Effect of SCH 37224 on anti-IgE-induced formation of sulfidopeptide leukotrienes in human lung parenchyma

European Journal qf Pharrnacology, 219 (1992) 159-164 ~) 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.110

159

EJP 52592

Effect of SCH 37224 on anti-IgE-induced formation of sulfidopeptide leukotrienes in human lung parenchyma T. V i g a n 6 , M . T . Crivellari, M . R . A c c o m a z z o , A. M o n o p o l i ~, P.A. Belloni b, M. T o r r e b, S. N i c o s i a and G.C. Folco hlstitute of Pharmacolo,f,,ical Sciences. Via Balzaretti 9, 20133 Milan, Italy, ~' Research Laboratories, Schering-Plough, Comazzo, Milan, Italy and J~Centro de Ga~peris di Chirurgia Toracica, Ospedale Niguarda, Milan, Ita(v Received 10 December 1991, revised MS received 31 March 1992, accepted 26 May 1992

SCH 37224, 1-(1,2-dihydro-4-hydroxy-2-oxo-l-phenyl-l,8-naphthyridin-3-yl) pyrrolidinium, is a structurally novel compound that had been shown to inhibit leukotriene D 4 formation in guinea pig lung in vitro. We tested whether SCH 37224 is able to inhibit both the formation of eicosanoids from human lung parenchyma in vitro and the binding of sulfidopeptidc leukotrienes to membranes of lung parcnchyma and bronchi. SCH 37224, at a concentration of 30 and 100 ~xM, was able to inhibit antigen-induced formation of sulfidopeptide leukotrienes, measured as leukotriene E4, while it did not significantly affect the formation of prostaglandin D z. At concentrations up to 100 ~tM, it did not affect either the binding of [~H]lcukotriene C a to membranes of human lung parenchyma or human bronchi, or the binding of [3H]leukotriene D4 to the parenchyma. In conclusion, SCH 37224 is a selective inhibitor of leukotriene formation in human lung in vitro, which might be of potential therapeutic interest in the treatment of asthma. SCH 37224; Sulfidopeptide leukotrienes; Lung parenchyma (human); Leukotriene binding

1. Introduction

The sulfidopeptide leukotrienes (LT) LTC4, L T D 4 and L T E 4 are potent constrictors of airway smooth muscle in several species, including the human (Drazen and Austen, 1987; Dahldn et al., 1980). In addition, they are known to induce hypersecretion of mucus (Marom et al., 1982) and increase vascular permeability, resulting in mucosal edema (Hau et al., 1985). They are indeed formed in human airways upon immunological challenge (Vigan6 et al., 1986; Kumlin and Dahl6n, 1990). For these reasons, it is generally accepted that these metabolites of arachidonic acid may play an important role in hypersensitivity reactions such as asthma (Samuelsson, 19831. The evaluation in vivo of this hypothesis requires the development of either inhibitors of LT biosynthesis or LT receptor antagonists and it is likely that such compounds will prove to be interesting therapeutic agents for the treatment of asthma. The above mentioned considerations have prompted the search for LT inhibitors or antagonists.

Correspondence to: T. Vigan6, Institute of Pharmacological Sciences, Via Balzaretti 9, 20133 Milan, Italy.

SCH 37224, 1-(1,2-dihydro-4-hydroxy-2-oxo-l-phenyl-l,8-naphthyridin-3-yl) pyrrolidinium, is a structurally novel compound that inhibits L T D 4 formation in guinea pig lung in vitro, anaphylactic bronchospasm in guinea pig and rat (Kreutner et al., 1988), and airway hyperresponsiveness in allergic sheep (Abraham et al., 1988). We now report on the effect of SCH 37224 on sulfidopeptide LT formation in human lung parenchyma in vitro. In addition, in order to test the selectivity of the compound, we also assessed its ability to affect the formation of prostaglandin (PG)D2, a potent bronchoconstrictor (Giles and Left, 1988) which is formed in substantial amounts in human lung (Schulman et al., 1981; Vigan6 et al., 1986). We also tested whether the drug interacts with the specific binding sites for LTC 4 and L T D 4.

2. Materials and m e t h o d s

2.1. Challenge of human lung parenchyma Normal human lung parenchyma was obtained at the time of surgical resection for cancer of bronchiectasis. Tissue fragments of approximately 100 mg each,

16(1

wcrc extensively washed with Tyrodc buffer of the following composition in g I ~: NaCI 8.(t, KCI 0.25, N a H e P O 4 . H 2 0 0.066, glucose 1.0, N a H C O 3 1.0, CaCle 0.15, MgC1," 6H~O 0.01, pH 7.4 and preincubated for 1 h at 37°C in the same buffer in order to stabilize the tissue. At the cnd of the incubation, lung parenchymal fragments were washed again and resus3 °C pended in Tyrode (1 g 10 ml i each sample) at ,7 for 1(1 min with 50 /ll DMSO (control) or with SCH 37224 (1, 10, 30, 100 /,M). Stock suspension of SCH 37224 were prepared in DMSO and 50 p.l was added to the biological samples. Afterwards, the tissue was challenged with an anti-human IgE antibody and 15 min later aliquots of the incubation medium ((I.3 ml) were collected for immediate bioassay of slow reacting substance of anaphylaxis (SRS-A) activity to avoid further analytical processing of the samples which did not show the presence of sulfidopeptide LT. The remaining volume (approximately 4.1) ml) was used for P G D , and LTE 4 assays, as described (see below).

2.2. Enzyme immunoassay (EIA) of PGD e P G D : was measured by EIA (Pradelles ct al., 19851 as the stable derivative, the l l-methoxime-PGD, (PGD2-MO) (Maclouf et al., 1986). Aliquots of samples (50/,I) were evaporated under nitrogen and the residue was resuspended in 100 /xl of pyridine saturated with methoxamine-HCI. After 1 h at 35°C, the pyridine was evaporated under nitrogen and the samples were diluted with EIA incubation buffer of the following composition: NaC1 0.4 M, E D T A 1 mM, bovine serum albumin (BSA) 0.1%, and sodium azide 0.01%, in 0.1 M phosphate buffer, pH 7.4. The E I A apparatus was from Flow Laboratories (Helsinki, Finland) and includes an automatic plate washer (Microplate Washer 12(/), an automatic dispenser (Autodrop) and a spectrophotometer (Multiskan MC). For quantitative deterruination, a mouse monoclonal anti-rabbit IgG in 50 mM phosphate buffer was automatically dispensed into 96-well microtitre plates (Nunc-Denmark) with the Autodrop. After overnight incubation at room temperature, 100/xl of E I A buffer containing extra BSA (0.3%) and sodium azide (0.03%) was added to saturate any unbound plate areas. Coated plates were incubated for at least 18 h at 4°C prior to use. The plates were then washed with 10 mM phosphate buffer, pH 7.4, containing 0.05% Tween 20, using the Microplate Washer. Standard PGDe-MO or biological samples, enzymatic tracer (PGD2-acetylcholine esterase (ACHE)) and an appropriate dilution (1:100000) of specific antiserum were dispensed in a 50-#1 volume. After overnight incubation at room temperature, the plates were washed again and 200 #1 of Ellman's reagent was automatically dispensed with the Autodrop. After 1-2 h absorbance at 414 nm was measured with the Multi-

skan spcctrophotometer. Thc standard curve of PGD ,MO ranged from 15 pg ml ' to 1 ng ml ' The antibody against P G D , - M O demonstrated (I.2% crossreactivity with P G D , and less than 0.1~/c with othcr PG. The high percent coefficient of variation (CV%) was about 16% at 3.12 pg. The results were calculated in terms of percent of B Bo ~, where B and Bo represent absorbance measured for the bound fraction in the presence and absence of PGD2-MO competitor respectively. The standard curve and the results of quantitative determination of PGD,MO in biological samples were analyzed with an IBM computer using a linear log-logit transformation.

2.3. Quantitatiue determination of sulfidopeptide L T Sulfidopeptide LT were measured as LTE 4 following the conversion of LTC 4 and LTD 4 to LTE 4 according to Heavey et al. (19871. Total LTE 4 was resolved by reverse phase high performance liquid chromatography (RP-HPLC) and quantitated by EIA. More specifically, 20000 dpm of [3H]LTE4 was added to an exact volume of the biological sample (4 ml), which was incubated with 0.1 U ml-~ each of y-glutamyl transpeptidase (EC 2.3.2.2) and microsomal leucine aminopeptidase (EC 3.4.11.2), for 3 h at 37°C. Proteins were precipitated with three volumes of M e O H and, after centrifugation at 50(1 × g for 10 min, the supernatant was diluted further to 30% MeOH with phosphate buffer (1(10 raM, pH 7.4). The samples were loaded on a C~s Sep-Pak cartridge (Waters Assoc., Milford, MA, USA), preconditioned according to Powell (19821. LTEa was eluted in 8 ml of 80% M e O H - 2 0 % H 2 0 and recovery was 80%. These methanolic fractions were dried under vacuum with a rotary evaporator and reconstituted in 1 ml of 30% M e O H - 7 0 % HeO then injected into an HPLC system with a photodiode array detector. The analytical conditions were as follows: column (1511 x 4.6 mm) packed with Spherisorb ODS2 5/,tm (Phase Separation, UK); the mobile phase was MeOH : water : acetic acid, 7 0 : 3 0 : 0 . 0 2 ( v : v : v ) , pH 5.7 with NHaOH. The flow rate was 1 ml min ' Fractions, 0.5 ml, were collected with a Gilson fraction collector and the radioactivity of each fraction was evaluated to determine both recovery and exact retention time of LTE 4. The fractions corresponding to peaks of radioactivity as well as fractions before and after were assayed for the presence of LTE 4 by EIA with an LTEa-AChE tracer, following the same procedure as described for P G D , . The antibody raised against LTE 4 demonstrated 2 5 ~ cross-reactivity with LTD 4 and 20% with LTCa. The detection limit of the assay was 25 pg ml ~ of LTE4: the C V ~ was about 38% at 12.5 pg. The results were obtained by subtraction of the amount of radioactive tracer added and correction for recovery.

161

2.4. Bioassay of SRS-A actit,ity

2. 7. Materials

The SRS-A activity present in lung supernatants was bioassayed using a method based on the laminar flow technique according to Ferreira and Da Souza Costa (1976). Guinea pig ileum strips were pretreated with a mixture of receptor antagonists (0.05 ~ M scopolamine, 3 /.LM propranolol, 0.5 # M mepyramine, 0.5 /zM methysergide, and (1.5 /zM phenoxybenzamine) and 10 # M indomethacin, to obtain a specific response. LTC 4 (ranging from 0.25 to 2 ng) used as standard was added as a bolus in volumes not exceeding 0.1 ml. In order to verify the specificity of the assay, the same LTC 4 standard as well as the test solutions were assayed in the presence of a sulfidopeptide LT receptor antagonist (FPL 55712, 10 #M).

SCH 37224 was synthesized by the department of Medicinal Chemistry at Schering-Plough (Bloomfield, NJ, USA). [14,15-~H]LTE4 (specific activity 39 Ci m m o l - l), [14,15_,~H]LTC4 and [14,15-3H]LTD4 (40 Ci mmol ~) were from NEN Dupont (Boston, MA, USA). Ellman's reagent, mouse monoclonal anti-rabbit IgG, PGD~-AChE were purchased from Cayman Chemical Company, (Ann Arbor, MI, USA). y-Glutamyl transpeptidase, indomethacin, mepyramine, methoxamineHCI, methysergide, microsomal leucine aminopeptidase, P G D 2, phcnoxybenzamine, propranolo[, scopolamine, serine, glycine, cysteine, boric acid were obtained from Sigma Chemical Co. (St. Louis, MO, USA). The anti-human IgE antibody was supplied by Janssen Biochimica (Beerse, Belgium). PGD2-MO antiserum, LTE 4 anti-serum and LTE4-AChE were kindly provided by Dr. J. Maclouf, Unit6 150-1NSERM H6pital Lariboisiare (Paris). LTC 4, LTD4, LTE 4 and FPL 55712 were a generous gift from Dr. Ann F. Welton, Hoffman-La Roche (Nutley, N J, USA).

2.5. Binding studies Binding of [-~H]LTC 4 to m e m b r a n e s of human lung parenchyma and bronchi and of [~H]LTD 4 to parenehyma was performed essentially as previously described (Rovati et al., 1985; Civelli et al., 1987; Lewis et aI., 1985). Briefly, a crude m e m b r a n e fraction was prepared from macroscopically normal parenchymal fragments or bronchi. The tissue was minced, homogenized in 50 mM Tris-HCl, pH 7.4 (1"20, w ' v ) and centrifuged at 770 × g . The pellet was discarded and the supernatant was centrifuged again at 1 5 0 0 0 × g (parenchyma) or 27 000 × g (bronchi). The pellets were resuspended in the above mentioned buffer and stored at - 8 0 ° C . The incubation conditions, in a volume of 25(I #1, were as follows: (a) human lung parenchymaL T C 4 : 2 - 3 nM [~H]LTC4; 50 mM Tris-HCl; 20 mM serine-borate; 10 mM CaC12; 20 # M LTC 4 to determine non-specific binding; 0.04 mg protein sample ~; incubation for 20 rain at 4°C; (b) human bronchi-LTC4: 10-15 nM [~H]LTC4; 50 mM Tris-HC1; 20 mM serineborate; 1 mM CaCI2; 50 /.~M LTC 4 to determine non-specific binding; 0.04 mg protein sample ~; incubation for 15 rain at 4°C; (c) human lung parenchymaLTD 4 " 2 - 3 nM [~H]LTD4; 50 mM Tris-HCl; 20 mM serine-boratc; 10 mM glycine; 10 mM cysteinc; 5 mM MgCI2; 1 mM LY 171883 to determine non-specific binding; incubation tk~r 20 rain at 2_S°C. After the incubation, bound and free ligand were separated by rapid filtration through Whatman G F / C filters. The filters were washed twice with 4 ml of ice-cold 50 mM Tris-HCl, pH 7.4. The radioactivity was extracted from the filters with 10 ml of Filtercount (Packard) and was measured in the same scintillation fluid.

2.6. Statistical analysis Multiple group comparisons with basal values were performed according to Dunnett (1964).

3. Results

3.1. Effects of SCH 37224 on in t~itro anti-lgE induced formation of eicosanoids Normal human lung parenchyma responded to challenge with an anti-human IgE antibody with de novo synthesis of sulfldopeptide LT as well as of P G D 2. The data shown in fig. 1 indicate the mean amount of LTE 4 (as a measure of total sulfidopeptide leukotrienes, see Methods) formed in separate experiments, and the effect of increasing concentrations of SCH 37224. Pretreatment for 10 rain with 30 and 100 # M SCH 37224

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Fig. 1. Inhibition of antigen-induced formation of sulfidopeptide leukotrienes from normal human lung parenchyma by SCH 37224 (SCH). The values represent the means±S.E, of five different experiments and are expressed as net increase of LTE4. * P < 0.05: • * P < 0.01.

162

sis or receptor antagonists. Biosynthesis inhibitors should, in theory, have broader activity than selective antagonists because they block all forms of LT. SCH 37224 is a novel pyrrolidine derivative that has been shown to inhibit L T D 4 and thromboxane (TX)B, formation in guinea pig lung (Kreutner et al., 1988). However, the same authors indicate that this conch> sion cannot be generalized. Indeed, SCH 37224 was unable to inhibit LTC a formation in a mast cell line and differentiated granulocytes (Kreutner et al., 1998).

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induced a significant reduction in LT formation following immunological challenge. The basal levels of LTE 4 were not significantly affected by any of the concentrations of SCH 37224 (data not shown). SCH 37224 appeared to be ineffective against the formation of cyclooxygenase products. In fact, at 100 /,M the reduction in anti-IgE-induced formation of PGD 2 was not statistically significant (fig. 2). As already mentioned for LTE 4, the levels of P G D , in control (non challenged) samples were not modified, even with 100 p,M SCH 37224.

3.2. Effect of SCH 37224 on / ~ H / L T C 4 a n d / ~ H / L T D 4 binding Figure 3A shows that SCH 37224 elicited only a very slight inhibition of [-~H]LTC 4 specific binding to membranes of human lung parenchyma. Inhibition was approximately 30% at 100 p.M SCH 37224. Unlabelled LTCa was used as an inhibitor in the same experiments in order to have an internal control. In membranes of human bronchi, SCH 37224 was totally unable to interact with the sites labelled by [3H]LTC4 (fig. 3B). The inhibitory effect of LTC 4 is shown for comparison. No inhibition of [3H]LTD4 specific binding to human lung parenchyma by SCH 37224 was found (fig. 3C).

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163

Therefore, it seemed important to assess whether SCH 37224 displayed inhibition of the synthesis of sulfidopeptide LT in human airways. We now demonstrate that this compound is able to inhibit the formation of sulfidopeptide LT in human lung parenchyma in vitro, without affecting the synthesis of a cyclooxygenase product, namely P G D 2. We decided to assess the total formation of sulfidopeptide LT, rather than of only one of them. This can be accomplished by enzymatic conversion of both LTC 4 and L T D 4 to LTE 4 (Heavey et al., 1987), which is the main LT formed in vitro by human lung parenchyma Sautebin et al., 1985). It was then possible to show that SCH 37224 dose dependently inhibits sulfidopeptide LT formation and that this inhibition is statistically significant at 3(/and 100 ~M. Although it is not possible to calculate a precise ICsc~, the extent of the inhibition obtained in human lung was comparable to that reported by Kreutner et al. (1988) for the inhibition of LTD 4 formation in guinea pig lung (63% inhibition at 30 #M). Thus, there was no major difference between the inhibition of LT synthesis by SCH 37224 in human and in guinea pig lung. The concentrations active on LT formation are higher than that found in plasma of healthy volunteers after a single oral dose (Schering Corporation, unpublished study report), but might be attainable after chronic treatment. Although compounds more active than SCH 37224 as inhibitors of LT formation have been described, such as MK886 (Rouzer et al., 199(t) or A-64077 (Carter et al., 1989), most of the available natural or synthetic inhibitors display an IC50 in the order of 50-100 > M in animal or isolated cell models, showing a potency comparable to that of SCH 37224 in human tissue (Fitzsimmons and Rokach, 1989). In our experimental model, SCH 37224 did not inhibit the synthesis of PGD2; this indicates that the compound is not an inhibitor of either cyclooxygenase or phospholipase, in agreement with the results obtained with guinea pig and in cell cultures (Kreutner et al., 1988). However, it is interesting to note that, in the latter author's work, SCH 37224 abolished the antigen-induced formation of T X B , in guinea pig lung. We still cannot rule out that SCH 37224 is an inhibitor of T X A 2 biosynthesis, which works selectively without affecting the formation of other cyclooxygenase products, such as P G D , . However, it should be r e m e m b e r e d that in guinea pig lung, in contrast to human lung, sulfidopeptidc LT are able to induce the biosynthesis of cyclooxygenase products, particularly T X A 2 (Weichman et al., 1982). Thus, it is very likely that the inhibition of T X B 2 formation observed by Kreutner and coworkers (1988) is secondary to the inhibition of LT formation. It is not clear what might be the mechanism of the inhibitory action of SCH 37224 on LT biosynthesis.

Our data, as well as previous results (Kreutner ct al., 1988), indicate that it is not an inhibitor of phospholipases. On the other hand, Kreutner et al. (1988) claimed that it is not an inhibitor of 5-1ipoxygenasc in vitro, although this aspect has not been studied in detail. Indeed, these authors do not indicate whether the effect of SCH 37224 has been tested either in intact cells or in a homogenate. Therefore, it is not clear whether the possibility exists that SCH 37224 is an inhibitor of 5-1ipoxygenasc translocation from cytoso[ to membranes that would be effective only in intact cells, as was demonstrated for MK886 (Rouzer et al., 1990), although it might not be able to inhibit the isolated enzyme in a cell-free system. Should this not be the case, one should conclude that SCH 37224 is an inhibitor of glutathione-S-transferase, thus inhibiting LT formation at a step subsequent to 5-1ipoxygenasc activity. Our data show that SCH 37224 does not inhibit the binding of either LTC a or LTD 4 in human airways. This suggests that the compound is not an LT receptor antagonist, although the biological significance of the binding sites for LTC 4 is still a matter of debate (Sun et al., 1986; Norman et al., 1987; Sala et al., 1990). The lack of antagonistic activity is in accordance with the data obtained in guinea pig against LTC 4 (Kreutner et al., 1988). However, it was important to verify this possibility against both sulfidopeptide LT in human airways. Indeed, not only have LT receptors in guinea pig airways been demonstrated to be different from those in human airway, but LTC 4 receptors also differ from LTD 4 receptors (Gardiner, 1989). Although activity as a receptor antagonist would have been an additional favourable characteristic, the inhibition of LT formation in human lung parenchyma indicates that SCH 37224 is an interesting potential antiasthma drug.

Acknowledgements The skillful assistance of Ms. S. Carraro in preparing the manuscript is acknowledged. The authors are grateful to Dr. J. Maclouf (INSERM, Paris) and Dr. A.F. Welton (Hoffman-La Roche, Nutley, NJ) for the kind gifts of antisera and leukotrienes, respectively.

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Norman, P., T.S. Abram, t I . C Kluender, P.J. Gardiner and N..I. ('uthbert, 1987, The binding of [~H]leukotriene ('4 to guinea-pig lung membranes. The lack of correlation of LT(' 4 functional activity with binding affinity. Era-. J. Pharmacoh 143, 323. Powell, W.S., 1982, RapM extraction of arachMonic acM metaholites from biological samples using octadodecylsilyl silica, Meth. [£nzymol. 86, 467. Pradelles, P.. J. Grassi and ,1. Maclouf, 1085, Enzyme in/munoassay of eicosanoids using A c h e as labeh an ahcrnative to RIA, Anal. ('hem. 57. 1170. Rouzcr, C.A., A.W. Ford-llutchinson, I I.E. Morton and J.W. (;illard. 199(I, MK886, a potent and specific lcukotricnc biosynthesis inhibitor blocks and reverses the membrane association of 5-1ipoxygenase in ionophore-challenged leukocylcs, J. Biol. ('hem. 2~~5. 1436. Rovati, C,.E, D. OINa, L. Sautebin. G.C. Folco. A.F. Welton and S. Nicosia. 1985, Identification of specific binding sties for Icukotriene C~ in membranes from human hmg, 13iochem. Pharmacol. 34. 2831. Sala, A,, M. Civclli, I). Oliva, B. Spur, A.E.G. ('rca, G.('. Folco. and S. Nicosia, 1990, ('onlractile and binding activities of structural analogucs of ET(" 4 in the hmgitudinal nmscle of guinea-pig ileum, Eicosanoids 3. 1(15. Samuelsson, B., 1983, [,eukotrienes: mediators ot immediate hypersensitivity reactions and hlflamnlation, Science (Washington |)C) 22/), 5~8. Sautebhl, L., T. Vigano, E. Grassi, M.T. ('rivelhu'i, (]. (hllli. F. lJerti. M. Mezzetti and G.C. Folco, 1t~85, Release ol leukotricncs. indnced by the C a ' ionophorc A23187, from hunlan hmg parenchyma in vitro, J. Pharmacoh Exp. Ther. 234. 217. Schulman, E.S, I I.H. Newball, I,.M. l)emers. F.A. Fitzpatrick and N.F. Adkinson. 1981, Anaphylactic release of F X A , , P(}l),, and prostacyclin from humau lung parenchyma, Am. P,e~. Rcspir. Dis. 124, 402. Sun. F,F., I..-Y. ('hau, [:~. Spur, f=.,1. ('orey, R.A. I,ewis and K.|-. Auslen. 1986, Identification of high affinity Icukotricnc ('~-bind ing protein in rat liver cytosol as glutathione S transferasc, J. Biol. ('hem. 261, 8540. Vigani~, T.. 1.. Sautebin, F. Magni. M.T. Crivellari, R. Paganclli, G. Galli, F. Berti and (}.('. Folco, 1986, Stimulus-relatcd difference in thc formation of Icukotricnes and PGD, after immtmological and non-immunological challenge of human hlng parenchyma 'in vitro', Prostagh 1,¢ukotr. Med. 23, 10t). Weichman, B.M., R.M. Muccitelli, R.R. Osborn. D.A. ]|okJen, ,I.(}. Gleason and M.A. Wasserman. 1082, In vitro and in vivo mechanism of leukotriene mediated bronchoconstriction in lhc guinea pig, J. Pharmacol. Exp. Ther 222, 202.