Relative potencies of 5-lipoxygenase inhibitors on antigeninduced contractions of guinea pig tracheal strips

Relative Potencies of 54ipoxygenase Inhibitors on AntigenInduced Contractions of Guinea Pig Tracheal Strips

HWEI LING CHO, DAVID

PETER P. K. Ho,

EDWARD D.

MIHELICH,

AND

W. SNYDER

A quantitative method to assess relative potencies (I&,) of 54ipoxygenase (5-LO) enzyme inhibitors was established in antigen-induced contractions of tracheas isolated from actively sensitized guinea pigs (Schultz-Dale model). The relative potencies of four purported 5-LO inhibitors determined in this tissue assay were compared with those from a crude enzyme preparation isolated from guinea pig neutrophils. All compounds suppressed ovalbumin (OA)-induced tracheal contractions in a concentration-related manner in the presence of indomethacin and pyrilamine. lCSOValues, determined from the percent inhibition values obtained from responses at 30 ng/mL OA of these compounds ranged from 0.56-15 PM. A similar rank order of potency for inhibition of 5-HETE formation from a crude enzyme preparation was observed. This suggested that these agents had a common mechanism of action in the two assay systems and further validated the lCsO values determined in trachea assay. LY171883, an LTD4/LTE4 receptor antagonist, also suppressed OA-induced contractions concentration dependentlywith an I& of 4.9 FM determined by this method. LTD4 concentration-response curves were not altered by any of the four 5-LO inhibitors, ruling out the possibility that these agents were acting as LT receptor antagonists. Results of this study demonstrated that relative potencies of 5-LO inhibitors can be quantitatively assessed using this airway tissue model, which helps in identifying potential therapeutic agents for asthma. Leukotrienes; 5-Lipoxygenase inhibitors; Key Words: Antigen-induced contractions

Airway smooth muscle;

INTRODUCTION Antigen-induced sensitized

contractions

animals,

known

of smooth

muscle

as the Schultz-Dale

isolated

reaction

from

(Dale,

immunologically

1913; Schultz,

1910),

are mediated by the endogenous release of preformed substances, for example, histamine, and newly synthesized mediators, for example, cysteinyl leukotrienes (LT), thromboxane, and prostaglandins. In the presence of antihistamine and indomethacin, the LT-mediated component can be optimized, and the responses are amenable to blockade by LTD4/LTE4 receptor antagonists or 5-lipoxygenase (5-LO) inhibitors

(Adams

and Lichtenstein,

1979;

Hand

and Buckner,

1979;

Burka and Pa-

From the Lilly Research Laboratories, Pulmonary Research, Eli Lilly and Company, Indianapolis, Indiana 46285, U.S.A. Address reprint request to: Dr. David W. Snyder, MC931, 98C/4, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, U.S.A. Received February 15, 1991; revised and accepted August 7, 1991. 277 Journal of Pharmacological 0 1991 by H. L. Cho

Methods

26, 277-287 (1991)

278

H. 1. Cho et al.

0

-Ph AA 661

REV 6666

PH

N 'CH3 Compound 1

FIGURE 1.

terson,

1980,198l;

Compound 2

Chemical structure of the 5-lipoxygenase inhibitors.

Hay et al., 1987; Jones et al., 1988; Hand et al., 1986,1989;

Brown and Aharony, 1989). Guinea pig tracheal strips have served

as a functional

Redkar-

assay for the potency

as-

sessment of LTD4/LTE4 receptor antagonists (Fleisch et al., 1985; Jones et al., 1986, 1989; Hay et al., 1987; Snyder et al., 1987; Hand et al., 1989). 5-LO Inhibitors were also shown to suppress ovalbumin(OA) induced tracheal responses in a concentration-dependent Although

manner

attempts

(Jones et al., 1988;

have been

made

Redkar-Brown

to compare

and Aharony,

the activities

of 5-LO

1989).

inhibitors

(Hand et al., 1986; Jones et al., 19881, their potencies have never been quantitatively determined in the Schultz-Dale model. Earlier studies have used isolated enzyme or whole-cell

preparations

to assess biochemically

potencies

(Yoshimoto et al., 1982; Ashida et al., 1983; Summers 1989). Results from a cell assay may not be representative

of 5-LO

inhibitors

et al., 1987; Huang of a more complex

et al., tissue

preparation. Thus, the need for a tissue-based assay to assess potency of 5-LO inhibitors became evident. The purpose of this study was to develop a quantitative method utilizing the Schultz-Dale model to determine LO inhibitors selected from either structurally related

the relative potencies of 5or unrelated series (Figure

1). The rank orders of inhibitory potency of four selected pared on OA-induced contractions and on 5-HETE formation preparation.

compounds were comfrom the crude enzyme

METHODS Sensitization

Procedures

Male, Hartley strain guinea pigs (200-250 g) were injections of OA (IO mg/kg). The OA was administered

actively sensitized intraperitoneally

by three on days 1

5-10 Inhibitors Block Antigen-Induced

and 3 and subcutaneously the last injection.

on day 5. Experiments were performed

Contractions

21-26 days after

General On the day of the experiment, guinea pigs were killed by asphyxiation with COZ and the tracheas were removed, cleaned of surrounding connective tissue, and cut into spiral strips. Each strip was divided in half for paired experiments. Tissues were placed in IO-mL jacketed tissue baths maintained at 37°C and attached with cotton thread to Grass force-displacement transducers (FTO3C). Changes in isometric tension were displayed on a Grass polygraph (Model 7D). Tracheal strips were bathed in modified Krebs solution of the following composition (millimolar) NaCI, 118.2; KCI, 4.6; CaCI, - 2H20, 2.5; MgS04 . 7H20, 1.2; NaHC03, 24.8; KH2P04, 1.0; and dextrose, 10.0. Unless otherwise mentioned, the buffer contained indomethacin (5 PM), which potentiates the contractions of the cysteinyl LT by removing the influence of cyclooxygenase products (Krell et al., 1981). Tissue baths were aerated with 95% 02/5% COZ. Tracheal strips were placed under a resting tension of 2 g and allowed a minimal stabilization period of 60 min before undergoing experimentation. Bath fluid was changed at 15-min intervals during the stabilization period. OA and LTDI Concentration-Response

Curves

Initially, tissues were challenged with carbachol (IO ~.LM) following the 60-min stabilization period to ensure tissue viability. After recording the maximal response to the initial carbachol challenge, tissues were washed and reequilibrated for 60 min before starting the experimental protocol. Cumulative concentration-response curves were obtained from tracheal strips by increasing the agonist concentration in the organ bath by half-loglo increments, whereas the previous concentration remained in contact with the tissues (Van Rossum, 1963). Agonist concentration was increased after reaching the plateau of the contraction elicited by the preceding concentration. One concentration-response curve was obtained from each tissue. To minimize variability between tissues, contractile responses were expressed as a percentage of the maximal response obtained with carbachol (IO PM), added to the bath at the end of the concentration-response curve. Determination

of I&,

and ECso Values

To evaluate the properties of the selected test compounds to inhibit the antigeninduced responses, we incubated each compound with the tissues 30 min before the start of the curves. Vehicle [dimethylsulfoxide (DMSO)] was given to the paired control tissue. Depending on the nature of the experiment, we added pyrilamine (10 FM) to all baths at this time to block the actions of the released histamine. In the presence of test compound, the antigen-induced responses were suppressed in a concentration related, but not necessarily parallel, manner; thus dissociation constants for each compound could not be determined. Alternatively, responses obtained at the antigen concentration of 30 ng/mL were recorded in the absence and presence of drug, and percent inhibition was calculated for each pair of tissues. After several concentrations of each compound were tested, an KS0 value, as an

279

280

H. L. Cho et al. index of relative potency, was determined by linear regression. The maximal contractions were not used to calculate the percent inhibition values because the responses from vehicle-treated tissues tended to wane at the highest concentrations of OA. Preliminary results demonstrated that the OA concentration-response curves obtained from paired vehicle-treated tissues were nearly superimposable. With LTD4 concentration-response curves, the test compound was incubated as described above. Pyrilamine (IO ~J,M)was added to all baths. An ECsOvalue, which represents the molar concentration of agonist required to induce 50% of maximal response, was determined by linear regression. Differences in E& values and the maximal responses, in the presence and absence of test compound, were analyzed by Student’s t test with p < 0.05 regarded as significant. Isolated 5-10 Assay Guinea pigs weighing 250-300 g were injected intraperitoneally with 10 mL of 2% casein solution. After 16-18 hr, the animals were killed by asphyxiation with COZ. The peritoneal cavity was infused with 70 mL of saline, and 40-50 mL of fluid were recovered and subsequently centrifuged. The cell pellets were washed twice in Hanks’ balanced salt solution which lacked Ca2+ ions. The cells were then suspended in 5 mL of sodium phosphate buffer, pH 7.1, containing 1 mM of ethylenediaminetetraacetate (EDTA) and 0.1% gelatin. Approximately 20-30 x IO’ cells were isolated from each guinea pig. Analysis of their composition indicated that >95% of the cells were polymorphonuclear leukocytes (PMNL). The suspension of PMNL was sonicated with a Branson Sonifier (Model 350) with a microtip. The sonicates were combined and centrifuged at 30,000 g for IO min. The supernatant was aliquoted and kept frozen at -70°C until use. The 5-LO activity was assessed by measuring the formation of 5-HETE. An aliquot of 0.2 mL supernatant, obtained from PMNL sonicates, was incubated with 5 ~.LM14C-arachidonic acid, 1 mM CaC12, 2 mM adenosine triphosphate (ATP), and 1 mM glutathione for 15 min at 37°C in presence or absence of test compound. The reaction was stopped by the addition of 10 FL of 1 M citric acid and IO ~J,Lof alcohol solution containing 20 mg/mL each of indomethacin and butylated hydroxyanisole. The reaction products were separated by thin-layer chromatography in a solvent system that consisted of a mixture of ethyl acetate/2,2,4-trimethylpentane/glacial acetic acid/H20 (90:50:20:100). The mean I&, value was determined from at least three separate experiments. Drugs and Chemicals The following drugs were used: ovalbumin grade V, indomethacin, carbamylcholine chloride, histamine dihydrochloride, pyrilamine maleate, ATP, EDTA, Hanks’ balanced salt solution, butylated hydroxyanisole, gelatin, glutathione, and casein (Sigma Chemical Company, St Louis, MO); 2,2,4_trimethylpentane and ethylacetate (Aldrich Chemical Company, Milwaukee, WI); 14C-arachidonic acid (Amersham International, Amersham, UK); LY171883, LTD4, and the 5-LO inhibitors (Eli Lilly and Co, Indianapolis, IN). LTD, was greater than 95% isomerically pure as determined by reverse-phase high-performance liquid chromatography analysis.

5-10 Inhibitors Block Antigen-Induced

Contractions

RESULTS Effects of 5-10

Inhibitors

on OA-Induced

In the presence of indomethacin concentration-related contractions

Contractions

of Guinea

Pig Trachea

and pyrilamine, OA (0.3-10,000 ng/mL) produced of sensitized guinea pig tracheal strips. REV 6866

(0.3-3.0 FM) shifted the OA response curves to the right and suppressed imal contractions in a concentration-related manner (Figure 2). Complete sion of the OA-induced response was seen at 3 PM. Using the responses with 30 ng/mL of OA (see Methods),

the calculated I&,

was 0.56 PM.

the maxsuppresobtained

In the absence

of pyrilamine, REV 6866 (3 FM) suppressed the OA-induced maximal response only 12 k 2% (n = 5, p < 0.02). At this concentration, REV6866failed to alter the histamine concentration-response

curves.

Concentration-related served with

compounds

completely

suppressed

suppression

of the OA-induced

1, 2, and AA 861 (Figures the OA-induced

responses

were

3 and 4). Compound

contractions,

and an IC,,

of 1.3 FM

determined. The I& value for compound 2 was 15 PM, and complete was observed at 100 FM. At 100 PM, AA 861 completely suppressed contractions,

also ob-

1 at 10 PM was

suppression OA-induced

and an ICsO value of 7.4 FM was determined.

Effect of LY171BB3, An LTD,/LTE,,

Receptor

Responses

Pig Trachea

on Sensitized

Guinea

Antagonist,

Similar to results obtained with the 5-LO inhibitors, the OA response curves to the right and suppressed

on OA-Induced

LY171883 (1 .O-30.0 FM) shifted the maximal contractions in a

60 70 5 60 E ._ 2 I

50

2

40

# -e 30 0" &? 20 10 0 7

6

5

4

3

2

- log [Ovalbumin] (mglml)

FIGURE 2. Effect of REV 6866 (0.3-3.0 PM) on OA concentration-response curves on guinea pig tracheal strips. Compound or vehicle was incubated with the tissues for 30 min before beginning the curves. The control curves from paired experiments were combined for clarity. lCso value is the molar concentration of drug that reduced by 50% the contraction elicited by 30 q/ml of OA. The bath fluid contained indomethacin (5 FM) and pyrilamine (10 FM). Symbols represent the mean f SEM of number (n) of tissues.

281

282

H. L. Cho et al.

- log [Ovalbumin] (mglml)

60 70

ompound 2 N= 6 ompound 2 3OpM lOpA4 N = 6 ompound 2 1OOpM N = 4

E z60

E ._ ia 50 5 E 40 c

30

;

20 IO 0

i - log [Ovalbumin] (mglml)

FIGURE 3. Effects of compound 1 (1.0-10.0 t&t) and compound 2 (10-100 uM) on OA concentration-response curves on guinea pig tracheal strips. Compound or vehicle was incubated with the tissues for 30 min before beginning the curves. The control curves from paired experiments for each drug were combined for clarity. I&, value is the molar concentration of drug that reduced by 50% the contraction elicited by 30 ng/mL of OA. The bath fluid contained indomethacin (5 uM) and pyrilamine (10 FM). Symbols represent the mean f SEM of number (n) of tissues.

concentration-related manner (Figure 5). At 30 PM, the maximal concentration tested, LY171883 abolished responses elicited by concentrations of OA 130 ng/mL. As the OA concentration exceeded 30 ng/mL, a concentration-related contraction was observed; however, its maximal response did not plateau under the conditions of the experiment. At the maximal concentration of antigen (IO pg/mL), the response in the presence of LY171883 (30 ~J,M)was approximately 50% of that obtained from control tissues. The calculated I&,, value for LY171883 was 4.9 PM, employing the method developed for assessing potencies of S-LO inhibitors. Because similar re-

5-10 Inhibitors Block Antigen-Induced

Contractions

qAA86130fiMN=6 . AA 861 lOO~t.4

Es0

N = 4

E z 0 50 I 2

40

8 e 30 0 ;

20

- log [Ovalbumin] (mglml)

FIGURE 4. Effect of AA 861 (3.0-100.0 t&i) on OA concentration-response curves on guinea pig tracheal strips. Compound or vehicle was incubated with the tissues for 30 min before beginning the curves. The control curves from paired experiments were combined for clarity. KS0 value is the molar concentration of drug that reduced by 50% the contraction elicited by 30 @ml of OA. The bath fluid contained indomethacin (5 PM) and pyrilamine (10 PM). Symbols represent the mean 2 SEM of number (n) of tissues.

80 70 E z E = a

r

60 50

2

40

$ e

30

d r$ 20

10 0 -9,

7

1

6

1

I

5

I

4

I

3

I

2

- log [Ovalbumin] (mglml)

FIGURE 5. Effect of LY171BB3 (3.0-30.0 PM) on OA concentration-response curves on guinea pig tracheal strips. Compound or vehicle was incubated with the tissues for 30 min before beginning the curves. The control curves from paired experiments were combined for clarity. IC, value is the molar concentration of drug that reduced by 50% the contraction elicited by 30 ng/mL of OA. The bath fluid contained indomethacin (5 PM) and pyrilamine (10 @I). Symbols represent the mean rt SEM of number (n) of tissues.

283

284

H. L. Cho et al. TABLE 1 Effects of Selected 5-Lipoxygenase Inhibitors on LTD4 -log Response Obtained from Guinea Pig Tracheal Strips

EDw and Maximal

CONTROL MAXIMAL RESPONSES

COMPOUND - LOC E&b

@I” REV 6866: 10 PM (8) AA 861: 30 PM (4) Compound 1: 10 PM (6) Compound 2: 30 PM (4)

9.0 9.2 9.3 9.3

rt it rt t

TREATMENT

70.5 12.2 79.3 65.8

0.2 0.1 0.1 0.1

2 2 f 2

1.5 0.8 1.9 1.5

MAXIMAL RESPONSE

- LOG EC50 9.0 8.9 9.4 9.3

t + i t

69.0 71.1 77.6 66.1

0.1 0.1 0.1 0.1

a Number of tissues. b Values represent mean + SEM. ’ Values represent mean 2 SEM of maximal LTD4 response expressed as a percentage carbachol maximum response.

2 t 2 t

1.3 1.6 1.4 1.7

of the final

suits were obtained with this leukotriene receptor antagonist and 5-LO inhibitors, the need to show that the 5-LO inhibitors do not act as leukotriene receptor antagonists became apparent. Effects of S-LO Inhibitors

on LTDJnduced

Guinea Pig Tracheal Contractions

LTD, concentration-response curves were obtained in the presence and absence of each 540 inhibitor. Each inhibitor was tested at the concentration greater than its I&, or the concentration producing the greatest suppression of OA-induced responses. None of the compounds shifted the LTD4 response curves to the right as determined by the EDS0values or altered the LTD4 maximal responses (Table 1). Effects of Inhibitors

on Crude 5-10 Enzyme Preparation

All four selected inhibitors blocked in a concentration-related manner 5-HETE formation from the crude enzyme prepared from guinea pig neutrophils. As illustrated in Table 2, the I& values obtained from at least three separate experiments TABLE 2 Comparison of lnhibito~ Potencies of Selected Compounds on 5-Lipoxygenase Enzyme Activity and on OA-Induced Contraction of Guinea Pig Tracheal Strips

COMPOUND REV 6866 Compound Compound AA 861 a Mean * Value ’ Value d Value

1 2

INHIBITION0~ ISOLATED5LO ENZYMEACTIVITYFROM GUINEA PIG NEWTROPHILS UC50 nM)” 132 117 2210 1070

” r ‘rt

40 52 1260 590

+ SEM from three to four experiments. from Sweeney et al., 1987. from Summers et al., 1987. from Yoshimoto et al., 1982.

INHIBITIONOF OA-INDUCED COI\;TRACTIONS OF GUINEA PIG TRACHEA (ICso KM) 0.56 1.3 15.0 7.4

INHIBITION OF

RBL-1 5-LO ENZYMEACTIVITY (I&O nM) 1lOb 100’ 1300’ 800d

5-10 Inhibitors Block Antigen-Induced

Contractions

agree with those reported in the literature using rat basophilic leukemia cells (RBL1) assay system. The rank order of potency for these compounds, REV 6866 = compound 1 > AA 861 > compound 2, was similar in the tissue and the isolated enzyme assays. DISCUSSION The present study established a quantitative method to assess the relative potencies of four purported 5-LO inhibitors in the Schultz-Dale model. Because the OA-induced tracheal contractions defined in this study were mainly produced by cysteinyl LT, any molecule acting to block the action of or inhibit the formation of LTs may suppress the OA responses. Results with LY171883 and the four purported 5-LO inhibitors clearly indicated the involvement of cysteinyl LTs in this tissue model. Potencies of these compounds were determined from the OA concentration-response curves of control and drug-treated tissues. Calculation of percent inhibition and KS0 values (see Methods section) were made from responses obtained to OA 30 ng/mL. This was chosen because the responses were submaximal at this concentration and resided on the linear portion of the curves. With this method, potency values for the 5-LO inhibitors obtained from the tissue assay were compared with those from the enzyme assay. lCsO values obtained from the guinea pig neutrophils readily agreed with those previously reported in RBL-1 assays (Summers et al., 1987; Sweeney et al., 1987; Yoshimoto et al., 1982), indicating that similar potency ranking can be obtained from enzyme systems prepared from various sources. Likewise, a similar relative rank order of potencies, I& values, were obtained from tissue and enzyme preparations in the present investigation. Furthermore, the lack of effect of these compounds on LTD4 concentration-response curves indicated that these agents were acting by inhibiting 5-LO. However, activity in the tissue assay may not always relate to activity against the isolated enzyme because of the differences in the compounds’ physical and chemical properties or mechanism of action. Some inhibitors may be potent against the crude enzyme but poor in suppressing tissue responses due to limited access to the target enzyme in the more complex tissue environment. Agents that act by blocking the translocation of 5-LO from the cytosol to cellular membranes (Rouzer et al., 1990) would show activity only in the tissue assay. All compounds used in this study undoubtedly acted on the isolated enzyme preparation, excluding an inhibitory effect on the enzyme translocation process. Three of these four compounds shared a common hydroxamic acid moiety, and therefore, the variability in their physical and chemical properties could be greatly reduced. Interestingly, AA 861, a structurally diverse compound, showed intermediate potency in the two assay systems. The KS0 of 7.4 PM for AA 861 in the tissue assay approximates the I&, of 1.8 PM for inhibition of SRS-A release from guinea-pig chopped lung using a bioassay to measure SRS-A (Ashida et al., 1983). These results demonstrated the usefulness of the Schultz-Dale model in assessing the potency of novel 5-LO inhibitors. Hand et al. (1986) compared overall effect (percent inhibition + dose ratio) of four different 5-LO inhibitors in a similar Schultz-Dale model. However, only mod-

285

286

H. L. Cho et al. est differences (
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