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Biodisposition and catabolism of tissue-type plasminogen activator in rats and rabbits
Fhr~nolws (1988) 2. 31 36 c 1988 Longman Group UK Ltd
Biodisposition and Catabolism of Tissue-type Plasminogen Activator in Rats and Rabbits
C. Bakhit, D. Lewis, U. Busch, P. Tanswell,
M. Mohler
SUMMARY. The biodisposition and catabolism of 1251-rt-PA was investigated in rats and rabbits. Most of the ‘2sI-labelled rt-PA was sequestered by the liver following intravenous (i.v.) injection. Fractionation of supernatants obtained from liver homogenates by trichloroacetic acid (TCA) precipitation showed an increased, conversion of intact ‘2sI-rt-PA to TCA soluble material with time. In addition, there was a marked decrease of total radioactivity with time in the liver, and a concomittant increase in the blood, kidney and urine. This suggests a sequential process of degradation by the liver, secretion of degradation products into the blood and transport to the kidney for excretion in the urine. Autoradiographic localisation of 12SI-rt-PA showed results similar to the biochemical data with the added observation that ‘2sI-Labelled material does not appear in the fetuses of pregnant rats. KEYWORDS.
Recombinant
rt-PA. Biodisposition.
Hepatic elimination.
Autoradiography.
Recombinant t-PA was radiolabelled to high specific activity with Na’*‘I and used in the in vivo and in vitro experiments. The nature of rt-PA metabolites was examined by biochemical analysis of liver homogenates.
Use of recombinant tissue-type plasminogen activator (rt-PA) in thrombolytic therapy has generated considerable interest in understanding its physiological and pharmacological effects in vivo. Recent reports have shown that the clearance of rt-PA from the blood is rapid with an initial half-life (t-1/2) of approximately 2 min in rodents’,2’3 and 4-6 min in humans.4 Organ distribution studies of ‘2sI-rt-PA have also indicated that there is early uptake and rapid accumulation in the liver.‘,* At later times, trichloroacetic acid (TCA) soluble labeled material reappears in the circulation.2,s This material is cleared by the kidney with radioactivity appearing in the urine.* This apparent process of uptake and degradation of rt-PA by the liver has only been studied indirectly. The purpose of this study was to gain a better understanding of the biodisposition and fate of rt-PA in rats and rabbits; specifically to determine how effectively rt-PA is deactivated and whether active metabolites are formed in vivo. Having observed that rt-PA rapidly concentrates in the liver, the catabolism of this glycoprotein was studied in the liver which is the site of degradation of most endogenous and exogenous compounds. Biochemical and morphological studies were conducted to examine the organ distribution of rt-PA.
METHODS Preparation of ‘2SI-rt-PA Recombinant t-PA was labelled by the chloramine-T method.‘j The iodination reaction mixture consisted of 10 ~1 of 10mM sodium phosphate buffer (pH 8.0), 5 m Ci of Na-‘251 (Amersham, Arlington Heights, IL) in 10 ~1 of Phosphate Buffer, and a 100 ~1 sample containing 50 pg of rt-PA. The reaction was terminated after 3 min at room temperature with 10~1 of 20mM dithiothreitol, 50~1 of 24mM potassium iodide in 0.2% gelatin, and 10 pl of 1.5% Bovine Serum Albumin (BSA) Fraction V. The ‘251-labelled rt-PA was purified by column chromatography using a preequilibrated Sephadex G-25, PD-10 column (Pharmacia, Upsala, Sweden). This procedure provided a specific activity of 4(r50 pCi/pg (1.33 moles of lzsI per mole rt-PA). When ‘2sI-rt-PA was analysed by nonreduced SDS-PAGE gels, the radioactivity of the labelled preparations was associated with a single band corresponding to the molecular weight of rt-PA (Mr 65000). For the whole body autoradiography, rt-PA was labelled with Na’*9 (New England Nuclear) using the iodogen method,’ followed by
C. Bakhit, D. Lewis, M. Mohler, Department of Pharmacological Sciences. Genentech, Inc. 460 Point San Bruno, South San Francisco, CA 94080, USA. U. Busch, P. Tanswell, Biochemistry Department, Dr Karl Thomae GmbH, Postfach 1755, D-7950 Biberach, Federal Republic of Germany. 31
32
Biodisposition
and Catabolism
of Tissue-type
Plasminogen
Activator
separation of free “‘1 from ‘251-rt-PA on a Sephadex G-25 column equilibrated in 0.05M phosphate buffer pH 7.4 with 0.5% BSA. The specific activity of the labelled rt-PA was 30-60 uCi/ug protein. Amidolytic activity of the ‘251-labelled rt-PA was fully retained as measured in the spectrophotometic assay using the chromogenic substrate S 2288.
Biodisposition
packed beads and allowed to react overnight at 4°C. The unbound active sites on the Sepharose were blocked by incubating the gel with 0.9 M ethanolamine, pH 8.0, for 2 h at 4°C. The anti-rt-PA IgG Sepharose was washed extensively with PBS and stored at 4°C as 50% slurry in PBS and 0.05% sodium azide. A 75 ~1 aliquot of the 50% anti-rt-PA IgG Sepharose mixture had the capacity to bind 2 ug of rt-PA.
in Rabbits
Male rabbits (New Zealand White), weighing 2 to 3 kg were used to study the distribution and metabolism of ‘251-labelled rt-PA. All animals received a dose of 1 mg/kg rt-PA (the activity of the rt-PA used in this study was 500000 IU/mg) mixed with 30pCi/kg of 1251-rt-PA (diluted in PBS) as an infusion of 15 ml over 30 min via the ear vein. Blood samples were taken via a cannula in the contralateral ear vein every 5 min and just prior to sacrificing the animal. Whole blood was collected in a final concentration of 4.6 mM dipotassium ethylenediaminetetracetic acid (K, EDTA) and 3 units of heparin per ml of blood. The animals were sacrificed at 0, 10 and 60 min after the infusion by injection of pentobarbital into the ipsilateral ear vein. The animals were immediately disssected. Various organs were removed and samples of bile and urine were taken.
Biodisposition
in Rats and Rabbits
in Rats
Similar distribution studies were performed with male and female rats (Charles River) weighing 250 to 400 g. The animals were anesthetised with an intraperitoneal injection of pentobarbital (50mg/kg) before the experiment. The labelled rt-PA, 30uCi/kg mixed with 1 mg/kg unlabelled rt-PA, was injected into the femoral vein in a volume of 300 pl of PBS. Blood samples were taken 30 set, 5 and 10 min after the bolus injection by orbital sinus puncture. The blood was collected in a final concentration of 4.6mM Kz EDTA and 3 units of heparin per ml of whole blood. Ten and 60 min after the ‘251-rt-PA injection, the animals were dissected, the liver, lungs, heart, kidneys, spleen, gonads removed, and the urine collected from the bladder.
Preparation of Rabbit Anti-rt-PA IgG Bound Sepharose
Rabbit anti-rt-PA IgG was purified by loading 5 ml of rabbit immune serum onto a 1.6 cm x 5 cm proteinA Sepharose column (Pharmacia), equilibrated with PBS. The column was washed with 10 volumes of PBS to elute unbound protein and the IgG fraction was eluted with 0.15 M NaCl and 0.1 M acetic acid. Upon elution, the IgG fraction was immediately dialysed against 100 volumes of PBS at 4°C. The dialysed IgG was adjusted to 1 mg/ml by dilution with PBS and added to cyanogen bromide activated Sepharose (Pharmacia) at a ratio of 2 mg IgG per milliliter
Tissue Processing
After dissection, tissues were minced and homogenised in 10 volumes of ice cold 2 N acetic acid/O.2 N HCl with a Brinkman polytron. The homogenate was centrifuged at 12 000 x g for 45 min at 4°C. Plasma was prepared from whole blood by centrifugation at 10 000 x g for 5 min at 4°C. Plasma and urine were acidified with one volume of ice cold 4 N acetic acid/ 0.4 N HCl and mixed by vortex. The acidified samples and the tissue supernatant were frozen in liquid nitrogen and lyophilised in volumes of less than 25 ml for 48 h. Lyophilised material was resuspended in one fifth the original volume, homogenised and centrifuged at 12 000 x g for 45 min at 4°C. An aliquot of the resulting clear supernatant was immunoprecipitated with anti-rt-PA IgG bound sepharose as described below. A second aliquot of the same supernatant was fractionated with 100 ul of 100% (w/v) trichloroacetic acid (TCA) per ml of supernatant. The mixture was kept on ice 15 min, then centrifuged at 2000 x g for 2min. The supernatant was removed and the pellet washed with 1 ml of PBS and re-centrifuged at 10 000 x g for 5 min. The supernatant was removed and the pellet resuspended in 500 ul of 250 mM tris, ph 8.5, 5% sodium dodecyl sulfate (SDS). Aliquots of each homogenate and supernatant were taken and the radioactivity measured to determine organ distribution.
Immunoprecipitation
of rt-PA
A 100 pl aliquot of the cleared tissue supernatant was added to 500 pl of 10 mM tris, 150 mM NaCl, 0.1 percent gelatin and 0.05% Tween-80, ph 7.4 (TBSGT) and 75 ul of the anti-rt-PA IgG bound Sepharose and allowed to incubate overnight on a rocking platform at 4°C. The mixture was centrifuged for 2 min at 10000 x g, 4°C and the supernatant aspirated. The beads were then washed with 500 ul TBSGT and centrifuged again. The supernatant was aspirated and 100 ~1 of 20 mM tris, 5% SDS, pH 6.8, was added to the packed Sepharose beads and the mixture was heated to 100°C for 10min to elute the bound rt-PA from the Sepharose beads. The free rt-PA was separated from the Sepharose beads by passing the mixture through an Eppendorf pipet tip packed with a small glass wool plug.
Fibrinolysis
SDSPolyacrylamide PAGE)
Gel
Electropheresis
(SDS-
Aliquots of the clear tissue supernatant, the resuspended TCA pellet fraction and the immunoprecipitated rt-PA were loaded onto iOx, SDSpolycrylamide gels 0.75 mm thick, using the buffer system described by Laemmli.8 Electrophoreses were performed under reducing conditions (5% P-mercaptoethanol). Gels were dried on membrane backing (Bio-Rad, Richmond, CA) and used to expose Kodak X-omat-AR film at - 70°C for 2-30 days.
Whole Body Autoradiography Pregnant rats in 18 days of gestation (mean weight 270 g) were fasted 20 h with only water ad libitum before dosing. These rats received an i.v. bolus injection in the tail vein of 15 uCi 1251-rt-PA diluted with approximately 1000 fold excess of unlabelled rt-PA in formulation buffer to yield a total dose of 1 mg/kg body weight. Autoradiography was performed at 5 min after dosing. A blood sample from the retro-orbital plexus was obtained under halothane anaesthesia. Immediately thereafter, the rat was sacrificed in ethanol/dry ice at -80°C and embedded in a frozen aqueous suspension of carboxymethyl-cellulose. Thirty micron thick sagittal whole body slices were obtained using a cryomicrotome and exposed to X-ray film for 2&25 days. Samples of accessible tissues were gouged from the residual carcass for processing and counting as described.
RESULTS The biodisposition of ’251-rt-PA in rabbit organs was determined following an intravenous infusion. Unlabelled and ‘251-labelled rt-PA were mixed to provide a final dose of 1 mg/kg which was infused over a period of 30min. Table 1 shows that approximately 50% of the injected material was sequestered by the liver. The rest of the organs examined contained less than 1% of the labelled material with the exception of the lung and kidney which contained 1.0 and 2.6% respectively. There was also a significant amount of labelled material (17%) in the blood. When the data were expressed as CPM/gm wet tissue, it was apparent that highly vascularised organs such as the spleen, lung, kidney, pituitary and liver retain a larger amount of labelled material (Table 1). When the biodisposition of ‘*%rt-PA is examined at time points following the end of the infusion an interesting trend emerges. Table 2 shows the organ distribution of labelled material at 0, 10, and 60min post-infusion. The amount found in the liver diminishes considerably with time post-infusion, reaching 13% at 60 min. In contrast, the amounts found in the kidney, urine and blood increased considerably. A
33
Table 1 Biodisposition of Radioactivity Derived From ‘251-rt-PA* In Rabbit Organs Following an Intravenous Infusion Tissue
CPM/g
Percent
Thyroid Thymus Lung Heart
23001 15327 59100 35314 172249 146080 5638 54488 14605 81135 16306 352770 1505s1** 46i60**
0.05 + 0.01 O.lOkO.06 1.00+0.47 0.22 +0.01 0.20 * 0.04 2.56 kO.67 0.05 + 0.00 0.001 +o.OO 0.08 & 0.03 0.07 f 0.04 0.58kO.13 47.23 + 5.84 16.61 k 3.26 0.92 + 0.77 69.52 k 7.56
Spleen
Kidney Brain Pituitary Gonads Gall bladder Stomach Liver Plasma Urine Total
of total injected
* 30uCi/kg of ‘ZSI-rt-PA were mixed with unlabelled rt-PA to a final dose of 1 mg/kg and infused for l/2 h (see test for details). The rabbits (n =4) were sacrificed at the end of the infusion. Values are means + S.D. ** CPM/ml
Table 2 Time Course of the Biodisposition of Radioactivity Derived from ‘Z51-rt-PA in Rabbit Organs Following Intravenous Infusion for 30 min
Tissue
Percent of total injected Time, post-infusion (min) 0 10
60
Liver Spleen Kidney Lung Bile Urine Blood
40.8 f 9.38 0.29kO.22 2.19*0.83 0.93 f 0.49 0.07 * 0.03 0.13+0.14 16.20 + 4.69
13.01 k4.54 0.09 LO.02 4.22kO.30 3.82k3.51 0.24 + 0.07 13.79 + 5.80 25.88 f 1.73
36.70+ 16.33 0.38 + 0.08 4.1 I kO.52 1.55*0.54 0.04~004 1.35kO.47 22.27 +_1.94
Labelled and unlabeled rt-PA were mixed to provide a final dose of 1mg/kg. Values are meansf SD. of 4 rabbits.
Table 3 Biodisposition of Radioactivity Derived From “‘1-rt-PA in Rat Organs at 10 and 60 min Following an Intravenous Injection. Values are the Means & S.D. of 4 Rats
Tissue
Percent of total injected 10 min
60 min
Liver Spleen Kidney Lung Heart Gonads Blood Urine
54.33 * 15.33 1.42& 0.39 1.20+0.36 1.94+_202 5.14*7.01 1.93+204 21.08k5.31 5.35 * 2.00
15.73 + 2.69 2.29 kO.48 1.48+0.01 4.58 +0.95 6.6Ok4.14 4.58 * 0.95 17.79* 1.14 2.20~0.01
slight but significant increase of ‘251-Labelled material was also observed in the lung. The biodisposition of 1251-rt-PA in rat organs following an i.v. bolus injection was also examined. A similar distribution of radioactivity was found in rat organs as was seen in the rabbits, with the exception of slightly higher amounts in the major internal organs other than the liver (Table 3). The disappearance of
34
Biodisposition
and Catabolism
Fig. 1 Whole body autoradiography
of Tissue-type
following
Plasminogen
an i.v. injection
Activator
of “‘I-G-PA
Table 4 Biodisposition of Radioactivity Derived From iz51-rt-PA in Selected Organs of Pregnant Rats Following an i.v. Injection of a Dose of 1 mg/kg Time post-injection
Brain Lung Liver Muscle Blood (heart) Thymus Blood (venous) Salivary gland Placenta
cpmlg
(min) 5% of blood
6446 177656 863726 9958 329910 25012 408380 39876 217466
0.02 0.44 2.12 0.02 0.81 0.06 1.00 0.10 0.53
in Rats and Rabbits
labelled material from the liver with time followed a trend similar to that seen in the rabbits. The biodisposition of ‘2sI-rt-PA in pregnant rats determined by whole body autoradiography shows that 5 min after injection of 12’1-rt-PA, radioactivity levels in the dam were highest in the liver, followed by lung, kidney, bone marrow and red pulp of the spleen (Fig. 1). The radioactivity level in the placenta was comparable to blood (Table 4), suggesting that the placental radioactivity is attributable to perfusion. No radioactivity was present in the fetus; individual fetal organs (brain, lung, liver and blood) were readily identifiable in the sections. Radioactivity levels were quantified in several tissues (Table 4). The results confirm the visual observations of the autoradiographs. Five min after ‘251-rt-PA was injected the highest radioactivity levels per gram or ml were observed in the blood and liver with significant amounts in the lung. Negligible radioactivity was detected in brain, testes, thymus, muscle and fat. The radioactivity counts in the placenta was about 50% that of venous blood. Having determined that the liver is the major site of disposition of rt-PA, and considering that it is also the main site of catabolism of endogenous and exogenous substances, we decided to examine the fate of i2’I-rt-PA in the liver. At 0, 10, and 60 min following a 30 min i.v. infusion of ‘251-rt-PA, rabbit livers were dissected, minced on ice, homogenised and centri-
in pregnant
rats.
fuged. The resulting supernatant was found to contain more than 95% of the labelled material (data not shown). Fractionation of the supernatant into TCA soluble and precipitable material showed an increase in soluble labelled material from 30% of the total at 0 time (end of infusion) to about 60% of the total 1 h following the end of the infusion (Fig. 2). Considering that the TCA fails to precipitate substances having a molecular weight below about 5000 D, the above data suggest extensive degradation of ‘251-rt-PA. It should be noted that in Figure 2 the data were expressed as a percentage of the total present in the liver at the particular time indicated. Thus the total amount present at 60 min was about 18% of the amount present at 0 time (see Table 2). To further examine the nature of the degradation products, the initial supernatant was fractionated by either TCA precipitation or immunoprecipitation using Sepharose bound rt-PA antibody. Soluble and precipitable material from both procedures were analysed by SDS gel electrophoresis. As can be seen in Figure 3, there were three bands which were present in the standard and the test samples, one at 67 kd is intact rt-PA and the two at 3C~35 kd are the two chains of the reduced rt-PA. The three bands having a Mr around 22 kd or below can only be detected in the test samples and are likely to be fragments generated in vivo as intermediates in the degradation of rt-PA.
DISCUSSION The data presented in this study demonstrate that most of the injected 1251-rt-PA was rapidly concentrated and subsequently degraded by the liver. The degradation of rt-PA results in the generation of predominantly small molecular weight fragments that are secreted into the blood stream and transported to the kidney for excretion in the urine. These results suggest that rt-PA is sequentially taken up and degraded by the liver and the degradation products are secreted into the blood stream and
Fibrinolysis
35
??TCA Soluble 0
TCA Preciptable
'10
0
Time Post-infusion (min.) Fig. 2 Time course of the degradation of i*sI-rt-PA to low molecular weight (< 5000 D, TCA soluble) species. At 0, 10, and 60 min following a 30 min infusion, rabbit livers (n =4) were dissected, homogenised in ice cold 2N acetic acid/0.2N HCI, centrifuged and the resulting supernatant fractionated by 10% TCA, centrifuged and the amount of labeled material in the resulting supernatant and pellet was determined. Data is presented as percent of amount present in the liver at each time point.
Mf -
94
- 67
- 43
- 30 - 20
A
B
C
D
E
St
Fig. 3 Electrophoretic analysis of the supernatant and pellet resulting from either TCA precipitation or immunoprecipitation of the initial supernatant of the liver homogenate. Samples were analyzed by SDS-PAGE under reducing conditions (see text for detail). (A) Initial supernatant. (B) TCA supernatant C-TCA pellet. (D) Immunoprecipitation supcrnatant. (E) immunoprecipitation pellet. St.-standard rt-PA sample.
then by the kidney into the urine. The results are in agreement with the findings of other investigators.’ It has been shown by Hotchkiss, et al (unpublished observation) that degradation products having the proteolytic activity of the parent molecule are not released back into the circulation since the degraded material found in the plasma is of low molecular weight (less than 5000 D), relative to the parent molecule at 65 kd. The three fragments observed by SDSPAGE are likely to be fragments of degradation. Electrophoretic analysis of labelled material in the plasma at different times following injection of rt-PA have failed to show the presence of these fragments, indicating that only small molecular weight (< 5000 D) fragments are released back into the blood. The observation that not all of the ‘251-rt-PA taken up by the liver is degraded 1 h following the end of the infusion, especially since most of the labelled material was already in the liver at the end of the infusion, suggests that the degradation step is rate-limiting in the elimination of rt-PA. However, more detailed examination of the presumed binding, uptake and degradation of rt-PA by liver cells is needed to confirm the above contention. The results obtained from the whole body autoradiography confirm and extend the biochemical data. Intravenous injection of ’251-rt-PA to rats yields autoradiographs at early time points showing specific
36
Biodisposition
and Catabolism
of Tissue-type
Plasminogen
Activator
organ radioactivity, principally in the liver, which is attributable to intact rt-PA or large fragments. In conclusion, the present study demonstrates that rt-PA undergoes extensive degradation in the liver.
ACKNOWLEDGEMENTS We wish to thank H. Zipp and P. Foldenauer for performing the iodine labelling of the whole body autoradiography experiments, and J. Baierl for excellent technical assistance. We would also like to thank Dr Bryan Finkle for his encouragement and support.
REFERENCES 1. Korninger C, Stassen C M, Cohen D 1981 Turnover of human extrinsic (tissue-type) plasminogen activator in rabbits. Thromb. Haemostas 46: 658-661
OKprint orders to: C. Bakhit, Department of Pharmacological Sciences, Genentech, Inc. 460 Point San Bruno, South San Francisco, CA 94080, USA.
in Rats and Rabbits
2. Fuchs H E, Berger Jr H, Pizza S V 1985 Catabolism of human tissue plasminogen activator in mice. Blood 65: 539-544 3. Nilsson T, Wallen P, Mellbring G 1984 In vivo metabolism of human tissue-type plasminogen activator. Stand J Haematol 33:49-53 4. Verstraete M, Bounameaux H, de Cock F, deWerf F V, Collen I$ 1985 Pharmacokinetics and Systemic fibrinolytic effects of recombinant human tissue-type plasminogen activator (rt-PA) in humans. J Pharmacol Exp Therap 235:506-512 5. Mohler M, Hotchkiss A, Bringman T, Tate K, Vehar G, Circulatory metabolism of recombinant tissue-type plasminogen activator. In preparation of 6. Greenwood F, Hunter W, Glover J 1963 The preparation r3’I-Labeled human growth hormone of high specific radioactivity. Biochem J 89: 114-123 7. Mohler M, Refine C J, Chen S A, Chen A B, Hotchkiss A J 1986 D-Phe-Pro-Arg-Chloromethylketone: It’s potential use in inhibiting the formation of in vitro artifacts in blood collected during tissue-type plasminogen activator thrombolytic therapy. Thromb Haemost in press 8. Laemmli V K 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:68&685
Biodisposition and Catabolism of Tissue-type Plasminogen Activator in Rats and Rabbits
C. Bakhit, D. Lewis, U. Busch, P. Tanswell,
M. Mohler
SUMMARY. The biodisposition and catabolism of 1251-rt-PA was investigated in rats and rabbits. Most of the ‘2sI-labelled rt-PA was sequestered by the liver following intravenous (i.v.) injection. Fractionation of supernatants obtained from liver homogenates by trichloroacetic acid (TCA) precipitation showed an increased, conversion of intact ‘2sI-rt-PA to TCA soluble material with time. In addition, there was a marked decrease of total radioactivity with time in the liver, and a concomittant increase in the blood, kidney and urine. This suggests a sequential process of degradation by the liver, secretion of degradation products into the blood and transport to the kidney for excretion in the urine. Autoradiographic localisation of 12SI-rt-PA showed results similar to the biochemical data with the added observation that ‘2sI-Labelled material does not appear in the fetuses of pregnant rats. KEYWORDS.
Recombinant
rt-PA. Biodisposition.
Hepatic elimination.
Autoradiography.
Recombinant t-PA was radiolabelled to high specific activity with Na’*‘I and used in the in vivo and in vitro experiments. The nature of rt-PA metabolites was examined by biochemical analysis of liver homogenates.
Use of recombinant tissue-type plasminogen activator (rt-PA) in thrombolytic therapy has generated considerable interest in understanding its physiological and pharmacological effects in vivo. Recent reports have shown that the clearance of rt-PA from the blood is rapid with an initial half-life (t-1/2) of approximately 2 min in rodents’,2’3 and 4-6 min in humans.4 Organ distribution studies of ‘2sI-rt-PA have also indicated that there is early uptake and rapid accumulation in the liver.‘,* At later times, trichloroacetic acid (TCA) soluble labeled material reappears in the circulation.2,s This material is cleared by the kidney with radioactivity appearing in the urine.* This apparent process of uptake and degradation of rt-PA by the liver has only been studied indirectly. The purpose of this study was to gain a better understanding of the biodisposition and fate of rt-PA in rats and rabbits; specifically to determine how effectively rt-PA is deactivated and whether active metabolites are formed in vivo. Having observed that rt-PA rapidly concentrates in the liver, the catabolism of this glycoprotein was studied in the liver which is the site of degradation of most endogenous and exogenous compounds. Biochemical and morphological studies were conducted to examine the organ distribution of rt-PA.
METHODS Preparation of ‘2SI-rt-PA Recombinant t-PA was labelled by the chloramine-T method.‘j The iodination reaction mixture consisted of 10 ~1 of 10mM sodium phosphate buffer (pH 8.0), 5 m Ci of Na-‘251 (Amersham, Arlington Heights, IL) in 10 ~1 of Phosphate Buffer, and a 100 ~1 sample containing 50 pg of rt-PA. The reaction was terminated after 3 min at room temperature with 10~1 of 20mM dithiothreitol, 50~1 of 24mM potassium iodide in 0.2% gelatin, and 10 pl of 1.5% Bovine Serum Albumin (BSA) Fraction V. The ‘251-labelled rt-PA was purified by column chromatography using a preequilibrated Sephadex G-25, PD-10 column (Pharmacia, Upsala, Sweden). This procedure provided a specific activity of 4(r50 pCi/pg (1.33 moles of lzsI per mole rt-PA). When ‘2sI-rt-PA was analysed by nonreduced SDS-PAGE gels, the radioactivity of the labelled preparations was associated with a single band corresponding to the molecular weight of rt-PA (Mr 65000). For the whole body autoradiography, rt-PA was labelled with Na’*9 (New England Nuclear) using the iodogen method,’ followed by
C. Bakhit, D. Lewis, M. Mohler, Department of Pharmacological Sciences. Genentech, Inc. 460 Point San Bruno, South San Francisco, CA 94080, USA. U. Busch, P. Tanswell, Biochemistry Department, Dr Karl Thomae GmbH, Postfach 1755, D-7950 Biberach, Federal Republic of Germany. 31
32
Biodisposition
and Catabolism
of Tissue-type
Plasminogen
Activator
separation of free “‘1 from ‘251-rt-PA on a Sephadex G-25 column equilibrated in 0.05M phosphate buffer pH 7.4 with 0.5% BSA. The specific activity of the labelled rt-PA was 30-60 uCi/ug protein. Amidolytic activity of the ‘251-labelled rt-PA was fully retained as measured in the spectrophotometic assay using the chromogenic substrate S 2288.
Biodisposition
packed beads and allowed to react overnight at 4°C. The unbound active sites on the Sepharose were blocked by incubating the gel with 0.9 M ethanolamine, pH 8.0, for 2 h at 4°C. The anti-rt-PA IgG Sepharose was washed extensively with PBS and stored at 4°C as 50% slurry in PBS and 0.05% sodium azide. A 75 ~1 aliquot of the 50% anti-rt-PA IgG Sepharose mixture had the capacity to bind 2 ug of rt-PA.
in Rabbits
Male rabbits (New Zealand White), weighing 2 to 3 kg were used to study the distribution and metabolism of ‘251-labelled rt-PA. All animals received a dose of 1 mg/kg rt-PA (the activity of the rt-PA used in this study was 500000 IU/mg) mixed with 30pCi/kg of 1251-rt-PA (diluted in PBS) as an infusion of 15 ml over 30 min via the ear vein. Blood samples were taken via a cannula in the contralateral ear vein every 5 min and just prior to sacrificing the animal. Whole blood was collected in a final concentration of 4.6 mM dipotassium ethylenediaminetetracetic acid (K, EDTA) and 3 units of heparin per ml of blood. The animals were sacrificed at 0, 10 and 60 min after the infusion by injection of pentobarbital into the ipsilateral ear vein. The animals were immediately disssected. Various organs were removed and samples of bile and urine were taken.
Biodisposition
in Rats and Rabbits
in Rats
Similar distribution studies were performed with male and female rats (Charles River) weighing 250 to 400 g. The animals were anesthetised with an intraperitoneal injection of pentobarbital (50mg/kg) before the experiment. The labelled rt-PA, 30uCi/kg mixed with 1 mg/kg unlabelled rt-PA, was injected into the femoral vein in a volume of 300 pl of PBS. Blood samples were taken 30 set, 5 and 10 min after the bolus injection by orbital sinus puncture. The blood was collected in a final concentration of 4.6mM Kz EDTA and 3 units of heparin per ml of whole blood. Ten and 60 min after the ‘251-rt-PA injection, the animals were dissected, the liver, lungs, heart, kidneys, spleen, gonads removed, and the urine collected from the bladder.
Preparation of Rabbit Anti-rt-PA IgG Bound Sepharose
Rabbit anti-rt-PA IgG was purified by loading 5 ml of rabbit immune serum onto a 1.6 cm x 5 cm proteinA Sepharose column (Pharmacia), equilibrated with PBS. The column was washed with 10 volumes of PBS to elute unbound protein and the IgG fraction was eluted with 0.15 M NaCl and 0.1 M acetic acid. Upon elution, the IgG fraction was immediately dialysed against 100 volumes of PBS at 4°C. The dialysed IgG was adjusted to 1 mg/ml by dilution with PBS and added to cyanogen bromide activated Sepharose (Pharmacia) at a ratio of 2 mg IgG per milliliter
Tissue Processing
After dissection, tissues were minced and homogenised in 10 volumes of ice cold 2 N acetic acid/O.2 N HCl with a Brinkman polytron. The homogenate was centrifuged at 12 000 x g for 45 min at 4°C. Plasma was prepared from whole blood by centrifugation at 10 000 x g for 5 min at 4°C. Plasma and urine were acidified with one volume of ice cold 4 N acetic acid/ 0.4 N HCl and mixed by vortex. The acidified samples and the tissue supernatant were frozen in liquid nitrogen and lyophilised in volumes of less than 25 ml for 48 h. Lyophilised material was resuspended in one fifth the original volume, homogenised and centrifuged at 12 000 x g for 45 min at 4°C. An aliquot of the resulting clear supernatant was immunoprecipitated with anti-rt-PA IgG bound sepharose as described below. A second aliquot of the same supernatant was fractionated with 100 ul of 100% (w/v) trichloroacetic acid (TCA) per ml of supernatant. The mixture was kept on ice 15 min, then centrifuged at 2000 x g for 2min. The supernatant was removed and the pellet washed with 1 ml of PBS and re-centrifuged at 10 000 x g for 5 min. The supernatant was removed and the pellet resuspended in 500 ul of 250 mM tris, ph 8.5, 5% sodium dodecyl sulfate (SDS). Aliquots of each homogenate and supernatant were taken and the radioactivity measured to determine organ distribution.
Immunoprecipitation
of rt-PA
A 100 pl aliquot of the cleared tissue supernatant was added to 500 pl of 10 mM tris, 150 mM NaCl, 0.1 percent gelatin and 0.05% Tween-80, ph 7.4 (TBSGT) and 75 ul of the anti-rt-PA IgG bound Sepharose and allowed to incubate overnight on a rocking platform at 4°C. The mixture was centrifuged for 2 min at 10000 x g, 4°C and the supernatant aspirated. The beads were then washed with 500 ul TBSGT and centrifuged again. The supernatant was aspirated and 100 ~1 of 20 mM tris, 5% SDS, pH 6.8, was added to the packed Sepharose beads and the mixture was heated to 100°C for 10min to elute the bound rt-PA from the Sepharose beads. The free rt-PA was separated from the Sepharose beads by passing the mixture through an Eppendorf pipet tip packed with a small glass wool plug.
Fibrinolysis
SDSPolyacrylamide PAGE)
Gel
Electropheresis
(SDS-
Aliquots of the clear tissue supernatant, the resuspended TCA pellet fraction and the immunoprecipitated rt-PA were loaded onto iOx, SDSpolycrylamide gels 0.75 mm thick, using the buffer system described by Laemmli.8 Electrophoreses were performed under reducing conditions (5% P-mercaptoethanol). Gels were dried on membrane backing (Bio-Rad, Richmond, CA) and used to expose Kodak X-omat-AR film at - 70°C for 2-30 days.
Whole Body Autoradiography Pregnant rats in 18 days of gestation (mean weight 270 g) were fasted 20 h with only water ad libitum before dosing. These rats received an i.v. bolus injection in the tail vein of 15 uCi 1251-rt-PA diluted with approximately 1000 fold excess of unlabelled rt-PA in formulation buffer to yield a total dose of 1 mg/kg body weight. Autoradiography was performed at 5 min after dosing. A blood sample from the retro-orbital plexus was obtained under halothane anaesthesia. Immediately thereafter, the rat was sacrificed in ethanol/dry ice at -80°C and embedded in a frozen aqueous suspension of carboxymethyl-cellulose. Thirty micron thick sagittal whole body slices were obtained using a cryomicrotome and exposed to X-ray film for 2&25 days. Samples of accessible tissues were gouged from the residual carcass for processing and counting as described.
RESULTS The biodisposition of ’251-rt-PA in rabbit organs was determined following an intravenous infusion. Unlabelled and ‘251-labelled rt-PA were mixed to provide a final dose of 1 mg/kg which was infused over a period of 30min. Table 1 shows that approximately 50% of the injected material was sequestered by the liver. The rest of the organs examined contained less than 1% of the labelled material with the exception of the lung and kidney which contained 1.0 and 2.6% respectively. There was also a significant amount of labelled material (17%) in the blood. When the data were expressed as CPM/gm wet tissue, it was apparent that highly vascularised organs such as the spleen, lung, kidney, pituitary and liver retain a larger amount of labelled material (Table 1). When the biodisposition of ‘*%rt-PA is examined at time points following the end of the infusion an interesting trend emerges. Table 2 shows the organ distribution of labelled material at 0, 10, and 60min post-infusion. The amount found in the liver diminishes considerably with time post-infusion, reaching 13% at 60 min. In contrast, the amounts found in the kidney, urine and blood increased considerably. A
33
Table 1 Biodisposition of Radioactivity Derived From ‘251-rt-PA* In Rabbit Organs Following an Intravenous Infusion Tissue
CPM/g
Percent
Thyroid Thymus Lung Heart
23001 15327 59100 35314 172249 146080 5638 54488 14605 81135 16306 352770 1505s1** 46i60**
0.05 + 0.01 O.lOkO.06 1.00+0.47 0.22 +0.01 0.20 * 0.04 2.56 kO.67 0.05 + 0.00 0.001 +o.OO 0.08 & 0.03 0.07 f 0.04 0.58kO.13 47.23 + 5.84 16.61 k 3.26 0.92 + 0.77 69.52 k 7.56
Spleen
Kidney Brain Pituitary Gonads Gall bladder Stomach Liver Plasma Urine Total
of total injected
* 30uCi/kg of ‘ZSI-rt-PA were mixed with unlabelled rt-PA to a final dose of 1 mg/kg and infused for l/2 h (see test for details). The rabbits (n =4) were sacrificed at the end of the infusion. Values are means + S.D. ** CPM/ml
Table 2 Time Course of the Biodisposition of Radioactivity Derived from ‘Z51-rt-PA in Rabbit Organs Following Intravenous Infusion for 30 min
Tissue
Percent of total injected Time, post-infusion (min) 0 10
60
Liver Spleen Kidney Lung Bile Urine Blood
40.8 f 9.38 0.29kO.22 2.19*0.83 0.93 f 0.49 0.07 * 0.03 0.13+0.14 16.20 + 4.69
13.01 k4.54 0.09 LO.02 4.22kO.30 3.82k3.51 0.24 + 0.07 13.79 + 5.80 25.88 f 1.73
36.70+ 16.33 0.38 + 0.08 4.1 I kO.52 1.55*0.54 0.04~004 1.35kO.47 22.27 +_1.94
Labelled and unlabeled rt-PA were mixed to provide a final dose of 1mg/kg. Values are meansf SD. of 4 rabbits.
Table 3 Biodisposition of Radioactivity Derived From “‘1-rt-PA in Rat Organs at 10 and 60 min Following an Intravenous Injection. Values are the Means & S.D. of 4 Rats
Tissue
Percent of total injected 10 min
60 min
Liver Spleen Kidney Lung Heart Gonads Blood Urine
54.33 * 15.33 1.42& 0.39 1.20+0.36 1.94+_202 5.14*7.01 1.93+204 21.08k5.31 5.35 * 2.00
15.73 + 2.69 2.29 kO.48 1.48+0.01 4.58 +0.95 6.6Ok4.14 4.58 * 0.95 17.79* 1.14 2.20~0.01
slight but significant increase of ‘251-Labelled material was also observed in the lung. The biodisposition of 1251-rt-PA in rat organs following an i.v. bolus injection was also examined. A similar distribution of radioactivity was found in rat organs as was seen in the rabbits, with the exception of slightly higher amounts in the major internal organs other than the liver (Table 3). The disappearance of
34
Biodisposition
and Catabolism
Fig. 1 Whole body autoradiography
of Tissue-type
following
Plasminogen
an i.v. injection
Activator
of “‘I-G-PA
Table 4 Biodisposition of Radioactivity Derived From iz51-rt-PA in Selected Organs of Pregnant Rats Following an i.v. Injection of a Dose of 1 mg/kg Time post-injection
Brain Lung Liver Muscle Blood (heart) Thymus Blood (venous) Salivary gland Placenta
cpmlg
(min) 5% of blood
6446 177656 863726 9958 329910 25012 408380 39876 217466
0.02 0.44 2.12 0.02 0.81 0.06 1.00 0.10 0.53
in Rats and Rabbits
labelled material from the liver with time followed a trend similar to that seen in the rabbits. The biodisposition of ‘2sI-rt-PA in pregnant rats determined by whole body autoradiography shows that 5 min after injection of 12’1-rt-PA, radioactivity levels in the dam were highest in the liver, followed by lung, kidney, bone marrow and red pulp of the spleen (Fig. 1). The radioactivity level in the placenta was comparable to blood (Table 4), suggesting that the placental radioactivity is attributable to perfusion. No radioactivity was present in the fetus; individual fetal organs (brain, lung, liver and blood) were readily identifiable in the sections. Radioactivity levels were quantified in several tissues (Table 4). The results confirm the visual observations of the autoradiographs. Five min after ‘251-rt-PA was injected the highest radioactivity levels per gram or ml were observed in the blood and liver with significant amounts in the lung. Negligible radioactivity was detected in brain, testes, thymus, muscle and fat. The radioactivity counts in the placenta was about 50% that of venous blood. Having determined that the liver is the major site of disposition of rt-PA, and considering that it is also the main site of catabolism of endogenous and exogenous substances, we decided to examine the fate of i2’I-rt-PA in the liver. At 0, 10, and 60 min following a 30 min i.v. infusion of ‘251-rt-PA, rabbit livers were dissected, minced on ice, homogenised and centri-
in pregnant
rats.
fuged. The resulting supernatant was found to contain more than 95% of the labelled material (data not shown). Fractionation of the supernatant into TCA soluble and precipitable material showed an increase in soluble labelled material from 30% of the total at 0 time (end of infusion) to about 60% of the total 1 h following the end of the infusion (Fig. 2). Considering that the TCA fails to precipitate substances having a molecular weight below about 5000 D, the above data suggest extensive degradation of ‘251-rt-PA. It should be noted that in Figure 2 the data were expressed as a percentage of the total present in the liver at the particular time indicated. Thus the total amount present at 60 min was about 18% of the amount present at 0 time (see Table 2). To further examine the nature of the degradation products, the initial supernatant was fractionated by either TCA precipitation or immunoprecipitation using Sepharose bound rt-PA antibody. Soluble and precipitable material from both procedures were analysed by SDS gel electrophoresis. As can be seen in Figure 3, there were three bands which were present in the standard and the test samples, one at 67 kd is intact rt-PA and the two at 3C~35 kd are the two chains of the reduced rt-PA. The three bands having a Mr around 22 kd or below can only be detected in the test samples and are likely to be fragments generated in vivo as intermediates in the degradation of rt-PA.
DISCUSSION The data presented in this study demonstrate that most of the injected 1251-rt-PA was rapidly concentrated and subsequently degraded by the liver. The degradation of rt-PA results in the generation of predominantly small molecular weight fragments that are secreted into the blood stream and transported to the kidney for excretion in the urine. These results suggest that rt-PA is sequentially taken up and degraded by the liver and the degradation products are secreted into the blood stream and
Fibrinolysis
35
??TCA Soluble 0
TCA Preciptable
'10
0
Time Post-infusion (min.) Fig. 2 Time course of the degradation of i*sI-rt-PA to low molecular weight (< 5000 D, TCA soluble) species. At 0, 10, and 60 min following a 30 min infusion, rabbit livers (n =4) were dissected, homogenised in ice cold 2N acetic acid/0.2N HCI, centrifuged and the resulting supernatant fractionated by 10% TCA, centrifuged and the amount of labeled material in the resulting supernatant and pellet was determined. Data is presented as percent of amount present in the liver at each time point.
Mf -
94
- 67
- 43
- 30 - 20
A
B
C
D
E
St
Fig. 3 Electrophoretic analysis of the supernatant and pellet resulting from either TCA precipitation or immunoprecipitation of the initial supernatant of the liver homogenate. Samples were analyzed by SDS-PAGE under reducing conditions (see text for detail). (A) Initial supernatant. (B) TCA supernatant C-TCA pellet. (D) Immunoprecipitation supcrnatant. (E) immunoprecipitation pellet. St.-standard rt-PA sample.
then by the kidney into the urine. The results are in agreement with the findings of other investigators.’ It has been shown by Hotchkiss, et al (unpublished observation) that degradation products having the proteolytic activity of the parent molecule are not released back into the circulation since the degraded material found in the plasma is of low molecular weight (less than 5000 D), relative to the parent molecule at 65 kd. The three fragments observed by SDSPAGE are likely to be fragments of degradation. Electrophoretic analysis of labelled material in the plasma at different times following injection of rt-PA have failed to show the presence of these fragments, indicating that only small molecular weight (< 5000 D) fragments are released back into the blood. The observation that not all of the ‘251-rt-PA taken up by the liver is degraded 1 h following the end of the infusion, especially since most of the labelled material was already in the liver at the end of the infusion, suggests that the degradation step is rate-limiting in the elimination of rt-PA. However, more detailed examination of the presumed binding, uptake and degradation of rt-PA by liver cells is needed to confirm the above contention. The results obtained from the whole body autoradiography confirm and extend the biochemical data. Intravenous injection of ’251-rt-PA to rats yields autoradiographs at early time points showing specific
36
Biodisposition
and Catabolism
of Tissue-type
Plasminogen
Activator
organ radioactivity, principally in the liver, which is attributable to intact rt-PA or large fragments. In conclusion, the present study demonstrates that rt-PA undergoes extensive degradation in the liver.
ACKNOWLEDGEMENTS We wish to thank H. Zipp and P. Foldenauer for performing the iodine labelling of the whole body autoradiography experiments, and J. Baierl for excellent technical assistance. We would also like to thank Dr Bryan Finkle for his encouragement and support.
REFERENCES 1. Korninger C, Stassen C M, Cohen D 1981 Turnover of human extrinsic (tissue-type) plasminogen activator in rabbits. Thromb. Haemostas 46: 658-661
OKprint orders to: C. Bakhit, Department of Pharmacological Sciences, Genentech, Inc. 460 Point San Bruno, South San Francisco, CA 94080, USA.
in Rats and Rabbits
2. Fuchs H E, Berger Jr H, Pizza S V 1985 Catabolism of human tissue plasminogen activator in mice. Blood 65: 539-544 3. Nilsson T, Wallen P, Mellbring G 1984 In vivo metabolism of human tissue-type plasminogen activator. Stand J Haematol 33:49-53 4. Verstraete M, Bounameaux H, de Cock F, deWerf F V, Collen I$ 1985 Pharmacokinetics and Systemic fibrinolytic effects of recombinant human tissue-type plasminogen activator (rt-PA) in humans. J Pharmacol Exp Therap 235:506-512 5. Mohler M, Hotchkiss A, Bringman T, Tate K, Vehar G, Circulatory metabolism of recombinant tissue-type plasminogen activator. In preparation of 6. Greenwood F, Hunter W, Glover J 1963 The preparation r3’I-Labeled human growth hormone of high specific radioactivity. Biochem J 89: 114-123 7. Mohler M, Refine C J, Chen S A, Chen A B, Hotchkiss A J 1986 D-Phe-Pro-Arg-Chloromethylketone: It’s potential use in inhibiting the formation of in vitro artifacts in blood collected during tissue-type plasminogen activator thrombolytic therapy. Thromb Haemost in press 8. Laemmli V K 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:68&685