The Journal of Pathology

Vol. 108 No. 1

T H E E F F E C T O F ANTI- INFLAMMATORY D R U G S O N INCREASED VASCULAR PERMEABILITY I N D U C E D BY CHEMICAL MEDIATORS

JEAN CARR* Prince Henry Hospifat, Sydney, and the University of New South Wales, Sydney, Australia

IN the inflammatory reaction, leakage of fluid occurs from the blood vessels in two main phases (Miles, 1956; Burke and Miles, 1958; Wilhelm and Mason, 1958,1960; Spector and Willoughby, 1959n and b; Logan and Wilhelm, 1963; Wells and Miles, 1963).

This leakage can be determined experimentally, by assaying the leakage of a radioactive tracer or a coloured dye (Menkin and Menkin, 1930; Miles and Miles, 1952; Judah and Wjlloughby, 1962; Nitta, Hayashi and Norimatsu, 1963; Carr and Wilhelm, 1964), bound in each case to plasma albumin. The leakage can be measured quantitatively (Judah and Willoughby; Nitta et al.; Ascheim, 1964; Carr and Wilhelm).

Rather than use a complex biological stimulus, it is easier to produce a controlled inflam- matory lesion by injecting one of several known chemical mediators (Miles and Miles; Spector and Willoughby, 1962; Wilhelm, 1962). Though the clinical effects of anti- inflammatory reagents on the complex inflammatory situations of human disease are known, and the effect of these anti-inflammatory reagents on experimental situations have been investigated (Ungar, Damgaard and Hummel, 1952; Yourish et al., 1955; Spector and Willoughby, 1959a and b ; Winter, Risley and Nuss, 1962), the effects of matching anti- inflammatory reagents against pure chemical mediators has been little exploited (Trethewie, 1951, 1954, 1957; Kelemen, 1957; LaBelle and Tornaben, 1957; Sparrow and Wilhelm, 1957; Northover and Subramanian, 1961 ; Spector and Willoughby, 1962; Northover, 1963).

This paper is an account of the results of matching known anti-inflammatory reagents against known chemical mediators.

Six well-documented permeability factors (PFs) were chosen : histamine and its liberators, compound 48/80 and polymyxin B ; kallikrein, guinea-pig globulin PF and bradykinin. These agents were chosen for the following reasons. It has been shown that in the biphasic increase of permeability after mild injury, the initial phase is mediated by histamine or 5-hydroxytryptamine (5HT) (Wilhelm and Mason, 1960; Wilhelm; Logan and Wilhelm,

Received 23 Sept. 1971 ; accepted 25 Jan. 1972. * Present address: National Blood Transfusion Service, Regional Transfusion Centre, Longley

1. PATH.-VOL. 108 (1972) 1 A Lane, Sheffield S5 7JN.

2 JEAN CARR

1963, 1966). It has been alleged that the secondary phase of increased vascular permeability may also be mediated by histamine (Schayer and Ganley, 1959; Schayer, 1 9 6 0 ~ and b), but that the histamine cannot be assayed or inhibited because it is produced and utilised rapidly in the actual effector cells. Many other workers believe that the secondary phase, or indeed the whole inflammatory reaction, is mediated by proteases or their products, the polypeptides (reviews by Ungar, 1953; Feldberg, 1956; Spector, 1958; Lewis, 1960; Wilhelm). Since there are not yet any specific antagonists to the polypeptides, these theories are as yet unconfirmed.

Thus the first three PFs, histamine, and the two liberators compound 48/80 and poly- myxin B, which are said to act in different ways (Bushby and Green, 1955; Hogberg and Uvnas, 1957), may be taken to represent both phases of increased vascular permeability.

0.067 0.2 0.6 1.8

Dose of histamine ( p g )

Identical batches of tissue were prepared, with histamine as the test permeability factor. The first batch 0-0 was extracted on the day of the experiment. The second batch @--@ was extracted after storage for 1 mth at -40°C.

FIG. 1 .-Decrease in dye recovery from guinea-pig skin after storage for 1 mth.

Kallikrein is a protease that has been used extensively in this type of investigation. Brady- kinin is the end-result of the action of kallikrein on protein substrates, and guinea-pig globulin PF, which some have suggested has itself an enzymatic activity (Becker, Wilhelm and Miles, 1959; Miles and Wilhelm, 1960), has been suggested as the substrate for this reaction (Lewis, 1958; Margolis, 1962). The latter three agents thus represent the postulated mechanism of the second phase of increased vascular permeability.

The anti-inflammatory drugs tested were acetylsalicylic acid, phenylbutazone and its metabolite oxyphenbutazone, chlorpromazine, procaine hydrochloride and triprolidine.

In a previous experiment (Carr and Wilhelm), the six PFs were assayed quantitatively by extracting dye that exuded into the skin after intracutaneous injection. I t was shown that the previous methods of assay by the measurement of the superficial diameter and the intensity of colour of such lesions were less accurate than dye extraction. However, since dye extraction is laborious, the drugs to be tested were screened roughly by the old methods, to determine those that seemed to be most active. The active compounds were then assayed quantitatively by dye extraction.

ANTI-INFLAMMATORY DRUGS AND CHEMICAL MEDIATORS 3

20

15

h

.- s s - f

Q) Q

M i v

210- - n m C

> W

5 -

MATERIALS AND METHODS

Animal experiments Female adult guinea-pigs (500-600 g) were used. The animals were depilated and blued

with Evans’ blue (BDH) ; permeability factors were then injected as previously described (Carr and Wilhelm). The dye was extracted from the homogenised skin by Clausen and

-

-

i c

01 I 1 2 I

0.05 0.1 0.2 0.4 Injection volume (ml)

FIG. 2.-Constant dose titrations. The amount of dye extracted from the skin after constant dose titrations of six standard permeability factors, by the technique of intracutaneous injection in “blued ” guinea-pigs. The response to a standard dose in an increasing volume is recorded as the amount of dye extracted from the skin. B = 0.007 pg bradykinin; K = 48.6 pg kallikrein; C =5.4 pg compound 48/80; H = 0.06 pg histamine; P = 5.4 pg polymyxin B; G = 0.6 pg guinea-pig globulin PF.

Lifson’s technique (1956), as adapted by Carr and Wilhelm. The concentration of dye was estimated colorimetrically in pg per standard 13 cm2 skin. Tissues were usually extracted on the day of the experiment and were never kept for more than 48 hr. Even in the dark and under refrigeration, the amount of extractable dye decreases after a period (fig. 1). The original dye standard was used as a control throughout the experiment.

Subsequent to the work published in 1964 (Carr and Wilhelm), it was shown that an increase in the volume of fluid injected induced a variation in the amount of dye exuded, in response to the same dose of some permeability factors. However, at the standard injection level of 0.1 ml this variation is negligible (fig. 2).

4 JEAN CARR

The drugs Permeability factors. These were: (a) histamine acid phosphate (BDH), 0.067-1.8 pg

cited as base; (6) compound 48/80 (Wellcome Research Laboratories), 06-16.2 p g ; (c) polymyxin B sulphate (Burroughs Wellcome Limited), 0.6-26.2 pg ; (d ) guinea-pig globulin PF (serum fraction G2 prepared according to Wilhelm, Miies and Mackay, 1955), 0.2-5.4pg; (e) hog kallikrein (Winthrop Laboratories), 1.8-48.6 pg; and ( f ) bradykinin (Sandoz Ltd), 0-002-0067 pg.

All permeability factors were injected in a standard volume of 0.1 ml, as previously described (Carr and Wilhelm). Doses were chosen to give lesions of similar size. The same

TABLE The relative inhibitory effect of anti-inflammatory agents on mediators

of increased vascular permeability

Local inflammatory effect in the presence of parenterally injected in- hibitor (as percentage of that in control guinea-pigs) of local injection of

Inhibitor

None (control) Aspirin 1 mg per kg Phenylbutazone 1 mg

per kg Phenylbutazone

100 mg per kg Phenylbutazone

200 mg per kg Oxyphenbutazone

1 mg per kg Triprolidine 0.1 mg

per kg Procaine 2 mg per kg Chlor~romazine 1 m g

histamine

105 110 89

9"

0.2* g t

81

110

95 67

48/80

105 127

I*

25*

2*

80

31*

21* 5*

polymyxin I

100 100 28*

7*

0.7*

65

18"

22* 35*

GP G2 1 kallikrein I

111- I 4* 151-

9 t 17" 32t

68

53 I 66 61 1 93

I

bradykinin

100 43* 65

6*

12"

105

24

45 75

* Extrapolation of plot ) see text. t Direct dye calculation 0-30 per cent. = Marked inhibition; 30-50 per cent. = moderate inhibition; 50-70 per cent. =

mild inhibition; above 70 per cent. the results may be equivocal.

set of four randomisation charts was used in each experiment on four animals, in order to reduce biological variation. All injections were carried out in 30 min., and the animals were killed 20 min. after the last injection. Each animal had a control injection (0.1 ml) of the saline used to dilute the permeability factors. All permeability factors were injected into each animal, to allow simultaneous testing against each anti-inflammatory agent.

Anti-inflammatory agents. These, their sources, the dose, the route of injection and the time they were given before the injection of the permeability factors, were: (a) acetylsalicylic acid, Burroughs Wellcome and Co., 1 mg per kg intraperitoneally, 30 min.; (b) phenyl- butazone, Geigy Pharmaceuticals, 1 mg sodium salt per kg intravenously, 1 hr, or (c) 100 or 200 mg per kg intraperitoneally, 1 hr ; ( d ) oxyphenbutazone, Geigy Pharmaceuticals, 1 mg sodium salt per kg, intravenously, 1 hr; (e) triprolidine, Burroughs Wellcome and Co., 0 1 mg per kg intraperitoneally, 0 min.; ( f ) procaine HCI (BP), 2 mg per kg intra- peritoneally, 1 hr; and (8) chlorpromazine, May and Baker, 1 nig per kg intraperitoneally, 1 hr.

ANTI-INFLAMMATORY DRUGS AND CHEMICAL MEDIATORS 5

In the case of phenylbutazone, therapeutic to toxic doses were used to obtain maximum suppression. Generally, relatively small doses were used so that toxic reactions that might

Histamine C

0

A Pr

Ch

P I

P100

P200 T

I

0,067 0.2 0.6 1.8 Dose of histamine (g)

FIG. 3.-Relative inhibitory potency of some anti-inflammatory drugs on the permeability-increasing effect of histamine.

For this and subsequent figures, the factors were tested by the technique of intracutaneous injection in " blued " animals. All inhibitors were given parenterally. Results are expressed as the mean amount of dye exuded into the skin. C = Control values in untreated animals; 0 = oxyphenbutazone 1 mg per kg intravenously 1 hr before test; A = acetylsalicylic acid 1 mg per kg intraperitoneally 3 hr before test; Pr = procaine hydrochloride 2 mg per kg intra- peritoneally 1 hr before test; Ch = chlorpromazine 1 mg per kg intraperitoneally 1 hr before test; P1 = phenylbutazone 1 mg per kg intravenously 1 hr before test; P100, P200 = phenyl- butazone 100 mg per kg or 200 mg per kg intraperitoneally 1 hr before test; T = triprolidine 0.1 mg per kg intravenously immediately before test.

obscure small variations in response to the permeability factors would be reduced to a minimum.

6 JEAN CARR

-15 0 W ._ Lo - L W

M i

aJ 3

a

v

5 1 0 - *1 c m > u1

5 -

0 -

RESULTS The results are expressed quantitatively as the amount of Evans' blue ex-

tracted from the lesions (see the table and figs. 3-8).

-

Criteria of suppression In a previous paper (Carr and Wilhelm), the criterion for comparison of

the effects of individual permeability factors was the standard blueing dose

Polymyxin B

' O r

I / " co 0

Ch PI Pr

T

PI 00

P200 0 0

1 1 I I I

0.6 I .8 5.4 16.2 Dose of polyrnyxin B (pg)

FIG. 4.-Relative inhibitory potency of some anti-inflammatory drugs on the permeability-increasing effect of polymyxin B. CO = Additional control values in untreated animals for a group given a different batch of polymyxin B.

(SBD). The SBD was defined as that amount of a permeability factor in 0-1 ml that, when injected intracutaneously, would induce the exudation of 10 pg of Evans' blue. Comparisons were then made on the number of SBD per mg for an individual permeability factor.

In this experiment the standard means of comparison must be between the control and test values for a single permeability factor. Therefore, SBD per mg are no longer useful. The standard blueing dose of a factor required to

ANTI-INFLAMMATORY DRUGS AND CHEMICAL MEDIATORS 7

15

h

0

aJ

aJ a M

.- VI - I

a 10- v

a, 3 - n v) c > w

5 -

0 -

induce the exudation of 10 pg of dye was therefore expressed as a percentage of the SBD of the same factor required to induce the exudation of 10 pg of dye after the animal had been treated with an inhibitor.

Values lying between 70 and 100 per cent. or over were regarded as equi- vocal. Values lying between 50 and 70 per cent. were considered to indicate mild inhibition, between 30 and 50 per cent. moderate inhibition, and below 30 per cent. marked inhibition. Values below 5-10 per cent. were assessed from amounts of Evans’ blue no greater than those that could be extracted

-

Compound 48/80

A

PI 00 0 Pr Ch P I

T P200

0.6 1.8 5.4 16.2 Dose of compound 48/80 (pg)

FIG. 5.-Relative inhibitory potency of some anti-inflammatory drugs on the permeability-increasing effect of compound 48/80.

after control saline injections in normal animals. Control saline injections in treated animals yielded even smaller amounts than this.

In some instances, suppression was so great that the slopes could not be extrapolated to the 10 pg level. In these cases, the calculation was made on the basis of maximum exudation of dye in control animals over maximum exu- dation of dye in test animals. The calculation gives falsely high results. In some instances in the table, the results are given both in this way and by extrapolation of the slopes, to allow comparisons to be made.

Control values were obtained from a separate group of four untreated animals.

8 JEAN CARR

Acetylsalicylic acid This was a poor inhibitor when given at a dose of 1 mg per kg intraperi-

toneally 30 min. before the injection of the permeability factors. At this dose

25

20

h g . I5 ._ rn W

W c

- L

Y v

W 3

13 - -m 10 0 > w

5

C

Globulin PF

L I

0.2 0.6 1.8 5.4 Dose of globulin PF (pg)

FIG. 6.-Relative inhibitory potency of some anti-inflammatory drugs on the permeability-increasing effect of globulin PF.

level, only bradykinin was affected moderately. Polymyxin B and kallikrein were unaffected. Histamine and compound 48/80 were apparently potentiated. Owing to a suspected error in dilution, guinea-pig globulin PF was not accur- ately assayed, but also appeared to be potentiated.

ANTI-INFLAMMATORY DRUGS AND CHEMICAL MEDIATORS 9

Phenylbutazone This drug was given at three dose levels, 1, 100 and 200 mg per kg. The

latter two were tested in only two animals each.

Kallikrein

0 C

A

T Ch P I

Pr

P200

PI 00

I I

1.8 5.4 16.2 48.6 Dose of kallikrein (pg)

FIG. 7.-Relative inhibitory potency of some anti-inflammatory drugs on the permeability-increasing effect of kallikrein.

When phenylbutazone (1 mg per kg) was given intravenously as the sodium salt 1 hr before injecting the permeability factors, it was a potent inhibitor of histamine-release by compound 48/80 and polymyxin B, but did not inhibit histamine materially. It also inhibited the effects of guinea-pig globulin to a moderate degree, and affected bradykinin mildly.

At the higher dose levels (100 and 200 mg per kg), given intraperitoneally

10 JEAN CARR

as the sodium salt 1 hr before the injection of the permeability factors, it was a potent inhibitor of all the permeability factors tested.

FIG.

25

2c

1’: h c 0 aJ L a, Q

M 3.

aJ 3

.- VI -

v

ii 10 VI c m > w

5

0

Bradykinin

P

0.002 0.007 0.022 0.067 Dose of bradykinin (pg)

8 .-Relative inhibitory potency of some anti-inflammatory drugs on the permeability-increasing effect of bradykinin.

The sodium salt was in a highly alkaline vehicle, and itself caused shock to some of the animals at the time of intraperitoneal administration. The affected animals apparently recovered completely from this shock in a few minutes. However, when the Evans’ blue was injected just before the permeability factors were tested, many of the animals were profoundly shocked and some died. The sequence of administration of these two substances, phenylbutazone and Evans’ blue, was not significant. Blueing itself induced no shock, but previously blued animals often became shocked when these high doses of phenylbutazone were administered. The experiments reported were carried out in animals that had not shown this intense shock at any time in the experiment. They were, however, more subdued

ANTI-INFLAMMATORY DRUGS AND CHEMICAL MEDIATORS 11

than usual. At these dose levels there was marked or complete suppression of all permeability factors tested.

Oxyphenbutazone Oxyphenbutazone (1 mg per kg) was given intravenously as the sodium

salt 1 hr before the permeability factors were tested; although this metabolite of phenylbutazone is as effective clinically as the parent substance, it was a poor inhibitor of the permeability factors at this dose level. There was mild inhibition of histamine release by polymyxin B and possible inhibition of the guinea-pig- globulin permeability factor.

Triprolidine This substance (0.1 mg per kg) was given intravenously immediately before

the permeability factors were injected. It was a potent inhibitor of histamine and the histamine-releasing agents compound 48/80 and polymyxin B. It was also unexpectedly a potent inhibitor of bradykinin. Only the high doses of phenylbutazone were more effective. Triprolidine also affected guinea-pig- globulin permeability factor mildly.

Procaine hydrochloride This substance (2 mg per kg) was given intraperitoneally 1 hr before the

Permeability factors were injected. It was a potent inhibitor of histamine- release by both compound 48/80 and polymyxin B. It did not inhibit histamine at all, but was a moderate inhibitor of bradykinin and a mild inhibitor of guinea- pig-globulin permeability factor and kallikrein.

Chlorpromazine Chlorpromazine (1 mg per kg) was given intraperitoneally 1 hr before the

permeability factors were injected. It is a marked inhibitor of compound 48/80, a moderate inhibitor of polymyxin By and a mild inhibitor of guinea-pig- globulin permeability factor and histamine. Bradykinin and kallikrein were unaffected.

DISCUSSION The above results show clearly that the substances that are used in clinical

medicine to inhibit the inflammatory reaction all affect the action of the perme- ability factors used in experimental pathology. Not all have the same actions. All affect bradykinin or its liberators; not all affect histamine or its liberators. This suggests that the bradykinin mechanism is more likely to be operative in the secondary or delayed phase of inflammation.

These results disagree partially with those of Northover (1963) and Spector and Willoughby (1963). Northover found that, in mice, high doses of sali- cylates (200 mg per kg) did not inhibit large (40 pg) doses of bradykinin. His results with chlorpromazine were essentially similar to those reported here. Spector and Willoughby (1963) found non-specific suppression of a wide range of permeability factors that they tested, when high (600 mg per kg) doses of

12 JEAN CARR

salicylate were used. They also found suppression of histamine and globulin permeability factor by low doses of chlorpromazine.

These results provide a useful research tool in the investigation of the events of the inflammato,ry reaction. It is possible to obtain a quantitative estimate of the effect of any inhibitor on any permeability factor. It is also possible to make accurate comparisons of the effects of different inhibitors on the individual permeability factors.

SUMMARY A group of six inhibitors of inflammation used in clinical medicine was

tested against six standard chemically defined permeability factors. Aspirin inhibited bradykinin ; phenylbutazone in low dosage strongly in-

hibited 48/80 and polymyxin B, and in high dosage inhibited all the permeability factors tested. Oxyphenbutazone was relatively ineffective. Triprolidine strongly suppressed histamine, compound 48/80, polymyxin B and bradykinin. Chlorpromazine strongly suppressed compound 48/80, polymyxin B and brady- kinin.

These results suggest that there is strong evidence to support the hypothesis that bradykinin is significant in the mediation of the secondary phase of the inflammatory reaction.

My thanks are due to Miss Jan Lyall for unfailingly meticulous assistance, and to Professor D. L. Wilhelm for his help and encouragement during the course of the work. The work was supported by the Bushel1 Research Trust.

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