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Action of the Pentapeptide (ICI 50123) on Gastric Secretion in Man
GASTROENTEROLOGY Official Publication of the American Gastroenterological Association ©CoPYRIGHT 1966 THE WILLIAMs
VoLUME
51
& WILKINS Co.
October 1966
NuMBER
4
ACTION OF THE PENTAPEPTIDE (ICI 50123) ON GASTRIC SECRETION IN MAN G. M. M.-l.KHLOuF, M.B., PH.D., M.R.C.P., J.P. A. McMANus, M.B., M .R.C .P., AND w. I. CARD, M.D., F.R.C.P. Gastrointestinal Unit, Western General Hospital, Edinburgh, and the University of Edinburgh, Edinburgh, Scotland ·
Gregory and Tracy 1 • 2 have recently described the properties of a number of synthetic derivatives representing various portions of the total gastrin molecule and demonstrated a close correlation between function and molecular structure. They noted in particular that of the 17 residues of gastrin, only the C-terminal tetrapeptide sequence is required for the entire range of physiological activity displayed b~· the whole molecule and that the properties are radically changed by the absence from the sequence of the amide group \Yhich masks the C-terminal residue. The present report concerns the effect on gastric secretion in man of a substituted synthetic derivative of gastrin, the pentapepticle ICI 50123 (N-t-butyloxycarbonyl(3 Ala-Tryp-Met-Asp-Phe-NH 2 ). The Cterminal tetrapeptide amide portion of the Received April 18, 1966. Accepted June 1, 1966. Address requests for reprints to: Dr. G. M. J\Iakhlouf, Lemuel Shattuck Hospital, 170 Morton Street, Boston, Massachusetts 02130. This work was supported by a grant from the :tvledical Research Council. The authors thank Dr. J. D. Fitzgerald, Dr. S. J. Morley, and Dr. J. Raventos of Imperial Chemical Industries, Ltd., Pharmaceuticals Di,·ision, and Dr. Thomson of the Teaching and Research Centre, Western General Hospital, Edinburgh, for valuable advice, and Miss Irene Xewlands for technical assistance. The pentapeptide (ICI 50123) was the gift of of Imperial Chemical Industries, Ltd., Pharmaceuticals Division.
455
stimulant is the same as in porcine and human gastrin. The data obtained are interpreted in the light of a quantitative model of gastric secretion.
Methods In section I, under "Results," the secretory responses of 2 normal male subjects, G. M . and J. M., to increasing subcutaneous doses of gastrin II and the pentapeptide were analyzed. The same 2 subjects have been intensively studied over a period of 3 years and the results were reported in earlier communications:-• The detailed procedures for the collection of juice and avoidance of salivary contamination have been fully described in previous communications.3' •· • The juice was collected every 5 min and pooled into samples corresponding to 10-min periods for purposes of titration. One 20-min collection of spontaneous secretion and eight 10-min collections following stimulation were obtained in each case. Acid was titrated to the phenolphthalein end point, sodium and potassium were estimated by flame photometer and chloride electrometrically. Pepsin was measured by the method of Hunt7 and biuret-reacting nitrogen by a modification of the method of Gornall et al.8 The pentapeptide in a dose of 1000 p.g was diluted with distilled water to a volume of 3 ml and the appropriate dose injected subcutaneously. The range of doses used in this series was 1 to 13 p.g per kg subcutaneously. The results of these experiments are labeled section I. Observations were also extended to a series of 8 adult male volunteers, including subjects G. M. and J. M., and the relationship between the responses to subcutaneous histamine acid
456
M.AKHLOUF ET AL. TABLE
Vol . 51 , No.4
1. Outputs obtained dw·ing the hour following stimulation by the pentapeptide Output
Subject Volume
Dose, sc
a
H
Na
ml/hr
mEq/ltr
mEq/hr
G. M.
2.00 3. 24 6.30 9.28
111 .5 133. 0 159 .0 154. 0
11 .91 16. 05 18 .66 19.24
3.64 2.76 3.84 2.97
J. M.
1.06 4.23 6.68 6.77 8.59 13.05
150 .0 253.5 249.5 255.5 253.5 292.5
15 .01 31.17 31.59 31.61 31.38 33.73
4.08 4 .31 3 .30 3.37 3.63 7.36
use.
Two tests were done on each subject, one with t he pentapeptide and one with histamine acid phosphate. A 20-min basal collection and eight 10-min samples of stimulated juice were obtained in each case. A total of 144 samples were 140 120
'$
. .z
E
~
N itrogen
Peak ff+•
mEq/hr
tmits/lzr
mgfhr
mEq/ hr
17. 20 20.72 24.82 24.65
10 ,336 9,961 13,670 9,319
50.19 33. 08 40.03 51 .35
17.75 21.56 27. 18 25.90
21 .45 39 .69 39.35 39.28 39.48 44.32
19' 153 29,300 42,460 33, 120 19,995 19,500
76.05 96 .86 75.42 59.90 90.24
22.2i 38.55 41.29 40 .50 42 .53 45 .-!8
Cl
mEq/ltr
mEq/hr
1.62 1.94 2.43 2.46
17 .16 20.75 24 .93 24 .67
2.28 4. 18 4.24 4.52 4.64 4.85
21.37 39.66 39 .14 39. 10 39. 71 45.94
The last column lists the corresponding peak acid outputs.
phosphate (40 p..g per kg) and subcutaneous pentapeptide (6 p..g per kg) was studied in more detail. The choice of 6 p..g per kg was dictated by the observation that the responses of subjects G. M. and J . M . to this dose were closely similar to t he maximal subcutaneous histamine responses. Although higher responses equivalent to t he maximal subcutaneous gastrin response could be obtained wit h larger doses of the pentapeptide, t he possibility of encountering side effects, mentioned below, prechided their
_J
Pepsin
Cations
K
- -- - - -
p.gfkg
100 'tl
80
~'0" oe~.
60
0
40 20 _25
x-x-x
0
o -~~.-
20 40 60 80 100 120 140 160 H• mEq /L
Fra 1. Linear relationship between the mean concentrations of acid and sodium following histamine ( 0) and the pentapeptide ( • ) ; 1· 0.996, p < 0.001.
=
analyzed for acid, sodium, potassium, chloride, pepsin, and biuret-reacting nitrogen. These experiments are labeled section II.
Results
Section I The results of increasing doses of pentapeptide on G. M. and J. M. are summarized in table 1. Acid and volume output. Following stimulation, volume and acid output rose rapidly and simultaneously to a peak in the third and fourth 10-min periods. The time of appearance of peak output >vas the same over the whole range of doses used. The rates of rise and decline of acid secretion were similar to those observed with hist amine. Electrolyte concentration. Acid concentration reached high levels of around 138 mEq per liter coincident with the peaks of acid and volume output and tended to fall by the end of 1 hr. As with other stimulants, t he potassium concentration peak preceded that of acid by some 10 min, and its rate of decline was more rapid. No difference in t he behavior of electrolytes between histamine, gastrin, and the pentapeptide could be detected. The linear relationship observed in earlier studies between the mean concentrations of hydrogen and ~odium was maintained (fig. 1). Pepsin and nitrogen output. The secre-
PENTAPEPTIDE AND~GASTRIC SECRETION
October 1966
tion of pepsin and nitrogen followed similar patterns. The outputs of both products rose to an early peak in the second and third 10-min periods and thereafter de-
457
clined progressively and more rapidly than acid output. At the end of 80 min, however, the outputs of both pepsin and nitrogen were still about twice as high as the spontaneous output prior to the test. Relationship of dose and response. 1.
Two measures were used in this series to express acid output: (a) the output of acid during the first hour following stimulation, i.e., the poststimulatory hour output and (b) the peak hour output, derived from the second 20-min period and expressed as peak output per hour by multiplication by
w Vl
z
0 Q
3.
Vl
w
~
0 .02
In this study and over the ·whole range of doses used, peak and poststimulatory hour outputs were highly correlated (1· 0.992, n = 10). 2. It is clear from table 1 that both peak and poststimulatory hour outputs rose with increasing doses of the pentapeptide. The relationship between dose and response is illustrated in figures 2 and 3 by the use of the reciprocals of dose and response. The dose-response curves for gastrin were derived from data obtained in earlier studies on the 2 subjects. 4 • 8 Only subject G. M. was tested with increasing doses of histamine.
=
0 .01
04
08
1.2
1.6
2.0
YoosE FIG. 2. Relationship between the reciprocal of
peak acid response and the reciprocal of the subcutaneous dose in p.g per kg of gastrin (0), pentapeptide ( •), and histamine (X) (subject
G. ?vi.).
0.05
p 0.04
w
1.1)
z
0.03
./••
0
a_
1.1)
w
~
/.
0.02 ~-------------o----o---------0-- G
0.01
0.2
0.4 ',
0.6
0.8
1.0
1.2
YoosE FIG. 3. R elationship between the reciprocal of peak acid response and the reciprocal of the subcutaneous dose in p.g per kg of gastrin (0), and the pentapeptide (e) (subject J.M.).
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MAKHLOUF ET AL.
The reciprocals of dose and peak acid output were linearly related with correlation coefficients for all the plots over 0.98. The intercepts on the vertical or response axis were closely similar in each subject for the various stimulants. The calculated maximal responses are summarized in table 2. Similar results were obtained from doseresponse curves for the output of potassium (fig. 4). 3. The output of pepsin rose with inTABLE
creasing doses of the pentapeptide but showed a tendency to fall in both subjects with doses above 8 p.g per kg.
Section II The patterns of response to histamine and the pentapeptide were similar in all subj ects. The course of acid and pepsin secretion is shown in figure 5. Two points are worthy of emphasis: (a) peak volume and acid output invariably occurred in the second 20-min period, and (b) pepsin output rose to some 6 times the basal le-.;el in the first 20 min and was still nearly twice basal at the end of 80 min. Fractionally higher acid concentrations were observed in histamine-stimulated juice, i.e., above 140 mEq per liter in all subjects with histamine and 135 to 140 mEq per liter with the pentapeptide. It should, however, be noted, in this connection, that the juice in 4 subjects tested with t he pentapeptide was slightly bile-
2. Calculated maximal responses Subject G.M.
Ratio, G.M./ ].M.
Subject J.M.
I
%
mEq/ lzr
Pentapeptide, sc .. .. . . Gastrin, sc .. . . . . . . . . . . Gastrin, iv ... . . . . . .... Gastrin, infusion ... . . . Histamine, sc .. ... . .. .
32.0 34.7 36 .0 36.0 36 .5
49.5 51.5 54.0 54.0
65 67 66 66
0.024 Q/
/
0.020
/ .
0.016
w z 0 (j)
Q.
0 .012
•
••
o'o
(j)
w
~
;y6
0.008
0.004
_j_
0.2
0.4
0 .6
0.8
1.0
YoosE FIG. 4. Relationship between the reciprocals of peak potassium response and dose of pentapeptide. The response was expressed as a percentage of the respective maximal potassium responses of subject J. M. (0) and subject G. M. ( • ).
October 1966
~
.6"
PENTAPEPTIDE AND GASTRIC SECRETION
24
(\j
(J 0 0
16
•n
c
E
r:~:~
/:
12
0 (\j
"'" o-
E
8
0
u <(
~:~
4 i
B
0
20
40
60
80
MINUTES
FrG. 5. Mean acid output in mEq per 20 min and mean pepsin output in units per 20 min follo,Ying histamine (0) and the pentapeptide ( • ) . The course of secretion was similar in all subjects, the peak invariably occurring in the Eecond 20-min period. TABLE
459
contaminated. Peak potassium concentration either accompanied or just preceded peak a cid concentration. The mean concentration of all ions and of pepsin during the first hour were much the same for the two stimulants: For histamine: [H] = 129.2, [Na] 15.4, [K] = 17.5, [Cl] = 161.9, pepsin 183 units per mi. For the pentapeptide: [H] = 127.0, [Na] = 16.8, [K] = 17.0, [Cl] = 160.6, pepsin = 175 units per mi. The concentrations of nitrogen following histamine were somewhat higher, 363 mg per liter as compared with 322 mg per liter. No significance could be attributed to this finding because of the tendency to bloodstaining of the histamine-stimulated juice. The full data, summarized in table 3, displayed a number of highly significant correlations : 1. Both the peak and poststimulatory hour acid outputs following histamine were highly correlated with the corresponding outputs following the pentapeptide (r = 0.97, n = 16) , with a slope of unity and a small intercept not significantly different from zero (fig. 6). 2. The mean output of acid from the two tests on each individual was highly correlated with the corresponding mean output
3. The outputs of the secretory products during the first hour following stimulation by pentapeptide and histami nea G.M.
R. K.
J.N.
- - - - -- - - Acid output pentapeptide, mEq/ hr . Acid output hista mine, mEq/ hr . .. . . . .. Yolume output p entapept ide , ml/hr . . .. Yo!ume output histamine, ml / hr. ..... . Potassium output pentapeptide, mEq/ hr ... . . .. . . ... .. ... .. . .. . .... . ....... Potassium output histamine, mEq/ hr ... Sodium output pentapeptide, mEq/ hr. Sodium output hista mine, mEq/ hr . . .. . Chloride output pentapeptide, mEq/hr . . Chloride output histamine, inEq/hr . ... Pepsin output p ent apeptide, units/hr ... Pepsin output histamine , units/hr . Nitrogen output pentapeptide, mg/hr .. Nitrogen output histamine, mg/ hr. . . ..
P . L. J . M. --- ---
S. R .
E. F.
D. B.
--- --- ---
18 .66 21.44 25.75 31.61 32 .04 34.75 38 .52 58.93 23 .58 27.14 30.14 32.35 34.37 38 .50 48 .10 60.49 159 .0 168.5 218.0 255.5 242.5 263.0 306 .0 447.5 186.5 219.0 230.0 258.0 267.5 272.0 382.0 465.5 3.68 4.53 4.09 2.43 2 .87 4.66 3 . 10 4.07 3.98 4. 32 4.79 4 .78 3 .84 3 .30 5.30 3 .38 3 .23 4.09 3.32 3 .22 3. 76 5 .21 4.34 2 .86 24 .82 27.67 34 .54 39.28 39.32 43.40 30 .09 34.69 37.81 40.83 43.52 45.99 13,670 23,330 40,040 42,460 36,940 56,310 22,910 37,820 34,340 36,230 49,920 42,430 40.00 63 .90 73.10 75.40 82.10 75.40 52.80 89.10 92 .00 92 .20 89.00 112.00
5.30 7.59 7 .04 7.96 4.82 6 .70 5.51' 6.88 48 .64 73.26 60.65 75 .58 75,010 72,190 87,520 107' 180 127 .56 126 .80 145.22 157 . 10
" The outputs are listed in order of increasing magnitude from left to right. The highest secretors, E. F . a nd D. B., were male patients with duodenal ulcer; all the others were healthy male subjects .
460
MAKHLOUF ET AL.
70 0:
-s_
60
w 2 w
50
0
0
i= Q. w Q. <(
0
./
40
0
•!/
30
z w
•
.,
o/
IQ.
/
.
20 10 10
20 30 40 50 60 70 HISTAMINE MEo/HR
Fm. 6. Relationship between the acid output in mEq per hr following histamine acid phosphate (40 p.g per kg) and the corresponding output following the pentapeptide (6 p.g per kg): peak hour (0); poststimulatory hour ( e ) .
of potassium or chloride (r = 0.996). The correlation between acid and sodium outputs was somewhat obscured by biliary contamination in a few tests (r = 0.88). 3. Further, the mean output of acid was correlated with the mean outputs of pepsin (r = 0.95) and nitrogen (r = 0.92). Discussion of Section I
Pattern of Pentapeptide-Stimulated Secretion The features of the gastric secretory response to subcutaneous administration of the pentapeptide were in all respects similar to those for subcutaneous histamine. No difference could be detected in the behavior of the electrolytes following the pentapeptide or histamine apart from a slight tendency for peak concentration of acid following large doses of histamine to be somewhat higher. The inverse linear relationship between the hydrogen and sodium ion concentrations was maintained. The positive linear relationship between hydrogen and potassium ion concentrations was only seen during the rise of potassium concentration to a peak (fig. I). The curve relating hydrogen and sodium ion concen-
Vol. 51, No.4
trations in figure 1 was obtained from an analysis of 170 samples obtained from subject G. M. over the last 2 years, 9 and is clearly the best fit for the data (59 samples) obtained from the same subject in this study (1· = 0.996). Dose-Response Relationships 1. Theoretical considerations. Implicit in all accounts of secretory data, and in particular to the meaning that should be attributed to maximal response, maximal dose, and the difference in these respects between stimulants, is some model of secretion. A great deal of confusion has arisen because these models have largely remained unformulated. However accurate the data or good the fit obtained, it is not possible to infer the mechanism of gastric secretion from the shape of the dose-response curves. A number of models could be proposed, each leading to curves consistent with the data . On the other hand, some experimental observations, such as the inability of combined stimulants to potentiate maximal respome by the intact human stomach, 5 would tend to exclude some models. A model describing the dose-response relationship has been proposed in earlier papers, 3 • 4 • 10 in which a number of secretory units or cellular receptors form actiYe complexes with a number of molecules of the stimulant according to a reversible equation governed by the laws of mass action. It is assumed that the number of secretory units or receptor sites is proportional to the number of parietal cells and that the response is proportional to the number of cells activated. The assumption is reasonable, in view of the large number of cells involved even in the lowest responses and despite the scatter that can be expected in the number of receptors per cell. The following equation was derived to express the dose-response relationship: ( 1)
where c, the number of activated cells, is identified with the observed response, and Cmax, the total number of cells, is identified
PENT APEPTIDE AND GASTRIC SECRETION
October 1966
\rith maximal response. D is the concentration of the stimulant in the tissues, assumed to be a direct function of the administered close, and K is the value of the dose which elicits half the maximal response, that is the ED 50 (when K = D in equation 1, c = Cmax/2). The plot of observed response, c against log-dose, gives the symmetrical sigmoid curve known as the logistic. Further transformation of equation 1 uncovers a linear relationship between the reciprocals of dose and response:
~ C
=
_ 1_
Cmax
+ _!£___,!_
(2)
Cmax D
Since Cmnx or the total number of cells in any single stomach may be considered as constant for the period of testing, the relationship of 1/ c and 1/ D is linear. At infinitely high concentrations of the stimulant, 1/ c = 1/Cmnx , so that the observed response equals the maximal response. Following continuous infusion of gastrin3 or histamine, 10 the relationship of dose and response showed a good fit to a logistic function. The problem of establishing an accurate dose-response relationship following single intravenous or subcutaneous injections of a stimulant appears, at first sight, more complex than for continuous infusion. A measure of acid output must be devised that could be related to the dose of the stimulant, or, more accurately, to the concentration established in the tissues. From his study of the kinetics of distribution of an intravenous stimulant, Teorell 11 concluded (a) that the time of appearance of peak concentration of the stimulant in the tissues for a particular dose and by inference of peak response, was independent of the injected dose, and (b) that the magnitude of this tissue concentration and response was proportional to the dose. The general validity of both conclusions with respect to intravenous injections of gastrin was demonstrated in earlier studies on subjects G. M. and J . M. 3 • 4 and allowed a fit of the secretory data to the same logistic function. The values of the maximal responses as calculated from the asymptotes
461
of the dose-response curves following single intravenous injections for each subject were identical with the values obtained following continuous infusion. This was to be expected, if, as equation 1 predicts, maximal response is essentially determined by activation of all available secretory units independently of how this is achieved. This conclusion received further confirmation when it was subsequently shown in subject G. M. that combinations of gastrin and a synergistic cholinergic agent modified the value of K and D but were without effect on the calculated maximal response. 4 Although the establishment of ·a dose-response relationship following subcutaneous injections is complicated by the rate of resorption of the stimulant from the subcutaneous depot, it appears that, for simplified conditions, the same two conclusions with respect to the time of appearance and the magnitude of peak concentration and response apply as well. 11 If this conclusion is correct, it should allow a fit of the subcutaneous experimental data to the same logistic function and permit a test of the model described by equations 1 and 2, that maximal response in the intact human stomach is essentially determined by activation of all secretory units and is independent not only of the mode of stimulation (continuous infusion, intravenous or subcutaneous injections), but also of the nature of the stimulant (gastrin, pentapeptide, or histamine). The last statement concerning the nature of the stimulant needs amplification. Stimulants administered by the same route vary in their capacity to elicit a particular submaximal response. Their potency in this respect is described quantitatively by the value of J( or the En 50 . But at sufficiently high doses and provided no pharmacological effects , such as on gastric blood flow, intervene to disturb the dose-response relationship, it is clearly immaterial which stimulant is used, since the observed response c should equal the maximal response Crnax . For a particular stimulant, however, the ability to achieve a particular tissue concentration and therefore the value of the ED50 will vary with the route of administration.
462
MAKHLOUF ET AL .
2. Dose-response relationship following subcutaneous stimulation. The pattern of the secretory responses disclosed by the subcutaneous experiments with the pentapeptide appeared to follow closely the predicted pattern of distribution of the stimulant in the tissues. Peak acid output invariably occurred in the second 20-min period, thus confirming the theoretical deductions regarding the time of appearance of peak response. The close fit of the data to equation 2 confirmed the predicted relationship between dose and peak acid output. The close similarity of the intercepts on the vertical axis in each subject indicated that the same maximal response could be obtained following subcutaneous administration of a sufficiently large dose of all types of stimulants. Maximal response thus appeared to be determined by activation of all available secretory units and not by the nature of the stimulant employed. Although the calculated maximal response was obtained by extrapolation, it should be emphasized that this is not a theoretical value since it has been experimentally reproduced, under optimal conditions in both subjects, by the slow intravenous injection of a massive dose of gastrin. 4 -The values of the calculated maximal responses appeared to be somewhat higher for intravenous gastrin (table 2). In the experiments with intravenous gastrin, however, peak acid output was short-lived and was calculated from the peak 10-min output following direct titration of 5-min samples. In experiments with subcutaneous stimulants, peak response was flatter and more prolonged and could only be calculated over a period of 20 min. The technical difficulty of obtaining an absolute estimate of the subcutaneous peak which was probably of shorter duration was reflected in a slightly altered but not significantly different maximal response. It was found in an earlier study 4 that the ratio ofthe output of subject G. M. to subject J. M. following all modes of stimulation was 66%. It was concluded that this fixed ratio, under similar conditions of stimulation, reflected the respective sizes of
Vol. 51, No.4
their parietal cell masses. This ratio was maintained in the present study (table 2) and confirmed the earlier conclusion. 3. Potency of the various stimulants. The first crude estimate of the relative molar potencies of gastrin and histamine, obtained on the basis of equivalent responses from 0.5 p..g per kg of gastrin and 40 p..g per kg of histamine acid phosphate, 6 was given as 500 to 1. It appears from the more accurate data obtained in this study that this was an overestimate. The slopes of the lines in figures 2 and 3 indicated the relative potencies of the various stimulants. From the ED 50 calculated in micrograms, it appeared that gastrin 'ns 10 times more potent than the pentapeptide and 30 times more potent than histamine acid phosphate. On a molar basis, the difference was more pronounced. Gastrin was 30 times more potent than the pentapeptide and 240 times more potent than histamine. Thus the pentapeptide was 3 times more potent than histamine on a weight basis and 8 times more potent on a molar basis. When the potassium output responses were expressed as a percentage of the calculated maximum for potassium and the dose of the pentapeptide was expressed per kilogram of body weight, the curves for the 2 subjects could be exactly superimposed (fig. 4). This indicated that their ED 50 values were identical. The mean ED 50 of the 2 subjects calculated from the potassium dose-response curves was 1.35 ± 0.03 p..g per kg and that from the acid dose-response curves was 1.44 ± 0.15 p..g per kg. Discussion of Section II
The course of secretion in response to the pentapeptide was similar in all respects to that for histamine. Peak acid output invariably occurred in the second 20-min period. The relationship between peak and poststimulatory hour outputs following stimulation by the pentapeptide was identical to that demonstrated in earlier studies with gastrin and histamine. 6 • 9 The acid outputs following histamine (40 p..g per kg) and the pentapeptide (6 p..g per kg) were highly correlated and very nearly equal, with a slope of unity and an inter-
October 1966
PENTAPEPTIDE AND GASTRIC SECRETION
cept not significantly different from zero. The acid outputs following histamine (40 p.g per kg) and gastrin (2 p.g per kg) were also shown in a previous study 6 to be highly correlated, although a slightly higher output resulted from the administration of gastrin. The purpose of secretory tests is to provide a stable and discriminating measure of the functional capacity of the stomach. The emphasis in this statement is on the ability of a secretory test to provide a repeatable index rather than an absolute measure of the utmost capacity of the stomach to secrete. It is therefore immaterial which stimulant is used since the outputs elicited by all stimulants are highly correlated. It should, however, be recognized that these outputs are not strictly maximal, but are repeatable functions or fractions of the real maximum as expressed in the calculated maximal response of a dose-response curve. The similarity in the shape of the secretory curves for all individuals following the administration of a particular stimulant, together with the highly significant correlation between the outputs from different stimulants, suggests that the factors responsible for the establishment of tissue concentration of a stimulant, namely, its distribution, inactivation, and elimination, are similar in different individuals. This rejoins the finding that the ED 50 values for the pentapeptide or for gastrin 5 were similar in subjects G. M. and J. M. Both histamine and the pentapeptide were equally effective in stimulating pepsin in man, but the output levels achieved were not maximal. The fact that the pepsin output at the end of 80 min was still twice as high as the basal level precludes an interpretation on the basis of a washout. Cellular masses as determinants of output. Card and Marks 12 showed that acid output was linearly related to both the parietal cell mass and the mucosal volume of the stomach, while Huntl 3 showed that the outputs of the "parietal" and "nonparietal" components were related to each other and to the output of pepsin. These findings pointed to the probability that the cellular
463
and extracellular elements comprising the wall of the stomach and responsible for the secretory products were present in similar proportions in different individuals. Maximal stimulation by gastrin, histamine, or the pentapeptide introduces a standard situation for purposes of comparison. Under these conditions, the response of pepsin and other constituents of the juice, though not necessarily maximal, may be assumed to be proportional to the size of the cellular and extracellular masses concerned. This prediction was confirmed by the significant linear relationship between the output of acid in every subject and the corresponding output of all other electrolytes, pepsin, and nitrogen (table 3). A similar relationship was obtained in earlier studies on gastrin and histamine, 5 • 9 and may be calculated from data reported by other workers. 14 Side Effects
No side effects were encountered following subcutaneous injection of up to 6 p.g per kg of the pentapeptide. Doses above 8 and up to 13 p.g per kg were accompanied by a sinking abdominal sensation and awareness of intestinal movement lasting some 10 min. These effects seem attributable to enhanced gastrointestinal tone and motility. Since no such effects were encountered with maximal subcutaneous doses of gastrin, then it would appear that doses of gastrin and the pentapeptide which elicited nearly equivalent acid responses had different ef, fects on tone and motility. Intravenous or intramuscular injections of pentapeptide were not tried, but in view of the side effects encountered with the higher doses, administration by these routes should be approached with circumspection with especial care taken to inject the stimulant slowly by the intravenous route. Summary 1. A substituted derivative, structurally related to gastrin, the pentapeptide, ICI 50123, was tested for its action on gastric secretion in man. In particular, its effectiveness as compared with gastrin and histamine was studied in terms of a quantitative model of secretion.
464
MAKHLOUF ET AL.
2. The theoretical predictions from the model were verified experimentally. In 2 subjects, the maximal responses calculated from dose-response curves following subcutaneous gastrin or pentapeptide were similar to those obtained in earlier studies and calculated from dose-response curves following infusion or single intravenous injections of gastrin. In 1 subject, the same maximum was also obtained following subcutaneous histamine or a combination of gastrin and a cholinergic agent. It was concluded, in terms of the model, that maximal acid output was represented by activation of all available secretory units or parietal cells and could be achieved independently of the nature of the stimulant or the route through which it was administered. 3. The potency of a gastric secretory stimulant is properly described by its ED 50 for a particular route of administration and not in terms of the output it elicits. In this sense, gastrin may be described as more potent than the pentapeptide which in turn is more potent than histamine. 4. The similarity of the ED 50 values in all subjects for a particular stimulant is probably a reflection of similar patterns of distribution, elimination, and inactivation of the stimulant. 5. The responses of all 8 subjects to 6 p.g per kg of the pentapeptide were in all respects similar to their responses to 40 p.g per kg of histamine acid phosphate. 6. No side effects were encountered with 6 p.g per kg of the pentapeptide. In 2 subjects tested with doses above 8 and up to 13 p.g per kg, there was a sinking abdominal sensation lasting around 10 min.
D
concentration of the stimulant in the tissues, assumed to be a direct function of the administered dose The rate of activation or complex formation is proportional to the product of dose and number of cells in process of stimulation: k1· (Cmax - c) ·D
The rate of deactivation or cell-stimulant breakdown is proportional to the number of activated cells or formed complexes: kz·c
At equilibrium, both processes are proceeding at the same rate: kz·c = k1· (Cmax - c) ·D
Dividing both sides by lc1 , and replacing lc 2 /k 1 by K (a constant) and rearranging the equation: ( 1) REFERENCES 1. Gregory, H ., P. M. Hardy, D. S. Jones, G. W. K enner, and R. C. Sheppard. 1964. The antral hormone. Nature (London) 204: 931935. 2. Gregory, R. A., and H . J. Tracy. 1964.
3.
4.
Addendum Derivation of equation 1. 5.
Let Cma.x
c Cmax-
C
total number of cells available for activation number of cells activated by cell-stimulant complex formation number of cells in process of activation
Vol. 51, No.4
6.
Physiological properties of a series of synthetic peptides structurally related to gmtrin I. Nature (London) B04: 935-938 . Makhlouf, G. M., J. P. A. McManus, and W. I. Card. 1964. Dose-response curves for the effect of gastrin II on acid gastric secretion in man. Gut 5: 379-384. Makhlouf, G. M., J. P. A. McManus, and W. I. Card. 1965. A comparative stud~· of the effects of gastrin, histamine, Histalog, and mechothane on the secretory capacity of the human stomach in two normal subjects over 20 months. Gut 6: 525-534. Makhlouf, G. M., J . P. A. McManus, and W. I. Card. 1965. The action of gastrin II on the gastric secretion of electrolytes and pepsin in man. In Proceedings of the symposium on gastric secretion: l\1echanisms and control, Edmonton, Alberta. In press. Makhlouf, G. M., J. P. A. McManus, and W. I. Card. 1964. The action of gastrin II
October 1966
7. 8.
9.
10.
PENTA PEPTIDE AND GASTRIC SECRETION
on gastric acid secretion in man. Lancet 2: 485-490. Hunt, J. N. 1948. A method for estimating peptic activity in gastric contents. Biochem. J. 42: 104-109. Gornall, A. C., C. J. Bardawill, and M. M. D avid. 1949. D etermination of serum proteins by means of biuret re action. J. Bioi. Chern . 177 : 751-766. i\Iakhlouf, G . M ., J. P. A. M cManus, and IV. I. Card . 1966. A quantitative statement of t he two-component hypothesis of gastric secretion. Gastroenterology 51: 149171. Adam, H. M., W. I. Card, M. J. Riddell, M. Roberts, J. Strong, and B. Wolf. 1954. Dose-response curves for the effect of his-
11.
12.
13.
14.
465
tamine on acid gastric secretion in man. Brit. J. Pharmacal. 9: 329-334. T eorell, T. 1937. Kinetics of distribution of substances administered to the body. Arch. Int. Pharmacodyn. 57: 205- 225. Card, W. I., and I. N. Marks. 1960. The relationship between the acid output of the stomach follo wing "maximal" histamine stimulation and the parietal cell mass. Clin. Sci. 19 : 147-163. Hunt, J. N. 1950. An interpretation of the histamine test of gastric secretion. Gastroenterology 16: 231-240. Hirschowitz, B. I. 1961. Electrolytes in human gastric secretion. Observations and a theory. Amer. J. Dig. Dis. (N.S.) 6: 199228.
VoLUME
51
& WILKINS Co.
October 1966
NuMBER
4
ACTION OF THE PENTAPEPTIDE (ICI 50123) ON GASTRIC SECRETION IN MAN G. M. M.-l.KHLOuF, M.B., PH.D., M.R.C.P., J.P. A. McMANus, M.B., M .R.C .P., AND w. I. CARD, M.D., F.R.C.P. Gastrointestinal Unit, Western General Hospital, Edinburgh, and the University of Edinburgh, Edinburgh, Scotland ·
Gregory and Tracy 1 • 2 have recently described the properties of a number of synthetic derivatives representing various portions of the total gastrin molecule and demonstrated a close correlation between function and molecular structure. They noted in particular that of the 17 residues of gastrin, only the C-terminal tetrapeptide sequence is required for the entire range of physiological activity displayed b~· the whole molecule and that the properties are radically changed by the absence from the sequence of the amide group \Yhich masks the C-terminal residue. The present report concerns the effect on gastric secretion in man of a substituted synthetic derivative of gastrin, the pentapepticle ICI 50123 (N-t-butyloxycarbonyl(3 Ala-Tryp-Met-Asp-Phe-NH 2 ). The Cterminal tetrapeptide amide portion of the Received April 18, 1966. Accepted June 1, 1966. Address requests for reprints to: Dr. G. M. J\Iakhlouf, Lemuel Shattuck Hospital, 170 Morton Street, Boston, Massachusetts 02130. This work was supported by a grant from the :tvledical Research Council. The authors thank Dr. J. D. Fitzgerald, Dr. S. J. Morley, and Dr. J. Raventos of Imperial Chemical Industries, Ltd., Pharmaceuticals Di,·ision, and Dr. Thomson of the Teaching and Research Centre, Western General Hospital, Edinburgh, for valuable advice, and Miss Irene Xewlands for technical assistance. The pentapeptide (ICI 50123) was the gift of of Imperial Chemical Industries, Ltd., Pharmaceuticals Division.
455
stimulant is the same as in porcine and human gastrin. The data obtained are interpreted in the light of a quantitative model of gastric secretion.
Methods In section I, under "Results," the secretory responses of 2 normal male subjects, G. M . and J. M., to increasing subcutaneous doses of gastrin II and the pentapeptide were analyzed. The same 2 subjects have been intensively studied over a period of 3 years and the results were reported in earlier communications:-• The detailed procedures for the collection of juice and avoidance of salivary contamination have been fully described in previous communications.3' •· • The juice was collected every 5 min and pooled into samples corresponding to 10-min periods for purposes of titration. One 20-min collection of spontaneous secretion and eight 10-min collections following stimulation were obtained in each case. Acid was titrated to the phenolphthalein end point, sodium and potassium were estimated by flame photometer and chloride electrometrically. Pepsin was measured by the method of Hunt7 and biuret-reacting nitrogen by a modification of the method of Gornall et al.8 The pentapeptide in a dose of 1000 p.g was diluted with distilled water to a volume of 3 ml and the appropriate dose injected subcutaneously. The range of doses used in this series was 1 to 13 p.g per kg subcutaneously. The results of these experiments are labeled section I. Observations were also extended to a series of 8 adult male volunteers, including subjects G. M. and J. M., and the relationship between the responses to subcutaneous histamine acid
456
M.AKHLOUF ET AL. TABLE
Vol . 51 , No.4
1. Outputs obtained dw·ing the hour following stimulation by the pentapeptide Output
Subject Volume
Dose, sc
a
H
Na
ml/hr
mEq/ltr
mEq/hr
G. M.
2.00 3. 24 6.30 9.28
111 .5 133. 0 159 .0 154. 0
11 .91 16. 05 18 .66 19.24
3.64 2.76 3.84 2.97
J. M.
1.06 4.23 6.68 6.77 8.59 13.05
150 .0 253.5 249.5 255.5 253.5 292.5
15 .01 31.17 31.59 31.61 31.38 33.73
4.08 4 .31 3 .30 3.37 3.63 7.36
use.
Two tests were done on each subject, one with t he pentapeptide and one with histamine acid phosphate. A 20-min basal collection and eight 10-min samples of stimulated juice were obtained in each case. A total of 144 samples were 140 120
'$
. .z
E
~
N itrogen
Peak ff+•
mEq/hr
tmits/lzr
mgfhr
mEq/ hr
17. 20 20.72 24.82 24.65
10 ,336 9,961 13,670 9,319
50.19 33. 08 40.03 51 .35
17.75 21.56 27. 18 25.90
21 .45 39 .69 39.35 39.28 39.48 44.32
19' 153 29,300 42,460 33, 120 19,995 19,500
76.05 96 .86 75.42 59.90 90.24
22.2i 38.55 41.29 40 .50 42 .53 45 .-!8
Cl
mEq/ltr
mEq/hr
1.62 1.94 2.43 2.46
17 .16 20.75 24 .93 24 .67
2.28 4. 18 4.24 4.52 4.64 4.85
21.37 39.66 39 .14 39. 10 39. 71 45.94
The last column lists the corresponding peak acid outputs.
phosphate (40 p..g per kg) and subcutaneous pentapeptide (6 p..g per kg) was studied in more detail. The choice of 6 p..g per kg was dictated by the observation that the responses of subjects G. M. and J . M . to this dose were closely similar to t he maximal subcutaneous histamine responses. Although higher responses equivalent to t he maximal subcutaneous gastrin response could be obtained wit h larger doses of the pentapeptide, t he possibility of encountering side effects, mentioned below, prechided their
_J
Pepsin
Cations
K
- -- - - -
p.gfkg
100 'tl
80
~'0" oe~.
60
0
40 20 _25
x-x-x
0
o -~~.-
20 40 60 80 100 120 140 160 H• mEq /L
Fra 1. Linear relationship between the mean concentrations of acid and sodium following histamine ( 0) and the pentapeptide ( • ) ; 1· 0.996, p < 0.001.
=
analyzed for acid, sodium, potassium, chloride, pepsin, and biuret-reacting nitrogen. These experiments are labeled section II.
Results
Section I The results of increasing doses of pentapeptide on G. M. and J. M. are summarized in table 1. Acid and volume output. Following stimulation, volume and acid output rose rapidly and simultaneously to a peak in the third and fourth 10-min periods. The time of appearance of peak output >vas the same over the whole range of doses used. The rates of rise and decline of acid secretion were similar to those observed with hist amine. Electrolyte concentration. Acid concentration reached high levels of around 138 mEq per liter coincident with the peaks of acid and volume output and tended to fall by the end of 1 hr. As with other stimulants, t he potassium concentration peak preceded that of acid by some 10 min, and its rate of decline was more rapid. No difference in t he behavior of electrolytes between histamine, gastrin, and the pentapeptide could be detected. The linear relationship observed in earlier studies between the mean concentrations of hydrogen and ~odium was maintained (fig. 1). Pepsin and nitrogen output. The secre-
PENTAPEPTIDE AND~GASTRIC SECRETION
October 1966
tion of pepsin and nitrogen followed similar patterns. The outputs of both products rose to an early peak in the second and third 10-min periods and thereafter de-
457
clined progressively and more rapidly than acid output. At the end of 80 min, however, the outputs of both pepsin and nitrogen were still about twice as high as the spontaneous output prior to the test. Relationship of dose and response. 1.
Two measures were used in this series to express acid output: (a) the output of acid during the first hour following stimulation, i.e., the poststimulatory hour output and (b) the peak hour output, derived from the second 20-min period and expressed as peak output per hour by multiplication by
w Vl
z
0 Q
3.
Vl
w
~
0 .02
In this study and over the ·whole range of doses used, peak and poststimulatory hour outputs were highly correlated (1· 0.992, n = 10). 2. It is clear from table 1 that both peak and poststimulatory hour outputs rose with increasing doses of the pentapeptide. The relationship between dose and response is illustrated in figures 2 and 3 by the use of the reciprocals of dose and response. The dose-response curves for gastrin were derived from data obtained in earlier studies on the 2 subjects. 4 • 8 Only subject G. M. was tested with increasing doses of histamine.
=
0 .01
04
08
1.2
1.6
2.0
YoosE FIG. 2. Relationship between the reciprocal of
peak acid response and the reciprocal of the subcutaneous dose in p.g per kg of gastrin (0), pentapeptide ( •), and histamine (X) (subject
G. ?vi.).
0.05
p 0.04
w
1.1)
z
0.03
./••
0
a_
1.1)
w
~
/.
0.02 ~-------------o----o---------0-- G
0.01
0.2
0.4 ',
0.6
0.8
1.0
1.2
YoosE FIG. 3. R elationship between the reciprocal of peak acid response and the reciprocal of the subcutaneous dose in p.g per kg of gastrin (0), and the pentapeptide (e) (subject J.M.).
458
Vol. 51, No.4
MAKHLOUF ET AL.
The reciprocals of dose and peak acid output were linearly related with correlation coefficients for all the plots over 0.98. The intercepts on the vertical or response axis were closely similar in each subject for the various stimulants. The calculated maximal responses are summarized in table 2. Similar results were obtained from doseresponse curves for the output of potassium (fig. 4). 3. The output of pepsin rose with inTABLE
creasing doses of the pentapeptide but showed a tendency to fall in both subjects with doses above 8 p.g per kg.
Section II The patterns of response to histamine and the pentapeptide were similar in all subj ects. The course of acid and pepsin secretion is shown in figure 5. Two points are worthy of emphasis: (a) peak volume and acid output invariably occurred in the second 20-min period, and (b) pepsin output rose to some 6 times the basal le-.;el in the first 20 min and was still nearly twice basal at the end of 80 min. Fractionally higher acid concentrations were observed in histamine-stimulated juice, i.e., above 140 mEq per liter in all subjects with histamine and 135 to 140 mEq per liter with the pentapeptide. It should, however, be noted, in this connection, that the juice in 4 subjects tested with t he pentapeptide was slightly bile-
2. Calculated maximal responses Subject G.M.
Ratio, G.M./ ].M.
Subject J.M.
I
%
mEq/ lzr
Pentapeptide, sc .. .. . . Gastrin, sc .. . . . . . . . . . . Gastrin, iv ... . . . . . .... Gastrin, infusion ... . . . Histamine, sc .. ... . .. .
32.0 34.7 36 .0 36.0 36 .5
49.5 51.5 54.0 54.0
65 67 66 66
0.024 Q/
/
0.020
/ .
0.016
w z 0 (j)
Q.
0 .012
•
••
o'o
(j)
w
~
;y6
0.008
0.004
_j_
0.2
0.4
0 .6
0.8
1.0
YoosE FIG. 4. Relationship between the reciprocals of peak potassium response and dose of pentapeptide. The response was expressed as a percentage of the respective maximal potassium responses of subject J. M. (0) and subject G. M. ( • ).
October 1966
~
.6"
PENTAPEPTIDE AND GASTRIC SECRETION
24
(\j
(J 0 0
16
•n
c
E
r:~:~
/:
12
0 (\j
"'" o-
E
8
0
u <(
~:~
4 i
B
0
20
40
60
80
MINUTES
FrG. 5. Mean acid output in mEq per 20 min and mean pepsin output in units per 20 min follo,Ying histamine (0) and the pentapeptide ( • ) . The course of secretion was similar in all subjects, the peak invariably occurring in the Eecond 20-min period. TABLE
459
contaminated. Peak potassium concentration either accompanied or just preceded peak a cid concentration. The mean concentration of all ions and of pepsin during the first hour were much the same for the two stimulants: For histamine: [H] = 129.2, [Na] 15.4, [K] = 17.5, [Cl] = 161.9, pepsin 183 units per mi. For the pentapeptide: [H] = 127.0, [Na] = 16.8, [K] = 17.0, [Cl] = 160.6, pepsin = 175 units per mi. The concentrations of nitrogen following histamine were somewhat higher, 363 mg per liter as compared with 322 mg per liter. No significance could be attributed to this finding because of the tendency to bloodstaining of the histamine-stimulated juice. The full data, summarized in table 3, displayed a number of highly significant correlations : 1. Both the peak and poststimulatory hour acid outputs following histamine were highly correlated with the corresponding outputs following the pentapeptide (r = 0.97, n = 16) , with a slope of unity and a small intercept not significantly different from zero (fig. 6). 2. The mean output of acid from the two tests on each individual was highly correlated with the corresponding mean output
3. The outputs of the secretory products during the first hour following stimulation by pentapeptide and histami nea G.M.
R. K.
J.N.
- - - - -- - - Acid output pentapeptide, mEq/ hr . Acid output hista mine, mEq/ hr . .. . . . .. Yolume output p entapept ide , ml/hr . . .. Yo!ume output histamine, ml / hr. ..... . Potassium output pentapeptide, mEq/ hr ... . . .. . . ... .. ... .. . .. . .... . ....... Potassium output histamine, mEq/ hr ... Sodium output pentapeptide, mEq/ hr. Sodium output hista mine, mEq/ hr . . .. . Chloride output pentapeptide, mEq/hr . . Chloride output histamine, inEq/hr . ... Pepsin output p ent apeptide, units/hr ... Pepsin output histamine , units/hr . Nitrogen output pentapeptide, mg/hr .. Nitrogen output histamine, mg/ hr. . . ..
P . L. J . M. --- ---
S. R .
E. F.
D. B.
--- --- ---
18 .66 21.44 25.75 31.61 32 .04 34.75 38 .52 58.93 23 .58 27.14 30.14 32.35 34.37 38 .50 48 .10 60.49 159 .0 168.5 218.0 255.5 242.5 263.0 306 .0 447.5 186.5 219.0 230.0 258.0 267.5 272.0 382.0 465.5 3.68 4.53 4.09 2.43 2 .87 4.66 3 . 10 4.07 3.98 4. 32 4.79 4 .78 3 .84 3 .30 5.30 3 .38 3 .23 4.09 3.32 3 .22 3. 76 5 .21 4.34 2 .86 24 .82 27.67 34 .54 39.28 39.32 43.40 30 .09 34.69 37.81 40.83 43.52 45.99 13,670 23,330 40,040 42,460 36,940 56,310 22,910 37,820 34,340 36,230 49,920 42,430 40.00 63 .90 73.10 75.40 82.10 75.40 52.80 89.10 92 .00 92 .20 89.00 112.00
5.30 7.59 7 .04 7.96 4.82 6 .70 5.51' 6.88 48 .64 73.26 60.65 75 .58 75,010 72,190 87,520 107' 180 127 .56 126 .80 145.22 157 . 10
" The outputs are listed in order of increasing magnitude from left to right. The highest secretors, E. F . a nd D. B., were male patients with duodenal ulcer; all the others were healthy male subjects .
460
MAKHLOUF ET AL.
70 0:
-s_
60
w 2 w
50
0
0
i= Q. w Q. <(
0
./
40
0
•!/
30
z w
•
.,
o/
IQ.
/
.
20 10 10
20 30 40 50 60 70 HISTAMINE MEo/HR
Fm. 6. Relationship between the acid output in mEq per hr following histamine acid phosphate (40 p.g per kg) and the corresponding output following the pentapeptide (6 p.g per kg): peak hour (0); poststimulatory hour ( e ) .
of potassium or chloride (r = 0.996). The correlation between acid and sodium outputs was somewhat obscured by biliary contamination in a few tests (r = 0.88). 3. Further, the mean output of acid was correlated with the mean outputs of pepsin (r = 0.95) and nitrogen (r = 0.92). Discussion of Section I
Pattern of Pentapeptide-Stimulated Secretion The features of the gastric secretory response to subcutaneous administration of the pentapeptide were in all respects similar to those for subcutaneous histamine. No difference could be detected in the behavior of the electrolytes following the pentapeptide or histamine apart from a slight tendency for peak concentration of acid following large doses of histamine to be somewhat higher. The inverse linear relationship between the hydrogen and sodium ion concentrations was maintained. The positive linear relationship between hydrogen and potassium ion concentrations was only seen during the rise of potassium concentration to a peak (fig. I). The curve relating hydrogen and sodium ion concen-
Vol. 51, No.4
trations in figure 1 was obtained from an analysis of 170 samples obtained from subject G. M. over the last 2 years, 9 and is clearly the best fit for the data (59 samples) obtained from the same subject in this study (1· = 0.996). Dose-Response Relationships 1. Theoretical considerations. Implicit in all accounts of secretory data, and in particular to the meaning that should be attributed to maximal response, maximal dose, and the difference in these respects between stimulants, is some model of secretion. A great deal of confusion has arisen because these models have largely remained unformulated. However accurate the data or good the fit obtained, it is not possible to infer the mechanism of gastric secretion from the shape of the dose-response curves. A number of models could be proposed, each leading to curves consistent with the data . On the other hand, some experimental observations, such as the inability of combined stimulants to potentiate maximal respome by the intact human stomach, 5 would tend to exclude some models. A model describing the dose-response relationship has been proposed in earlier papers, 3 • 4 • 10 in which a number of secretory units or cellular receptors form actiYe complexes with a number of molecules of the stimulant according to a reversible equation governed by the laws of mass action. It is assumed that the number of secretory units or receptor sites is proportional to the number of parietal cells and that the response is proportional to the number of cells activated. The assumption is reasonable, in view of the large number of cells involved even in the lowest responses and despite the scatter that can be expected in the number of receptors per cell. The following equation was derived to express the dose-response relationship: ( 1)
where c, the number of activated cells, is identified with the observed response, and Cmax, the total number of cells, is identified
PENT APEPTIDE AND GASTRIC SECRETION
October 1966
\rith maximal response. D is the concentration of the stimulant in the tissues, assumed to be a direct function of the administered close, and K is the value of the dose which elicits half the maximal response, that is the ED 50 (when K = D in equation 1, c = Cmax/2). The plot of observed response, c against log-dose, gives the symmetrical sigmoid curve known as the logistic. Further transformation of equation 1 uncovers a linear relationship between the reciprocals of dose and response:
~ C
=
_ 1_
Cmax
+ _!£___,!_
(2)
Cmax D
Since Cmnx or the total number of cells in any single stomach may be considered as constant for the period of testing, the relationship of 1/ c and 1/ D is linear. At infinitely high concentrations of the stimulant, 1/ c = 1/Cmnx , so that the observed response equals the maximal response. Following continuous infusion of gastrin3 or histamine, 10 the relationship of dose and response showed a good fit to a logistic function. The problem of establishing an accurate dose-response relationship following single intravenous or subcutaneous injections of a stimulant appears, at first sight, more complex than for continuous infusion. A measure of acid output must be devised that could be related to the dose of the stimulant, or, more accurately, to the concentration established in the tissues. From his study of the kinetics of distribution of an intravenous stimulant, Teorell 11 concluded (a) that the time of appearance of peak concentration of the stimulant in the tissues for a particular dose and by inference of peak response, was independent of the injected dose, and (b) that the magnitude of this tissue concentration and response was proportional to the dose. The general validity of both conclusions with respect to intravenous injections of gastrin was demonstrated in earlier studies on subjects G. M. and J . M. 3 • 4 and allowed a fit of the secretory data to the same logistic function. The values of the maximal responses as calculated from the asymptotes
461
of the dose-response curves following single intravenous injections for each subject were identical with the values obtained following continuous infusion. This was to be expected, if, as equation 1 predicts, maximal response is essentially determined by activation of all available secretory units independently of how this is achieved. This conclusion received further confirmation when it was subsequently shown in subject G. M. that combinations of gastrin and a synergistic cholinergic agent modified the value of K and D but were without effect on the calculated maximal response. 4 Although the establishment of ·a dose-response relationship following subcutaneous injections is complicated by the rate of resorption of the stimulant from the subcutaneous depot, it appears that, for simplified conditions, the same two conclusions with respect to the time of appearance and the magnitude of peak concentration and response apply as well. 11 If this conclusion is correct, it should allow a fit of the subcutaneous experimental data to the same logistic function and permit a test of the model described by equations 1 and 2, that maximal response in the intact human stomach is essentially determined by activation of all secretory units and is independent not only of the mode of stimulation (continuous infusion, intravenous or subcutaneous injections), but also of the nature of the stimulant (gastrin, pentapeptide, or histamine). The last statement concerning the nature of the stimulant needs amplification. Stimulants administered by the same route vary in their capacity to elicit a particular submaximal response. Their potency in this respect is described quantitatively by the value of J( or the En 50 . But at sufficiently high doses and provided no pharmacological effects , such as on gastric blood flow, intervene to disturb the dose-response relationship, it is clearly immaterial which stimulant is used, since the observed response c should equal the maximal response Crnax . For a particular stimulant, however, the ability to achieve a particular tissue concentration and therefore the value of the ED50 will vary with the route of administration.
462
MAKHLOUF ET AL .
2. Dose-response relationship following subcutaneous stimulation. The pattern of the secretory responses disclosed by the subcutaneous experiments with the pentapeptide appeared to follow closely the predicted pattern of distribution of the stimulant in the tissues. Peak acid output invariably occurred in the second 20-min period, thus confirming the theoretical deductions regarding the time of appearance of peak response. The close fit of the data to equation 2 confirmed the predicted relationship between dose and peak acid output. The close similarity of the intercepts on the vertical axis in each subject indicated that the same maximal response could be obtained following subcutaneous administration of a sufficiently large dose of all types of stimulants. Maximal response thus appeared to be determined by activation of all available secretory units and not by the nature of the stimulant employed. Although the calculated maximal response was obtained by extrapolation, it should be emphasized that this is not a theoretical value since it has been experimentally reproduced, under optimal conditions in both subjects, by the slow intravenous injection of a massive dose of gastrin. 4 -The values of the calculated maximal responses appeared to be somewhat higher for intravenous gastrin (table 2). In the experiments with intravenous gastrin, however, peak acid output was short-lived and was calculated from the peak 10-min output following direct titration of 5-min samples. In experiments with subcutaneous stimulants, peak response was flatter and more prolonged and could only be calculated over a period of 20 min. The technical difficulty of obtaining an absolute estimate of the subcutaneous peak which was probably of shorter duration was reflected in a slightly altered but not significantly different maximal response. It was found in an earlier study 4 that the ratio ofthe output of subject G. M. to subject J. M. following all modes of stimulation was 66%. It was concluded that this fixed ratio, under similar conditions of stimulation, reflected the respective sizes of
Vol. 51, No.4
their parietal cell masses. This ratio was maintained in the present study (table 2) and confirmed the earlier conclusion. 3. Potency of the various stimulants. The first crude estimate of the relative molar potencies of gastrin and histamine, obtained on the basis of equivalent responses from 0.5 p..g per kg of gastrin and 40 p..g per kg of histamine acid phosphate, 6 was given as 500 to 1. It appears from the more accurate data obtained in this study that this was an overestimate. The slopes of the lines in figures 2 and 3 indicated the relative potencies of the various stimulants. From the ED 50 calculated in micrograms, it appeared that gastrin 'ns 10 times more potent than the pentapeptide and 30 times more potent than histamine acid phosphate. On a molar basis, the difference was more pronounced. Gastrin was 30 times more potent than the pentapeptide and 240 times more potent than histamine. Thus the pentapeptide was 3 times more potent than histamine on a weight basis and 8 times more potent on a molar basis. When the potassium output responses were expressed as a percentage of the calculated maximum for potassium and the dose of the pentapeptide was expressed per kilogram of body weight, the curves for the 2 subjects could be exactly superimposed (fig. 4). This indicated that their ED 50 values were identical. The mean ED 50 of the 2 subjects calculated from the potassium dose-response curves was 1.35 ± 0.03 p..g per kg and that from the acid dose-response curves was 1.44 ± 0.15 p..g per kg. Discussion of Section II
The course of secretion in response to the pentapeptide was similar in all respects to that for histamine. Peak acid output invariably occurred in the second 20-min period. The relationship between peak and poststimulatory hour outputs following stimulation by the pentapeptide was identical to that demonstrated in earlier studies with gastrin and histamine. 6 • 9 The acid outputs following histamine (40 p..g per kg) and the pentapeptide (6 p..g per kg) were highly correlated and very nearly equal, with a slope of unity and an inter-
October 1966
PENTAPEPTIDE AND GASTRIC SECRETION
cept not significantly different from zero. The acid outputs following histamine (40 p.g per kg) and gastrin (2 p.g per kg) were also shown in a previous study 6 to be highly correlated, although a slightly higher output resulted from the administration of gastrin. The purpose of secretory tests is to provide a stable and discriminating measure of the functional capacity of the stomach. The emphasis in this statement is on the ability of a secretory test to provide a repeatable index rather than an absolute measure of the utmost capacity of the stomach to secrete. It is therefore immaterial which stimulant is used since the outputs elicited by all stimulants are highly correlated. It should, however, be recognized that these outputs are not strictly maximal, but are repeatable functions or fractions of the real maximum as expressed in the calculated maximal response of a dose-response curve. The similarity in the shape of the secretory curves for all individuals following the administration of a particular stimulant, together with the highly significant correlation between the outputs from different stimulants, suggests that the factors responsible for the establishment of tissue concentration of a stimulant, namely, its distribution, inactivation, and elimination, are similar in different individuals. This rejoins the finding that the ED 50 values for the pentapeptide or for gastrin 5 were similar in subjects G. M. and J. M. Both histamine and the pentapeptide were equally effective in stimulating pepsin in man, but the output levels achieved were not maximal. The fact that the pepsin output at the end of 80 min was still twice as high as the basal level precludes an interpretation on the basis of a washout. Cellular masses as determinants of output. Card and Marks 12 showed that acid output was linearly related to both the parietal cell mass and the mucosal volume of the stomach, while Huntl 3 showed that the outputs of the "parietal" and "nonparietal" components were related to each other and to the output of pepsin. These findings pointed to the probability that the cellular
463
and extracellular elements comprising the wall of the stomach and responsible for the secretory products were present in similar proportions in different individuals. Maximal stimulation by gastrin, histamine, or the pentapeptide introduces a standard situation for purposes of comparison. Under these conditions, the response of pepsin and other constituents of the juice, though not necessarily maximal, may be assumed to be proportional to the size of the cellular and extracellular masses concerned. This prediction was confirmed by the significant linear relationship between the output of acid in every subject and the corresponding output of all other electrolytes, pepsin, and nitrogen (table 3). A similar relationship was obtained in earlier studies on gastrin and histamine, 5 • 9 and may be calculated from data reported by other workers. 14 Side Effects
No side effects were encountered following subcutaneous injection of up to 6 p.g per kg of the pentapeptide. Doses above 8 and up to 13 p.g per kg were accompanied by a sinking abdominal sensation and awareness of intestinal movement lasting some 10 min. These effects seem attributable to enhanced gastrointestinal tone and motility. Since no such effects were encountered with maximal subcutaneous doses of gastrin, then it would appear that doses of gastrin and the pentapeptide which elicited nearly equivalent acid responses had different ef, fects on tone and motility. Intravenous or intramuscular injections of pentapeptide were not tried, but in view of the side effects encountered with the higher doses, administration by these routes should be approached with circumspection with especial care taken to inject the stimulant slowly by the intravenous route. Summary 1. A substituted derivative, structurally related to gastrin, the pentapeptide, ICI 50123, was tested for its action on gastric secretion in man. In particular, its effectiveness as compared with gastrin and histamine was studied in terms of a quantitative model of secretion.
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2. The theoretical predictions from the model were verified experimentally. In 2 subjects, the maximal responses calculated from dose-response curves following subcutaneous gastrin or pentapeptide were similar to those obtained in earlier studies and calculated from dose-response curves following infusion or single intravenous injections of gastrin. In 1 subject, the same maximum was also obtained following subcutaneous histamine or a combination of gastrin and a cholinergic agent. It was concluded, in terms of the model, that maximal acid output was represented by activation of all available secretory units or parietal cells and could be achieved independently of the nature of the stimulant or the route through which it was administered. 3. The potency of a gastric secretory stimulant is properly described by its ED 50 for a particular route of administration and not in terms of the output it elicits. In this sense, gastrin may be described as more potent than the pentapeptide which in turn is more potent than histamine. 4. The similarity of the ED 50 values in all subjects for a particular stimulant is probably a reflection of similar patterns of distribution, elimination, and inactivation of the stimulant. 5. The responses of all 8 subjects to 6 p.g per kg of the pentapeptide were in all respects similar to their responses to 40 p.g per kg of histamine acid phosphate. 6. No side effects were encountered with 6 p.g per kg of the pentapeptide. In 2 subjects tested with doses above 8 and up to 13 p.g per kg, there was a sinking abdominal sensation lasting around 10 min.
D
concentration of the stimulant in the tissues, assumed to be a direct function of the administered dose The rate of activation or complex formation is proportional to the product of dose and number of cells in process of stimulation: k1· (Cmax - c) ·D
The rate of deactivation or cell-stimulant breakdown is proportional to the number of activated cells or formed complexes: kz·c
At equilibrium, both processes are proceeding at the same rate: kz·c = k1· (Cmax - c) ·D
Dividing both sides by lc1 , and replacing lc 2 /k 1 by K (a constant) and rearranging the equation: ( 1) REFERENCES 1. Gregory, H ., P. M. Hardy, D. S. Jones, G. W. K enner, and R. C. Sheppard. 1964. The antral hormone. Nature (London) 204: 931935. 2. Gregory, R. A., and H . J. Tracy. 1964.
3.
4.
Addendum Derivation of equation 1. 5.
Let Cma.x
c Cmax-
C
total number of cells available for activation number of cells activated by cell-stimulant complex formation number of cells in process of activation
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6.
Physiological properties of a series of synthetic peptides structurally related to gmtrin I. Nature (London) B04: 935-938 . Makhlouf, G. M., J. P. A. McManus, and W. I. Card. 1964. Dose-response curves for the effect of gastrin II on acid gastric secretion in man. Gut 5: 379-384. Makhlouf, G. M., J. P. A. McManus, and W. I. Card. 1965. A comparative stud~· of the effects of gastrin, histamine, Histalog, and mechothane on the secretory capacity of the human stomach in two normal subjects over 20 months. Gut 6: 525-534. Makhlouf, G. M., J . P. A. McManus, and W. I. Card. 1965. The action of gastrin II on the gastric secretion of electrolytes and pepsin in man. In Proceedings of the symposium on gastric secretion: l\1echanisms and control, Edmonton, Alberta. In press. Makhlouf, G. M., J. P. A. McManus, and W. I. Card. 1964. The action of gastrin II
October 1966
7. 8.
9.
10.
PENTA PEPTIDE AND GASTRIC SECRETION
on gastric acid secretion in man. Lancet 2: 485-490. Hunt, J. N. 1948. A method for estimating peptic activity in gastric contents. Biochem. J. 42: 104-109. Gornall, A. C., C. J. Bardawill, and M. M. D avid. 1949. D etermination of serum proteins by means of biuret re action. J. Bioi. Chern . 177 : 751-766. i\Iakhlouf, G . M ., J. P. A. M cManus, and IV. I. Card . 1966. A quantitative statement of t he two-component hypothesis of gastric secretion. Gastroenterology 51: 149171. Adam, H. M., W. I. Card, M. J. Riddell, M. Roberts, J. Strong, and B. Wolf. 1954. Dose-response curves for the effect of his-
11.
12.
13.
14.
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tamine on acid gastric secretion in man. Brit. J. Pharmacal. 9: 329-334. T eorell, T. 1937. Kinetics of distribution of substances administered to the body. Arch. Int. Pharmacodyn. 57: 205- 225. Card, W. I., and I. N. Marks. 1960. The relationship between the acid output of the stomach follo wing "maximal" histamine stimulation and the parietal cell mass. Clin. Sci. 19 : 147-163. Hunt, J. N. 1950. An interpretation of the histamine test of gastric secretion. Gastroenterology 16: 231-240. Hirschowitz, B. I. 1961. Electrolytes in human gastric secretion. Observations and a theory. Amer. J. Dig. Dis. (N.S.) 6: 199228.