anesthetized with ether and suspended by the upper incisors on a dissecting table at a 75-degree angle from horizontal. A high intensity light was positioned at the throat area, which allowed visualization of the larynx from the mouth. The [35S]SLPI solution (0.3 nCi/mg, 8.6 mg/kg body weight), eglin (8.6 mg/kg), a mixture of SLPI and eglin (8.6rug/kg each), or saline (control) was introduced into the upper trachea by a modified gavage needle. Instillation volume was 0.15 ml/IOO g body weight. Lung lavage was performed at I, 15, and 30 min and at I, 2, 4, and 8 h after intratracheal administration. The animals were anesthetized with sodium thiobutabarbital (100 mg/kg) approximately 30 min before lavage, and a cannula was secured in the trachea. For the early time points (lessthan I h) the animals wereanesthetized and cannulated before intratracheal administration. Saline (10 ml) was slowly introduced into the lung, and 6 ml of fluid were immediately withdrawn. In a second experiment, this procedure was repeated three times for each animal, and the maximal volume of lavage fluid was collected. The volume of each lavage was measured, stored separately on ice, and centrifuged at 2,000 rpm for 15min at 4° C. Recoveryranged from 6 to 10 ml for each lavage. The supernatants were kept frozen until analysis. Gel Filtration Studies BALF from rats lavaged I min and I h after intratracheal administration of SLPI were concentrated tenfold by lyophilization and analyzed on a FPLC gel-filtration Superose 12 column equilibrated with 50 mM sodium phosphate, 0.5 M NaCI (pH, 6.8) at a flow rate of 0.5 ml-min". A control rat lavage spiked with SLPI wastreated in the same manner and served as a reference. The presence of SLPI was assessed in each protein fraction by the ELISA procedure. The functional activi-
ty of SLPI was measured with the elastase inhibitory capacity as described above.
Calculation of Pharmacokinetic Parameters Plasma concentrations (c) of SLPI at time t after dosing can be described by a two-compartment open model: c = A·e- at + B·e-~t. The parameters B and ~ were determined by linear regression analysis of the logarithm of the plasma SLPI concentration-time relationship during the terminal phase of elimination of the compound. Parameters A and II were determined by the method of residuals. The area under the plasma concentrationtime curve (AU C) was calculated by the trapezoidal rule. Calculation oftotal plasma clearance (CIT)and volume of distribution of the central compartment (Vdc) was performed using CIT = D/AUC, Vdc = D/(A + B) (18). The group values for immunoreactive lung lavage fluid SLPI, radioactive, and SLPI antielastase activity were compared by a single-tailed t test for paired groups. Differences between groups were considered to be significant at p < 0.05. Results
Pharmacokinetics after Intravenous Administration Five minutes after intravenous administration of 2 mg/kg [35S]SLPI, 30070 of the total radioactivity wasdetected in kidneys, 20% in blood, and approximately 15070 in liver, muscle, and skin. Only 5% was found in the lung (figure 1). The serum concentration of unlabeled SLPI after a bolus injection of 2 mg/kg decreased in a biphasic fashion (figure 2) with halftimes of 6 min for the distribution phase and 51 ± 4 min for the elimination phase (table 1). Approximately 2% of the administered dose (labeled and unlabeled SLPI) was detected in both the zero to 8-h and 8- to 24-h urine and about 5% of the injected radioactivity was found in the zero to 24-h feces. The volume of distribution of the central compartment (Vdc) was 67 ± 3 ml- kg", and the total clearance (Ch) was 4.9 ± 0.2 ml-min? kg? (table 1). Pharmacokinetics after Intraperitoneal Administration The SLPI appearance in plasma after intraperitoneal administration of 12mg/kg SLPI is shown in figure 3. In two rats SLPI reached a maximal concentration of 6 to 8 ug/ml 30 min after injection, whereas in one rat a peak concentration of 10 ug/ml was observed 2 h after administration. The apparent half-time of elimination in plasma was about 1 h. When the AVC/dose was compared with the AVC/dose after intravenous injec-
891
PHARMACOKINETICS OF THE SECRETORY LEUKOCYTE PROTEINASE INHIBITOR
Blood Liver KidneyMuscle Skin Pancreas Fat
Lung Spleen Brain
Organ/Tissue Fig. 1. Distribution of radioactivity in organs and tissues of rats 5 min after intravenous administration of 2 mg/kg 35S-labeled SLPI.
100
Fig. 2. Plasma SLPI concentrations after intravenous administration of recombinant SLPI (2 mg/kg) to two rats as measured with ELISA. Each point corresponds to the mean value of three dilutions measured in triplicate (see table 1).
.1
.01
+--....----,---.--.,..-........-,.----.-~- ........-..., o 2 4 3 5 Time after administration (hours)
tion, an absolute bioavailability of 80070 was calculated.
Pharmacokinetics after Intratracheal Administration After intratracheal administration of SLPI (8.6 mg/kg), the concentration of immunoreactiveand activeSLPI in BALF (one lavage) was measured at set times.
Within the first hour after instillation the mean immunoreactive level remained at a high value and then decreased, with a half-time of 4 h (figure 4 and table 2). No significant difference was observed between the concentrations of immunoreactive and active SLPI as assessed by t test (p < 0.05) (figure 4). The concentration of active SLPI parallels the im-
TABLE 1 PHARMACOKINETIC PARAMETERS AFTER INTRAVENOUS ADMINISTRATION OF SLPI
Rat No.
1 2
Central Volume of Distribution (ml·kg-' body wt)
Tv, Distribution (min)
70 64
6 6
Total Clearance (ml-min-"kg-' body wt)
T'h Elimination (min)
5.1
55
4.7
47
munoreactive levelover the whole observation period. The half-time of elimination of active SLPI was 4.5 h, a value similar to that observed for immunoreactive SLPI (table 2). In a separate experiment, wemeasured total radioactivity and immunoreactive SLPI in sequential lung lavages and in plasma after intratracheal administration of 8.6 mg/kg unlabeled SLPI complemented with 35S-labeled SLPI. The results obtained for the first lavagesby both methods are shown in figure 5. The concentrations of SLPI determined by ELISA or calculated from the radioactivity data (figure 5A) were not significantly different (p < 0.05). Furthermore, the radioactivity decreased with a half-time of 5.3 h, a value similar to that of immunoreactive and active SLPI (table 2). Plasma immunoreactive levels of SLPI after intratracheal administration increased in parallel with the radioactivity concentrations and reached a peak concentration of 2 ug/ml l h after injection (figure 5B) and decreased thereafter. In contrast, the radioactivity concentrations continued to increase linearly to reach the equivalent of 5.5 ug SLPI!ml 8 h after injection (figure 5B). The AVe was compared with the AVe obtained after intravenous injection, and an absolute bioavailability of 45% was calculated. About 3% of the instilled radioactivity was recovered in the zero to 8-h urine, a value similar to that observed after intravenous administration. Toimprove the recoveryof intratracheally administered SLPI, weperformed sequential lavages; table 3 shows that 90 to 100% of the injected dose could be recovered after three lavages within the first hour. About 63% was recovered in the first lavage, whereas about 26.5 and 10.5% were recovered in the second and third lavages, respectively. We compared the recovery of SLPI with that of eglin c 1 min and 1 h after simultaneous administration of the same dose (8.6 mg/kg), The same percentage of the injected dose of SLPI and eglin c wererecoveredin the three lavages(results not shown), and these values weresimilar to that obtained for SLPI alone (table 3).
Chromatographic Separation of SLPI from Rat Lung Lavage Fluids To confirm that SLPI is not degraded in the rat lung, we performed gel filtration studies with two BALE All the detectable immunoreactive SLPI was present in a fraction eluting at the same position as cytochrome c (molecular weight, 12,3(0)
892
GAST, ANDERSON, PROIJST, NICK, THOMPSON, EISENBERG, AND SCHNEBLI
TABLE 2
12
LUNG LAVAGE HALF-TIME VALUES FOR SLP' AFTER INTRATRACHEAL ADMINISTRATION IN RATS
10
I
8
-I
Fig. 3. Plasma SLPI concentrations after intraperitoneal administrationof 12mglkg SLPlto rats. Each point corresponds to the mean value (± 1 SO) of three dilutions measured in triplicate with an ELISA procedure.
6
I'"
4
~
Method of Analysis Radiometry 35S ELISA (antigenic level of SLPI) Elastase inhibitory activity
Elimination Half-time (h)
5.3 4
4.5
2 0 2
0
4 6 Time afteradministration (h)
8
A
,,200
gp
~
E
! c
.S! 100
I ~
O+--~--r--~--.--~---r-~---,,..--~
o
4
2
6
8
B
200
Fig. 4. A. Immunoreactive concentrations of SLPI in rat lung lavage fluids after intratracheal administration of SLPI (8.6 mgt kg). The mean values (± 1 SO) of four animals are shown. B. Antielastase activities in lung lavage fluids. The inhibitory capacity has been measured as described in METHODS. The mean antielastase activity of 13 control rats (2.1 l1g/ml) is subtracted from the individual total antielastase values. The mean values (± 1 SO) of four animals are shown.
.....-~---r--"----,-
O+--~--r--~-
o
2
4
6
8
Time after administration (hours)
and correlated quantitatively with the inhibitory activity. Control experiments with a BALF spiked with SLPI and lyophilized showed that the concentration procedure had little effect on the activity of SLPI. Discussion Numerous studies suggest that emphysema results from an imbalance in the protease-antiprotease profile in the lung. Thus, a therapeutic approach would be
to prevent elastolytic degradation of the lung tissue by enhancing the antiprotease screen. For example, n.Pl has been used successfully to restore the balance in patients with inherited u.Pl deficiency (19, 20). SLPI, another naturally occurring human inhibitor, could play the same role since it is a very potent inhibitor of leukocyte elastase and is able to fully inhibit elastase already bound to elastin fiber (21). To profile SLPI as a therapeutic agent, we determined the pharmacoki-
netic parameters of recombinant SLPI after intravenous, intraperitoneal, and intratracheal administration in rats. When SLPI is administered intravenously, plasma immunoreactive levels decreased, with a half-time of 6 min for the distribution phase and 50 min for the elimination phase. These values are similar to values obtained after administration of eglin c (22) and aprotinin, the trypsinkallikrein bovine inhibitor (Ttasylolv) in rats (23). The very low amounts of SLPI recovered in urine and feces indicate that SLPI is not excreted. The catabolic pathway of SLPI remains to be clarified. Bioavailability after intraperitoneal administration of SLPI was 80070. This indicates a good peritoneal resorption of the protein. After intratracheal administration of SLPI, elastase inhibitory activity paralleled the immunoreactive SLPI levels in BALF, indicating that no significant inactivation of the molecule occurred during lung residence in the rats. This was confirmed by gel filtration analysis of BALF showing that all detectable immunoreactive SLPI was in a free and active form, with a molecular weight of 12,000. The half-time in the lung was between 4 and 5 h as assessed by radiometric, functional, and immunologic techniques. Because the half-life of SLPI in BALF is five to six times longer than in plasma, aerosol administration may be the preferred route if SLPI is to be used as an active therapeutic agent in lung diseases such as emphysema and ARDS. Recently, Smith and coworkers (24) demonstrated that aerosolization of human plasma u.Pl caused no loss of inhibitory activity in dogs. The chemical stability and structure of SLPI strongly support the idea that aerosolization would have little deleterious effect on the activity of the inhibitor. In addition, our preliminary results in rats show that SLPI remains biologically active in the lung for at least 8 h, suggesting that this inhibitor could significantly enhance the antielastase de-
893
PHARMACOKINETICS OF THE SECRETORY LEUKOCYTE PROTEINASE INHIBITOR
A
Lung lavage
300
200
100 Fig. 5. Immunoreactive and radioactive concentrations of SLPI in lung lavage fluid (A) and plasma (B) after intratracheal administration of labeled (20 !lCi/kg) and unlabeled (8.6mg/kg) SLPI. ELISA values are represented by the open circles, and radioactivity values are represented by the closed circles. The mean values(± 1 SD) ofthree animals are shown.
2
4
B
6
Plasma
4
2
2 4 6 Time after administration(hours)
fenses of the lower respiratory tract for a relatively long period. Some recent in vitro studies have shown that recombinant SLPI incubated with activated leukocytes remained active (> 90%) against elastase for at least 1 h and was able to block the elastolytic activity released from the phagocytes (25). However, other in vitro studies have demonstrated that SLPI can be inactivated by the phagocyte-derived myeloperoxidase system, as wellas by cigarette smoke (26, 27). Thus, the relevance of these findings remains to be established in vivo.
SLPI could be recovered almost quantitatively in three sequential lavages within the first hour after application, the period during which the SLPI concentration remains at a high level. Disappearance of SLPI from BALF is partly mirrored by the diffusion into the bloodstream as shown by the calculated bioavailability of 45%. These data suggest that SLPI has a relatively long residence time in the lung lumen and that a significant part of the low molecular weight inhibitor diffuses into the interstitium. In addition, immunohistochemistry studies clearly
TABLE 3 RECOVERY OF THE INTRATRACHEALLY INJECTED SLPI DOSE AFTER THREE LUNG LAVAGES'
Time after Administration (h) 0.017 0.25 0.5 1
2 4 8
Percentage of the Injected Radioactivity Recovered in the First Lavage 66.5 58.7 65.6 62.8 43.4 28.2 18.7
(11.1) (8.6) (12.7) (1) (10.9) (6) (4.7)
Second Lavage 20.6 22.2 24.1 26.5 19.8 17.1 8.7
(1.7) (6.2) (3.4) (2.8) (1.7) (0.7) (1.5)
• Values are mean ± SO of three rats; SO shown in parentheses.
Third Lavage 6.2 (1.3) 8.2 (2.1) 9.0 (2.1) 10.5 (0.9) 8.0 (1.44) 7.7 (0.7) 3.61 (3)
Total Recovery (% injected dose) 93.4 89.1 98.6 99.9 71.2 53 31
(8.4) (12.9) (14.2) (3.5) (6.6) (5.5) (5.3)
showed the presence of SLPI in the connective tissue matrix of the human lung (28). The low molecular weight of the inhibitor may facilitate the diffusion into the viscous lung interstitium when compared with n.Pl, The recovery yields observed for SLPI (pI> 10.5) and eglin c (pI of 5.5) 1 min and I h after simultaneous intratracheal administration were not significantly different, suggesting that possible ionic interactions of the highly positivelycharged SLPI with some acidic components of the epithelial lining did not occur. Gel filtration studies confirmed that SLPI recovered by lavage was in a free form. In rats that received [35S]SLPI intratracheally, the plasma levels of unchanged substance and total radioactivity correlated well for as long as 2 h. The immunoreactive concentration declined in parallel with BALF levels, with a half-time of 3.5 h. However, the low amounts of inhibitor found in rat plasma after intratracheal injection suggests a slow diffusion from the interstitium into the bloodstream. The trace amounts of SLPI found in normal human plasma support this hypothesis (29). The linear increase of the radioactivity can be ascribed to the degradation of SLPI occurring in plasma. It is not possible to functionally measure the plasma levels of the inhibitor. In conclusion, this study demonstrates that SLPI when administered intratracheally to rats can significantly augment the lung antiprotease levels for at least 8 h. The observation that SLPI is located on elastic fibers at the bronchial and bronchiolar level (28), in conjunction with the reported presence of leukocyte elastase on the human lung elastin fibers (30), strongly suggests that SLPI could act as an active inhibitor in vivo at the alveolar level. The fact that SLPI can fully inactivate elastase already bound to elastin supports this concept (21).Taken together, these findings indicate that SLPI could be a potent therapeutic agent able to restore the lung antiprotease defense in certain lung diseases such as emphysema, ARDS, and cystic fibrosis. Acknowledgment The writers thank Heidemarie HUgH, Roland Schneider, Matthias Kuhn, and Hansjoerg Wiegand for skillful technicalassistance.They are grateful to Dr. John Childs and Karin Hale for assistance in preparing the radioactive SLPI. The help of Dr. Jean-Michel Cardot for statistical analysis of data is greatly appreciated.
894
GAST, ANDERSON, PROBST, NICK, THOMPSON, EISENBERG, AND SCHNEBLI
References 1. Janoff A. Elastases and emphysema. Current assessment of the protease-antiprotease hypothesis. Am Rev Respir Dis 1985; 132:417-33. 2. Boudier C, Pelletier A, Gast A, Tournier JM, Pauli G, Bieth JG. The elastase inhibitory capacity and the at-proteinase inhibitor and bronchial inhibitor content of bronchoalveolar lavage fluids from healthy subjects. Bioi Chern Hoppe Seyler 1987; 368:981-90. 3. Morrison HM, Kramps JA, Burnett D, Stockley RA. Lung lavage fluid from patients with a t proteinase inhibitor deficiency or chronic obstructive bronchitis: anti-elastase function and cell profile. Clin Sci 1987; 72:373-81. 4. Kramps JA, Franken C, Meijer CJLM, Dijkman JH. Localization of low molecular weight inhibitor in serous secretory cells of the respiratory tract. J Histochem Cytochem 1981; 29:712-9. 5. De Water R, Willems LNA, van Muijen GNP, et al. Ultrastructural localization of bronchial antileukoprotease in central and peripheral human airways by a gold-labeling technique using monoclonal antibodies. Am Rev Respir Dis 1986; 133: 882-90. 6. Gauthier F, Frysmark U, Ohlsson K, Bieth JG. Kinetics of the inhibition of leucocyte elastase by the bronchial inhibitor. Biochim BiophysActa 1982; 700:178-83. 7. Griitter M, Fendrich G, Huber R, Bode W.The 2.5 A X-ray crystal structure of the acid stable proteinase inhibitor from human mucous secretions analysed in its complexwith bovine a-chymotrypsin. EMBO J 1988; 7:345-51. 8. Thompson RC, Ohlsson K. Isolation, properties and amino acid sequence of human secretory leukocyte proteinase inhibitor, a potent inhibitor ofleukocyte elastase. Proc Natl Acad SciUSA 1986; 83:6692-6. 9. Stetler G, Brewer MT, Thompson RC. Isolation and sequence of a human gene encoding a potent inhibitor of leukocyte proteases. NucleicAcids Res 1986; 14:7883-96.
10. Gast A, Probst A, Nick HP, Schnebli HP. Distribution and pharmacokinetics of the rDNA secretory leukocyte proteinase inhibitor (SLPI). Experientia 1988; 44:A38. 11. Kohno T, Carmichael DF, Sommer A, Thompson RC. Refolding of recombinant proteins. In: Goeddel D, ed. Methods in Enzymology. Vol. 185. New York: Academic Press, 1990; 187-95. 12. Rink A, Liersch M, Sieber P, Meyer F. A large fragment approach to DNA synthesis: total synthesis of a gene for the protease inhibitor eglin c from the leech Hirudino medicinalis and its expressionin£. coli. Nucleic Acids Res 1984; 12:6369-87. 13. Kramps JA, Franken C, Dijkman JH. ELISA for quantitative measurement of low-molecularweight bronchial protease inhibitor in human sputum. Am Rev Respir Dis 1984; 129:959-63. 14. Muller-Ester! W, Oettl A, Trutscheit E, Fritz H. Monitoring of aprotinin plasma levels by an enzyme-linked immunosorbent assay (ELISA). Fresenius Z Anal Chern 1984; 317:718-9. 15. Baugh RJ, 'Travis J. Human leukocyte granule elastase: rapid isolation and characterization. Biochemistry 1976; 15:836-41. 16. Botta L, Gerber H-U, Schmid K. Measurement of radioactivity in biological experiments. In: Garrett ER, Hirtz JL, eds. Drug fate and metabolism. Vol. 5. New York: Marcel Dekker, 1985; 99-134. 17. Brain JD, Knudson DE, Sorokin SP, Davis MA. Pulmonary distribution of particles given by intratracheal instillation or by aerosol inhalation. Environ Res 1976; 11:13-33. 18. Gibaldi M, Perrier D, eds. Pharmacokinetics. 2nd ed. New York: Marcel Dekker, 1982. 19. Gadek JE, KleinHG, Holland PV,Crystal RG. Replacement therapy of alpha-l-antitrypsin deficiency.Reversalof protease-antiprotease imbalance within the alveolar structures of PiZ subjects. J Clin Invest 1981; 68:1158-65. 20. Wewers MD, Casolaro A, Sellers SE, Swayze SC, McPhaul KM, Crystal RG. Replacement therapy for alpha-l-antitrypsin deficiency associated with emphysema. N Engl J Med 1987;361:1055-62.
21. Bruch M, Bieth JG. Influence of elastin on the inhibition of leukocyte elastase by u-proteinase inhibitor and bronchial inhibitor. Biochem J 1986; 238:269-73. 22. Nick HP, Probst A, Schnebli HP. Development of eglin c as a drug: pharmacokinetics, In: Horl WH, Heidland A, eds. Proteases II. Potential role in health and disease. New York and London: Plenum Press, 1988; 83-8. 23. Fritz H, Oppitz KH, Meckl D, et al. The distribution and excretion of naturally occurring and chemically modified protease inhibitors following intravenous injection in rat, dog (and human). Hoppe Seylers Z Physiol Chern 1969;350:1541-50. 24. Smith RM, Spragg RG, Moser KM. Biologic availability of injected or aerosolized alpha, proteinase inhibitor. Prog Clin Bioi Res 1987; 236A: 203-10. 25. Axelsson L, Linder C, Ohlsson K, Rosenberg M. The effect of the secretory leukocyte protease inhibitor on leukocyte proteases released during phagocytosis. Bioi Chern Hoppe Seyler 1988; 369 (Suppl:89-93). 26. Carp H, Janoff A. Inactivation of bronchial mucous proteinase inhibitor by cigarette smoke and phagocyte-derived oxidants. Exp Lung Res 1980; 1:225-37. 27. Kramps JA, Van Twisk C, Klasen EC, Dijkman JH. Interactions among stimulated human polymorphonuclear leucocytes, released elastase and bronchial antileucoprotease. Clin Sci 1988; 75:53-62. 28. Willems LNA, Otto-Verberne CJM, Kramps JA, Ten Haveopbroek AAW,Dijkman JM. Detection of antileukoprotease in connective tissue of lung. Histochemistry 1986; 86:165-8. 29. Tournier JM, Jacquot J, Sadoul P, Bieth JG. Noncompetitive enzyme immunoassay for the measurement of bronchial inhibitor in biological fluids. Anal Biochem 1983; 131:345-50. 30. Damiano VV, Tsang A, Kucich U, et al. Immunolocalization of elastasein human emphysematous lungs. J Clin Invest 1986; 78:482-93.
ALAIN GAST, WAYNE ANDERSON, ALESSANDRO PROBST, HANSPETER NICK, ROBERT C. THOMPSON, STEPHEN P. EISENBERG, and HANSPETER SCHNEBLI
Introduction
It is believed that an imbalance between proteases and their naturally occurring inhibitors results in an abnormal degradation of connective tissue, thus playing a major role in the development of lung diseases such as emphysema (1) and cystic fibrosis. The local concentration of leukocyte elastase exceeds that of active inhibitors, namely, at-proteinase inhibitor (u.Pl), secretory leukocyte proteinase inhibitor (SLPI), and some not-yet-characterized proteins (2, 3), leading to tissue damage. SLPI is synthesized in the central airways by serous glandular cells and in the lower respiratory tract by Clara cells and goblet cells (4, 5). This potent human leukocyte elastase inhibitor (6) is a single polypeptide chain with a molecular weight of 11,700 containing two highly homologous domains. Site-directedmutagenesis of the SLPI gene (in preparation) and X-ray crystallography of the SLPIchymotrypsin complex (7) indicate that the C-terminal domain inhibits trypsinlike enzymes, chymotrypsinlike enzymes, and leukocyte elastase (8). A striking feature of this molecule is its unusual resistance against denaturation by heat and acid, probably because of the presence of eight disulfide bridges. In order to develop SLPI as a therapeutic agent, the gene for this polypeptide was cloned and expressed in Escherichia coli (9). Here, we report the pharmacokinetics and distribution of SLPI after intravenous, intraperitoneal, and intratracheal administration in rats. Some preliminary results have been presented elsewhere (10). Methods Recombinant SLPI and Eglin c Recombinant SLPI was produced in E coliand purified by affinity chromatography on anhydrochymotrypsin AffigellO (11). [3SS]SLPI was produced by either of two methods. In the first method, a 17Q-ml culture of the strain containing the SLPI gene was grown at 37° C to a density of rv 2 x 108cells/ml in M9 medium containing 15 J.1g tetracycline/ml. The
SUMMARY secretory leukocyte proteinase inhibitor (SLPI) is a potent elastase, trypsin, and chymotrypsin inhibitor occurring in all mucous secretions. Its inhibitory potency and profile suggested that it may become a therapeutic adjuvant In diseases where protelnases play a pathogenetic role. In the course of developing recombinant SLPI for therapeutic purposes, we studied Its pharmacokinetics after intravenous, Intraperitoneal, and Intratracheal application to rats. In plasma, SLPI was determined with an ELISA or by following a radlotracer ((35S1SLPI). In bronchoalveolar lavage fluid (BALF), SLPI was determined additionally by a functiQl'I"j assay (elastase inhibitory capaclty).lntr. venously applied SLPI (2 mg/kg) was rapidly cleared, with half-times o! distribution of 6 min and half-times of elimination of 50 min. Very little « 5%) appeared in the urirJ~ even after 24 h. ApproXImately 80% of Intraperitoneally Inlected SLPI (12mg/kg) was absorbed and generated maximal plasma concentration of 6 to 10 I!g/ml 30 to 120 min after administration. When given Intratracheally (8.6 mg/kg), SLPI disappeared from the lungs, with a half-time of 4 to 5 h. This value was the same whether the remaining SLPlln BALF was determined radiometrically, by ELISA or by the functional assay, indicating minimal metabolism in the lung. As in the case of Intraperitoneal application, SLPI was absorbed systemically, resulting In a maximal plasma level of about 2 I!g/ml1 to 2 h after application. In contrast to the measurements in BALF, the ELISA and radlotracer measurements In plasma correlated only for the first 2 h after application and diverged progressively after that, suggesting breakdown of the molecule once it reaches the plasma. The data provided demonstrate that in the rat, intratracheally applied SLPI remains in the lung in active form for several hours and may thus provide effective protection against proteinase-mediated lung damage. AM REV RESPIR DIS 1990; 141:889-894
SLPI gene was induced with IPTG (l mM), and incubation was continued for 80 min. At this point, 5.6 mCi of [35S]methionine (rv 1,000 Ci/mmol) (New England Nuclear, Boston, MA) was added to the culture, and incubation was continued for an additional 10min. Cells wereharvested by centrifugation at 5,000 x g for 10 min at 4° C, washed with 0.18 M cold NaCl, repelleted, and suspended in 0.4 ml of 0.1 M TRIS-HCl at pH 7.2. Lysis of cells and purification of PSS]SLPIwas performed as previously described (9, ll). In the alternate method, the bacterial cells were grown in a medium similar to the one described above, except that the sulfate ion concentration was reduced to 0.2 mM. This culture was grown to a density of 3 x 108 cells/ml. The cells were pelleted by centrifugation at 5,000 x g for 10min at 25° C, and then resuspended in medium as described above with the sulfate ion concentration now at 0.05 mM. The SLPI gene was induced as above and onetwentieth volume of [35S]sulfuric acid (1,300 Ci/mmol) (New England Nuclear) was immediatelyadded. The culture wasincubated with aeration for 4 h, the cells were harvested, and the [3SS]SLPI was purified as above. The gene of eglin c was synthesized and expressed in E. coli (12). Apart from its acetylated N-terminus the purified recombinant
protein is indistinguishable from the nati...e eglin isolated from the leech as demonstrated by inhibitory activity, dodecyl sulfate-polyacrylamide gel electrophoresis, and amino acid analysis (12).
Immunologic Assays Recombinant SLPI was measured in rat plasma and lung lavage fluid using the sandwich ELISA technique described by Kramps and coworkers (13) with the following modifications. PVC micro titer plates (Nunc-Immune plate I; Roskilde, Copenhagen, Denmark) were coated for 1 h at 37° C and then overnight at 4° C with 100 ul of a 5-ltg/ml solution of rabbit immunoglobulin raised against recombinant SLPI. Samples and SLPI standards were applied in triplicate and allowed to bind to the antibody for 3 h at 37° C in a humid chamber. Rabbit anti-SLPI conjugated
(Received in original form April 10, 1989) 1 From the Pharmaceutical Division, Ciba-Geigy Ltd, Basel, Switzerland, and Synergen Inc., Boulder, Colorado. 2 Correspondence and requests for reprints should be addressed to Hanspeter Nick, Pharmaceutical Division, Ciba-Geigy Ltd, 4002 Basel, Switzerland.
889
890 to horseradish peroxidase (Boehringer, Mannheim, FRG) diluted 1:1,500 was added and allowed to complex for 3 h at room temperature. The antigen-antibody complex was visualized with the addition of o-phenylenediamine (4 mg/ml) in citrate phosphate buffer at pH 5 containing 0.012% H 202 • The plates were placed in the dark for 40 min, and the absorbance was read at 490 nm after the color reaction was terminated with 2.5 M H 2S04 , The standard curve was similar to that obtained by Kramps and coworkers (13) and could accurately measure SLPI at concentrations between 0.4 and 1.5 ng/ml, An internal standard (human EDTA plasma diluted 1:50) was run in triplicate on each plate, and the experiment was repeated if the concentration was not 50 ± 10 ng/ml (50 ng/ml being the mean value of SLPI in the plasma). At the dilutions of the samples used in the assay, rat plasma and rat lung lavage did not interfere with the assay. Eglin concentrations in lung lavage were quantified with a competitive ELISA similar to that described for aprotinin (14)except that the eglin-horseradish conjugate was diluted 1:20,000.
Elastase Inhibitory Capacity The elastase inhibitory activity of BALF was assessed by measuring its ability to inhibit the hydrolysis of methoxysuccinyl-(alanylj.-prolyl-valyl-p-nitroanilide by human leukocyte elastase. Leukocyteelastase was purified using the method described by Baugh and Travis (15). Increasing amounts of unconcentrated lavage fluids were incubated with a constant amount of active elastase (20 nM) at 37° C for 15 min in 50 mM TRIS, 100 mM NaCl, and 0.05070 Triton XlOO at pH 7.7 (total reaction volume: I ml). Then, 10 IIIof a lOO-mM substrate solution in dimethyl sulfoxide were added, and after 10min at 37° C the reaction was stopped by the addition of 100 11112.5% acetic acid, and the absorbance at 405 nm was immediately read. An SLPI solution of known anti elastase activity was used as a standard. Radioactivity Measurement The "S activityof the samples was measuredin a liquid scintillationcounter (Packard CD 460; Canberra Packard, Downers Grove, IL) or LKB counter (Wallach 1215; Wallach, Turku, Finland) according to published methods (16). Blood and Urine Collection After intravenous and intraperitoneal administration, the animals were anesthetized with ether, and blood samples of about 0.5 ml were withdrawn from the retro-orbital plexus into small, heparinized glass tubes using capillary pipettes at set time points after dosing. Plasma was obtained by centrifugation at 2,000 rpm for 15 min at 4° C and stored frozen. Urine and feces were collected in standard metabolic cages after intravenous injection of 35S-labeled SLPI. After intratracheal administration, blood
GAST, ANDERSON, PROBST, NICK, THOMPSON, EISENBERG, AND SCHNEBLI
wascollected bycardiac puncture into syringes containing heparin (20IE/ml blood). Animals in the 8-h treatment group wereplaced in standard metabolic cages with free access to water. Urine was collected during this period.
Intravenous and Intraperitoneal Administration SLPI (unlabeled or 35S-labeled) was diluted in PBS to a concentration of 0.93 mg/ml. Approximately 500 III of this was injected into healthy random-bred male rats (Tif: RAIF [SPFj raised on the premises of Ciba-Geigy, weighing 200 to 230 g) intravenously into a tail vein or intraperitoneally. The rats were housed singly in metabolic cages designed to separate urine and feces. For distribution studies the animals were exsanguinated under ether anesthesia, and organs and tissues were obtained by dissection. Intratracheal Administration Intratracheal instillation was performed as described by Brain and coworkers (17). Male rats (weighing 300 to 350 g) were lightly anesthetized with ether and suspended by the upper incisors on a dissecting table at a 75-degree angle from horizontal. A high intensity light was positioned at the throat area, which allowed visualization of the larynx from the mouth. The [35S]SLPI solution (0.3 nCi/mg, 8.6 mg/kg body weight), eglin (8.6 mg/kg), a mixture of SLPI and eglin (8.6rug/kg each), or saline (control) was introduced into the upper trachea by a modified gavage needle. Instillation volume was 0.15 ml/IOO g body weight. Lung lavage was performed at I, 15, and 30 min and at I, 2, 4, and 8 h after intratracheal administration. The animals were anesthetized with sodium thiobutabarbital (100 mg/kg) approximately 30 min before lavage, and a cannula was secured in the trachea. For the early time points (lessthan I h) the animals wereanesthetized and cannulated before intratracheal administration. Saline (10 ml) was slowly introduced into the lung, and 6 ml of fluid were immediately withdrawn. In a second experiment, this procedure was repeated three times for each animal, and the maximal volume of lavage fluid was collected. The volume of each lavage was measured, stored separately on ice, and centrifuged at 2,000 rpm for 15min at 4° C. Recoveryranged from 6 to 10 ml for each lavage. The supernatants were kept frozen until analysis. Gel Filtration Studies BALF from rats lavaged I min and I h after intratracheal administration of SLPI were concentrated tenfold by lyophilization and analyzed on a FPLC gel-filtration Superose 12 column equilibrated with 50 mM sodium phosphate, 0.5 M NaCI (pH, 6.8) at a flow rate of 0.5 ml-min". A control rat lavage spiked with SLPI wastreated in the same manner and served as a reference. The presence of SLPI was assessed in each protein fraction by the ELISA procedure. The functional activi-
ty of SLPI was measured with the elastase inhibitory capacity as described above.
Calculation of Pharmacokinetic Parameters Plasma concentrations (c) of SLPI at time t after dosing can be described by a two-compartment open model: c = A·e- at + B·e-~t. The parameters B and ~ were determined by linear regression analysis of the logarithm of the plasma SLPI concentration-time relationship during the terminal phase of elimination of the compound. Parameters A and II were determined by the method of residuals. The area under the plasma concentrationtime curve (AU C) was calculated by the trapezoidal rule. Calculation oftotal plasma clearance (CIT)and volume of distribution of the central compartment (Vdc) was performed using CIT = D/AUC, Vdc = D/(A + B) (18). The group values for immunoreactive lung lavage fluid SLPI, radioactive, and SLPI antielastase activity were compared by a single-tailed t test for paired groups. Differences between groups were considered to be significant at p < 0.05. Results
Pharmacokinetics after Intravenous Administration Five minutes after intravenous administration of 2 mg/kg [35S]SLPI, 30070 of the total radioactivity wasdetected in kidneys, 20% in blood, and approximately 15070 in liver, muscle, and skin. Only 5% was found in the lung (figure 1). The serum concentration of unlabeled SLPI after a bolus injection of 2 mg/kg decreased in a biphasic fashion (figure 2) with halftimes of 6 min for the distribution phase and 51 ± 4 min for the elimination phase (table 1). Approximately 2% of the administered dose (labeled and unlabeled SLPI) was detected in both the zero to 8-h and 8- to 24-h urine and about 5% of the injected radioactivity was found in the zero to 24-h feces. The volume of distribution of the central compartment (Vdc) was 67 ± 3 ml- kg", and the total clearance (Ch) was 4.9 ± 0.2 ml-min? kg? (table 1). Pharmacokinetics after Intraperitoneal Administration The SLPI appearance in plasma after intraperitoneal administration of 12mg/kg SLPI is shown in figure 3. In two rats SLPI reached a maximal concentration of 6 to 8 ug/ml 30 min after injection, whereas in one rat a peak concentration of 10 ug/ml was observed 2 h after administration. The apparent half-time of elimination in plasma was about 1 h. When the AVC/dose was compared with the AVC/dose after intravenous injec-
891
PHARMACOKINETICS OF THE SECRETORY LEUKOCYTE PROTEINASE INHIBITOR
Blood Liver KidneyMuscle Skin Pancreas Fat
Lung Spleen Brain
Organ/Tissue Fig. 1. Distribution of radioactivity in organs and tissues of rats 5 min after intravenous administration of 2 mg/kg 35S-labeled SLPI.
100
Fig. 2. Plasma SLPI concentrations after intravenous administration of recombinant SLPI (2 mg/kg) to two rats as measured with ELISA. Each point corresponds to the mean value of three dilutions measured in triplicate (see table 1).
.1
.01
+--....----,---.--.,..-........-,.----.-~- ........-..., o 2 4 3 5 Time after administration (hours)
tion, an absolute bioavailability of 80070 was calculated.
Pharmacokinetics after Intratracheal Administration After intratracheal administration of SLPI (8.6 mg/kg), the concentration of immunoreactiveand activeSLPI in BALF (one lavage) was measured at set times.
Within the first hour after instillation the mean immunoreactive level remained at a high value and then decreased, with a half-time of 4 h (figure 4 and table 2). No significant difference was observed between the concentrations of immunoreactive and active SLPI as assessed by t test (p < 0.05) (figure 4). The concentration of active SLPI parallels the im-
TABLE 1 PHARMACOKINETIC PARAMETERS AFTER INTRAVENOUS ADMINISTRATION OF SLPI
Rat No.
1 2
Central Volume of Distribution (ml·kg-' body wt)
Tv, Distribution (min)
70 64
6 6
Total Clearance (ml-min-"kg-' body wt)
T'h Elimination (min)
5.1
55
4.7
47
munoreactive levelover the whole observation period. The half-time of elimination of active SLPI was 4.5 h, a value similar to that observed for immunoreactive SLPI (table 2). In a separate experiment, wemeasured total radioactivity and immunoreactive SLPI in sequential lung lavages and in plasma after intratracheal administration of 8.6 mg/kg unlabeled SLPI complemented with 35S-labeled SLPI. The results obtained for the first lavagesby both methods are shown in figure 5. The concentrations of SLPI determined by ELISA or calculated from the radioactivity data (figure 5A) were not significantly different (p < 0.05). Furthermore, the radioactivity decreased with a half-time of 5.3 h, a value similar to that of immunoreactive and active SLPI (table 2). Plasma immunoreactive levels of SLPI after intratracheal administration increased in parallel with the radioactivity concentrations and reached a peak concentration of 2 ug/ml l h after injection (figure 5B) and decreased thereafter. In contrast, the radioactivity concentrations continued to increase linearly to reach the equivalent of 5.5 ug SLPI!ml 8 h after injection (figure 5B). The AVe was compared with the AVe obtained after intravenous injection, and an absolute bioavailability of 45% was calculated. About 3% of the instilled radioactivity was recovered in the zero to 8-h urine, a value similar to that observed after intravenous administration. Toimprove the recoveryof intratracheally administered SLPI, weperformed sequential lavages; table 3 shows that 90 to 100% of the injected dose could be recovered after three lavages within the first hour. About 63% was recovered in the first lavage, whereas about 26.5 and 10.5% were recovered in the second and third lavages, respectively. We compared the recovery of SLPI with that of eglin c 1 min and 1 h after simultaneous administration of the same dose (8.6 mg/kg), The same percentage of the injected dose of SLPI and eglin c wererecoveredin the three lavages(results not shown), and these values weresimilar to that obtained for SLPI alone (table 3).
Chromatographic Separation of SLPI from Rat Lung Lavage Fluids To confirm that SLPI is not degraded in the rat lung, we performed gel filtration studies with two BALE All the detectable immunoreactive SLPI was present in a fraction eluting at the same position as cytochrome c (molecular weight, 12,3(0)
892
GAST, ANDERSON, PROIJST, NICK, THOMPSON, EISENBERG, AND SCHNEBLI
TABLE 2
12
LUNG LAVAGE HALF-TIME VALUES FOR SLP' AFTER INTRATRACHEAL ADMINISTRATION IN RATS
10
I
8
-I
Fig. 3. Plasma SLPI concentrations after intraperitoneal administrationof 12mglkg SLPlto rats. Each point corresponds to the mean value (± 1 SO) of three dilutions measured in triplicate with an ELISA procedure.
6
I'"
4
~
Method of Analysis Radiometry 35S ELISA (antigenic level of SLPI) Elastase inhibitory activity
Elimination Half-time (h)
5.3 4
4.5
2 0 2
0
4 6 Time afteradministration (h)
8
A
,,200
gp
~
E
! c
.S! 100
I ~
O+--~--r--~--.--~---r-~---,,..--~
o
4
2
6
8
B
200
Fig. 4. A. Immunoreactive concentrations of SLPI in rat lung lavage fluids after intratracheal administration of SLPI (8.6 mgt kg). The mean values (± 1 SO) of four animals are shown. B. Antielastase activities in lung lavage fluids. The inhibitory capacity has been measured as described in METHODS. The mean antielastase activity of 13 control rats (2.1 l1g/ml) is subtracted from the individual total antielastase values. The mean values (± 1 SO) of four animals are shown.
.....-~---r--"----,-
O+--~--r--~-
o
2
4
6
8
Time after administration (hours)
and correlated quantitatively with the inhibitory activity. Control experiments with a BALF spiked with SLPI and lyophilized showed that the concentration procedure had little effect on the activity of SLPI. Discussion Numerous studies suggest that emphysema results from an imbalance in the protease-antiprotease profile in the lung. Thus, a therapeutic approach would be
to prevent elastolytic degradation of the lung tissue by enhancing the antiprotease screen. For example, n.Pl has been used successfully to restore the balance in patients with inherited u.Pl deficiency (19, 20). SLPI, another naturally occurring human inhibitor, could play the same role since it is a very potent inhibitor of leukocyte elastase and is able to fully inhibit elastase already bound to elastin fiber (21). To profile SLPI as a therapeutic agent, we determined the pharmacoki-
netic parameters of recombinant SLPI after intravenous, intraperitoneal, and intratracheal administration in rats. When SLPI is administered intravenously, plasma immunoreactive levels decreased, with a half-time of 6 min for the distribution phase and 50 min for the elimination phase. These values are similar to values obtained after administration of eglin c (22) and aprotinin, the trypsinkallikrein bovine inhibitor (Ttasylolv) in rats (23). The very low amounts of SLPI recovered in urine and feces indicate that SLPI is not excreted. The catabolic pathway of SLPI remains to be clarified. Bioavailability after intraperitoneal administration of SLPI was 80070. This indicates a good peritoneal resorption of the protein. After intratracheal administration of SLPI, elastase inhibitory activity paralleled the immunoreactive SLPI levels in BALF, indicating that no significant inactivation of the molecule occurred during lung residence in the rats. This was confirmed by gel filtration analysis of BALF showing that all detectable immunoreactive SLPI was in a free and active form, with a molecular weight of 12,000. The half-time in the lung was between 4 and 5 h as assessed by radiometric, functional, and immunologic techniques. Because the half-life of SLPI in BALF is five to six times longer than in plasma, aerosol administration may be the preferred route if SLPI is to be used as an active therapeutic agent in lung diseases such as emphysema and ARDS. Recently, Smith and coworkers (24) demonstrated that aerosolization of human plasma u.Pl caused no loss of inhibitory activity in dogs. The chemical stability and structure of SLPI strongly support the idea that aerosolization would have little deleterious effect on the activity of the inhibitor. In addition, our preliminary results in rats show that SLPI remains biologically active in the lung for at least 8 h, suggesting that this inhibitor could significantly enhance the antielastase de-
893
PHARMACOKINETICS OF THE SECRETORY LEUKOCYTE PROTEINASE INHIBITOR
A
Lung lavage
300
200
100 Fig. 5. Immunoreactive and radioactive concentrations of SLPI in lung lavage fluid (A) and plasma (B) after intratracheal administration of labeled (20 !lCi/kg) and unlabeled (8.6mg/kg) SLPI. ELISA values are represented by the open circles, and radioactivity values are represented by the closed circles. The mean values(± 1 SD) ofthree animals are shown.
2
4
B
6
Plasma
4
2
2 4 6 Time after administration(hours)
fenses of the lower respiratory tract for a relatively long period. Some recent in vitro studies have shown that recombinant SLPI incubated with activated leukocytes remained active (> 90%) against elastase for at least 1 h and was able to block the elastolytic activity released from the phagocytes (25). However, other in vitro studies have demonstrated that SLPI can be inactivated by the phagocyte-derived myeloperoxidase system, as wellas by cigarette smoke (26, 27). Thus, the relevance of these findings remains to be established in vivo.
SLPI could be recovered almost quantitatively in three sequential lavages within the first hour after application, the period during which the SLPI concentration remains at a high level. Disappearance of SLPI from BALF is partly mirrored by the diffusion into the bloodstream as shown by the calculated bioavailability of 45%. These data suggest that SLPI has a relatively long residence time in the lung lumen and that a significant part of the low molecular weight inhibitor diffuses into the interstitium. In addition, immunohistochemistry studies clearly
TABLE 3 RECOVERY OF THE INTRATRACHEALLY INJECTED SLPI DOSE AFTER THREE LUNG LAVAGES'
Time after Administration (h) 0.017 0.25 0.5 1
2 4 8
Percentage of the Injected Radioactivity Recovered in the First Lavage 66.5 58.7 65.6 62.8 43.4 28.2 18.7
(11.1) (8.6) (12.7) (1) (10.9) (6) (4.7)
Second Lavage 20.6 22.2 24.1 26.5 19.8 17.1 8.7
(1.7) (6.2) (3.4) (2.8) (1.7) (0.7) (1.5)
• Values are mean ± SO of three rats; SO shown in parentheses.
Third Lavage 6.2 (1.3) 8.2 (2.1) 9.0 (2.1) 10.5 (0.9) 8.0 (1.44) 7.7 (0.7) 3.61 (3)
Total Recovery (% injected dose) 93.4 89.1 98.6 99.9 71.2 53 31
(8.4) (12.9) (14.2) (3.5) (6.6) (5.5) (5.3)
showed the presence of SLPI in the connective tissue matrix of the human lung (28). The low molecular weight of the inhibitor may facilitate the diffusion into the viscous lung interstitium when compared with n.Pl, The recovery yields observed for SLPI (pI> 10.5) and eglin c (pI of 5.5) 1 min and I h after simultaneous intratracheal administration were not significantly different, suggesting that possible ionic interactions of the highly positivelycharged SLPI with some acidic components of the epithelial lining did not occur. Gel filtration studies confirmed that SLPI recovered by lavage was in a free form. In rats that received [35S]SLPI intratracheally, the plasma levels of unchanged substance and total radioactivity correlated well for as long as 2 h. The immunoreactive concentration declined in parallel with BALF levels, with a half-time of 3.5 h. However, the low amounts of inhibitor found in rat plasma after intratracheal injection suggests a slow diffusion from the interstitium into the bloodstream. The trace amounts of SLPI found in normal human plasma support this hypothesis (29). The linear increase of the radioactivity can be ascribed to the degradation of SLPI occurring in plasma. It is not possible to functionally measure the plasma levels of the inhibitor. In conclusion, this study demonstrates that SLPI when administered intratracheally to rats can significantly augment the lung antiprotease levels for at least 8 h. The observation that SLPI is located on elastic fibers at the bronchial and bronchiolar level (28), in conjunction with the reported presence of leukocyte elastase on the human lung elastin fibers (30), strongly suggests that SLPI could act as an active inhibitor in vivo at the alveolar level. The fact that SLPI can fully inactivate elastase already bound to elastin supports this concept (21).Taken together, these findings indicate that SLPI could be a potent therapeutic agent able to restore the lung antiprotease defense in certain lung diseases such as emphysema, ARDS, and cystic fibrosis. Acknowledgment The writers thank Heidemarie HUgH, Roland Schneider, Matthias Kuhn, and Hansjoerg Wiegand for skillful technicalassistance.They are grateful to Dr. John Childs and Karin Hale for assistance in preparing the radioactive SLPI. The help of Dr. Jean-Michel Cardot for statistical analysis of data is greatly appreciated.
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