Tetronic 701 — A novel hypocholesterolaemic agent

549

Atherosclerosis, 23 (1976) 549-558 @ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

TETRONIC

701-

A NOVEL

HYPOCHOLESTEROLAEMIC

J. GREEN, MONIQUE HEALD, K.H. BAGGALEY,

AGENT

R.M. HINDLEY and B. MORGAN

Beecham Pharmaceuticals, Research Division, Nutritional Research Centre, Walton Oaks, Tadworth, Surrey KT20 7NT (Great Britain) (Received 17th July, 1975) (Accepted 8th September, 1975)

Summary

The Tetronic series of polymeric surface-active agents were screened for hypocholesterolaemic activity in rats fed on a semi-synthetic hypercholesterolaemic diet. Only Tetronics 701 and 702 were active and the former was further investigated. Tetronic 701 lowered serum and liver cholesterol in rats fed on a semisynthetic diet, with or without cholesterol, but not in rats fed on a stock laboratory diet. A dose-related growth depression was observed. The compound was hypocholesterolaemic in chicks and rabbits fed on cholesterol-containing diets. The uptake of a single dose of cholesterol into liver and serum was inhibited in rats given Tetronic 701. Tetronics 701 and 702 were effective in precipitating cholesterol from mixed micelles in vitro. Non-hypocholesterolaemic Tetronits were inactive in this respect. A series of tetraesters of Tetronic 701 were prepared and tested in rats fed on a semi-synthetic hypercholesterolaemic diet. Several were hypocholesterolaemic and the tetrabenzoate was of especial interest in that it depressed growth less than did Tetronic 701 itself. Key words: Chick Tetronic

Hypocholesterolaemic

agents - Rabbit - Rat - Serum cholesterol

-

Introduction

One of the mechanisms by which hypocholesterolaemic agents act is by inhibition of cholesterol absorption from the gastro-intestinal tract. Such inhibition can be direct, as with p-sitosterol [l] or indirect, via the inhibition of bile acid absorption, as with cholestyramine [2] . This paper describes the hypocholes-

550

H(C$QC)y(C$6C)x

(C$6C)x(C$4C)yH / N-CH2-CH2-N

\

\

/ H(C$4C)y(Cf16C)x Fig. 1. Basic structure

(C$6C)x(C2H4C)yH

of Tetronics.

terolaemic properties of a novel agent (Tetronic 701) and certain of its esters, and it presents evidence suggesting that they act by inhibiting the absorption of cholesterol. The Tetronic series of surface-active agents (Wyandotte Chemicals Corporation, Michigan, U.S.A.) are block polymers prepared by sequential addition of ethylene oxide and propylene oxide (randomly) to ethylenediamine and have the basic structure as shown in Fig. 1. At least 21 members of the series are commercially available, with a molecular weight range of 1650 to over 26,000. They vary in the balance between the hydrophilic (polyoxyethylene) and hydrophobic (polyoxypropylene) side chains and this balance determines the physical properties of the individual Tetronic. Tetronic 701 is a liquid of mean molecular weight 3600 and contains about 10% by weight of polyoxyethylene.

TABLE

1

COMPOSITION

OF BASAL

SEMI-SYNTHETIC

Constituent

g/kg diet

a) Rats sucrose caaein hydrogenated coconut NaH2 PO4 . Hz 0 minerals a vitamins a

647 200 100 22 27 4

b) Rabbits casein maize starch cellophane flakes lard sugar glucose minerals b vitamins b cholesterol

oil

DIETS

250 221 180 100 88 60 65 16 20

a Diplock et al. [91. b Provides per kg diet: Fe. 25 mg: I. 3.2 mg; Mg, 48 mg; F, 68 mg; Mn. 0.6 mg; Cu. 1 mg; Zn. 0.75 mg: Na, 168 mg: K, 1560 mg; Ca, 654 mg; P. 453 mg: retinyl acetate, 1660 i.u.; cholecalciferol. 97 i.u.: menaphthone. 179 pg; a-tocopheryl acetate, 13 mg; biotin, 67 pg; thiamine, 1.8 mg; riboflavin, 1.8 mg; pyridoxine, 186 pg: cyanocobalamin, 5.5 fig: folic acid, 1 mg; Ca pantothenate, 4.5 mg: nicotinic acid, 22 mg; inositol. 223 mg: choline chloride, 400 mg.

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Materials and methods Animals Rats. Male rats of the CFY strain (Car-worth Europe, Alconbury, Hunts., U.K.) of initial body weight 80-100 g were used throughout. Animals were fed on a stock laboratory diet (Diet FFG; E. Dixon and Sons, Ware, Herts., U.K.) for 7 days, after which they were assigned to experimental groups (normally 10 rats/group, 5 rats/cage), so that the mean body weight of each group was the same. Experimental diets were either stock laboratory diet or a semi-synthetic diet of composition as shown in Table l(a). Diets were rendered hypercholesterolaemic by the addition of cholesterol (l%, w/w) plus ox-bile extract (0.5%, w/w). Food and water were allowed ad libitum. Substances under investigation were administered mixed with diets. Body weight and food consumption were recorded at regular intervals. At the termination of each experiment, rats were bled from the jugular vein and their livers were removed, weighed and frozen, pending subsequent analysis. Chicks. One-day-old Warren cockerels were fed on a normal breeders’ mash for 7 days, after which they were assigned to experimental groups (lo/group), so that the mean body weight of each group was the same. The basal semi-synthetic diet [3] was rendered hypercholesterolaemic by the addition of 0.5% (w/w) cholesterol. Food and water were allowed ad libitum. At the end of the 14-day experimental period, the chicks were bled from an alar vein. Rabbits. Male New Zealand White rabbits (initial body weight l-l.5 kg) were fed on a semi-synthetic cholesterol-containing diet, the composition of which is shown in Table l(b). Blood was obtained at weekly intervals from a marginal ear vein. Cholesterol absorption At the termination of the experiment in which rats were given Tetronic 701 in a stock diet supplemented with cholesterol and bile acid, five animals from each group were given an oral dose of 0.5 I.tCi [4-‘4C] cholesterol (Radiochemical Centre, Amersham, Bucks., U.K.) plus 5 mg carrier cholesterol, dissolved in 0.2 ml arachis oil. The animals were killed 4 h after dosing. Radioactivity was measured in total lipid extracts [4] of serum using scintillation counting [ 51. Micelle stability in vitro This was studied using the methods described by Thompson et al. [6]. Mixed micelles were prepared by dissolving [ 14C] cholesterol, oleic acid and sodium taurocholate in saline-phosphate buffer. The test substance was added and the mixture was incubated at 37°C for 15 min. The mixture was centrifuged at 3000 X g for 15 min, a portion of the supernatant removed and the 14C was measured. This gave the amount of cholesterol remaining in the micellar phase; i.e. not precipitated by the test substance. Analysis Serum measured

total cholesterol using Technicon

and cholesterol in total lipid extracts AutoAnalyzer Method N24a [ 71.

of liver were

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Tetronics Tetronics were obtained as gifts from the Wyandotte Chemical Corporation (Wyandotte, Michigan, U.S.A.). The compounds are described by 3 and 4 digit code numbers. When four digits are used, the first two indicate the average molecular weight of the hydrophobe and when three digits are used, only the first digit serves this purpose. The last digit used represents the weight percentage of the hydrophile to the nearest 10%. The last digit is always separated by a zero from the hydrophobic indicators. Tetronic tetraesters were prepared by standard methods of esterification as previously described [ 81. Results

Initial screening of Tetronics In an initial series of experiments, various members of the Tetronic series were tested for hypocholesterolaemic activity in rats fed on a semi-synthetic hypercholesterolaemic diet for 3 weeks. Substances were incorporated in the diet at a level of 0.5% w/w. Table 2 shows that, of all the compounds tested, only Tetronics 701 and 702 were hypocholesterolaemic. Tetronic 701 was chosen for more detailed investigation.

Hypocholesterolaemic activity in rats (1) Tetronic 701. Table 3 shows the effect of Tetronic

701 on serum choles-

TABLE 2 EFFECTS RATS a

OF VARIOUS

MEMBERS

OF THE TETRONIC

Tetronic

Serum cholesterol (% of control)

304 d 501 c 504 d 700 c 701 d 701 c 702 d 704d 900 c 901 c 1100 c 1101 c 1300 c 1501 d 1502 d 1504 d 1508 d 1704 d 1707 d

133 108 168b 129 24 b 51 b 34 b 93 147 b 80 135 ix 87 113 163 b 124b 115 191 b 108 130

a Animals were fed on semi-synthetic hypercholesterolaemic b Significantly different from control, P < 0.05. ’ Added to diet at 0.5%. d Added to diet at 0.05%.

SERIES

ON SERUM CHOLESTEROL

diets for 21 days.

IN

DIETS a

81 f 3.5 32 + 1.9 b 64 ? 3.0 b 56 + 4.1 51 * 2.9

0 0.5 0.2 0.1

0 0.1

0 0.25 0.1

0 0.1

cholesterol and bile acid

none

cholesterol

bile acid

cholesterol and bile acid

stock

Semi-synthetic ’

Semi-synthetic ’

Semi-synthetic ’

Semi-synthetic

f + i f

8.2 4.2 b 6.0 b 7.0 b

94 * 3.3 66 * 4.9 b 86 f 3.9

19 f 3.0 70 f 2.2 b

125 42 67 94

95 + 3.0 80 f 3.2 b

a Experimental diets were given for 21 days unless otherwise stated. b Significantly different from control, P < 0.05 ’ Experiment period 14 days.

0 0.1 0.05

0.5

0

none

Stock

gain (9)

Tetronic 701 (% of diet)

Supplement to diet

Body weight

701 IN RATS FED ON VARIOUS

Basal diet

Mean t SE of the mean.

EFFECTS OF TETRONIC

TABLE 3

0.26 0.20 0.25

0.30 0.29

-

0.31 0.36

0.25 0.12 0.15 0.19

0.24

0.29

Food eaten (g)

wt. gain (8)

0.12 0.14 b 0.10 b 0.17

5.8 + 0.12 5.5 f 0.21 5.4 ? 0.09 b

5.5 ? 0.15 5.9 + 0.15

5.3 r 0.12 6.3 + 0.19 b 5.8 f 0.13 b

5.1 * 0.11 5.6 f 0.22

f ? f f

239 + 26 102 f 12b 156 f 11 b

96 f 3 81?2b

92 + 6 45igb 61*ab

79 f 2 66h2b

300+21 97 ? 11 b 169 f 21 b 198 * 17 b

89 * 11 119 + 23

3.9 f 0.52 4.4 f 0.69 5.6 5.1 4.8 5.3

Serum cholesterol (mg/100ml)

Liver weight (% of body wt.)

* f f *

1.13 0.46 b 2.28 b 2.63 b

6.1 + 0.37 4.9 + 0.28 b

1.0 2 0.26 4.4 f 0.23 b 4.3 * 0.27 b

3.4 f 0.22 3.9 + 0.23

29.8 5.0 17.0 41.0

5.9 f 1.5 5.7 f 1.4

(mglg)

Liver cholesterol _~

554

TABLE

4

EFFECTS

OF

TAINING Mean

TETRONIC

701

CHOLESTEROL

AND

AND

ITS

BILE

ESTERS

ACID

IN

RATS

FED

ON

SEMI-SYNTHETIC

DIETS

CON-

a

f SE of the mean.

Supplement

to diet

b

Body

wt.

(g/l4

d)

gain

wt.

None

53 + 3.6 701

Tetronic

701

tetra(pheny1

Tetronic

701

tetra(ethyI

carbamate) carbamate)

Serum

(g)

cholesterol

(mg/100mI) Food

Tetronic

gain

e&en

(g)

0.30

397

t 52.8

22

? 3.2

=

0.15

206

f 33.0

35

i- 3.4

cd

0.24

241

* 36.4

0.19

123+

29i

5.6’

6.6

Tetronic

701

tetra(2,6-dimethylbenzoate)

52 i- 2.9

d

0.29

290

* 35.7

Tetronic

701

tetrabenzoate

36

cd

0.23

119

+

Tetronic

701

tetracyclo

0.18

128?

a Experimental bCompounds

diets under

hexanoate

were test

’ Different

from

control,

d Different

from

Tetronic

given were

+ 3.7

28k3.8’ for

added

’ ’ cd

7.8

cd

9.6

cd

14 days. to the diet

at a level

of 0.1%.

P < 0.05. P < 0.05.

group,

terol in rats fed on various diets. No activity was found in animals given the stock laboratory diet. However, when this diet was rendered hypercholesterolaemic with cholesterol and bile acid, the rise in serum cholesterol was inhibited by the Tetronic, at levels down to 0.1% of the diet. In experiments using the semi-synthetic diet, Tetronic 701 was hypocholesterolaemic in unsupplemented diets, or in diets supplemented with cholesterol alone, bile acid alone and cholesterol and bile acid in combination. In all experiments, the compound caused a growth depression that appeared to be dose-related. This growth depression was associated with a reduced efficiency of foodstuff utilisation. Tetronic 701 sometimes lowered relative liver weight, but this change was not consistently observed. When the compound showed hypocholesterolaemic activity, liver cholesterol concentration was also usually reduced. (2) Esters of Tetronic 701. It was thought that the detergent-like properties of Tetronic 701 might, in the long term cause toxic side effects resulting from irritation of the gastrointestinal tract. Therefore, a series of tetraesters were prepared and tested for hypocholesterolaemic activity. Table 4 shows the effects TABLE

5

TETRONIC Mean

701

* SE

Tetronic

AND

SERUM

CHOLESTEROL

IN CHICKS

a

of the mean.

701

Body

weight

gain (g/14

(% of diet)

Wt.

gain

(9)

Serum

d)

cholesterol

(mg/lOOml) Food’eaten

(9)

0

187

* 5.8

0.66

247

f

9

0.25

127

+ 8.0

b

0.55

198

+

9b

0.1

165

? 5.1

b

0.64

232

* 15

a Male

chicks

b Significantly

were

fed

different

on a semi-synthetic from

control,

cholesterol-containing

P < 0.05.

diet

for

14

days.

OF

TETRONIC

a Rabbits

were

different

(7/group)

b Significantly

from

63

? 257

f

a

control,

P <

0.05.

on a semi-synthetic

1541

1811

65

1657

1518+

fed

(g)

0

f 140

weight

0.25%

Body

IN RABBITS

5wk

in

701

3 wk

diet

6

? SE of the mean.

Tetronic

Mean

EFFECT

TABLE

diet

1572+

2321

7 wk

containing

52b

? 182

0.2%

1645?

2289

9 wk

cholesterol.

63b

f 239 565

813

3 wk I36

cholesterol

r 104

+

Serum

583?

1060

5wk

63

f 182

(mg/lOOml)

b

720

1480

7 WI? + 140 ? 106

b

709

1610

9 wk + 183 * 118

b

556

TABLE

I

CHOLESTEROL

ABSORPTION

IN RATS

FED WITH TETRONIC

701 a

Mean _+SE of the mean Tetronic 701 (% of diet)

Radioactivity

0 0.5 0.2 0.1

recovered

(% of dose)

Per ml serum

Per liver

0.87 0.13 0.17 0.38

13.8 4.6 5.3 11.1

f 0.15 t 0.01 ?r 0.04 f 0.07

b b b

+ f + *

2.4 0.7 b 0.6 b 0.9

a Rats (5/group) were fed on a stock diet containing cholesterol and bile acid for 21 days. Four hours before killing, each animal was given an oral dose of [4-’ 4 Cl cholesterol. b Significantly different from control. P < 0.05.

of the esters when given for 14 days at 0.1% synthetic diet containing cholesterol and bile the tetra (2,6-dimethyl-benzoate), showed a effect. Tetronic 701 tetrabenzoate not only olaemic effect than the parent compound but ly less than did Tetronic 701 itself.

in the diet of rats fed on a semiacid. All the esters tested, except significant hypocholesterolaemic showed a better hypocholesterinhibited weight gain significant-

Activity in chicks Table 5 shows that Tetronic 701 was hypocholesterolaemic in cholesterolfed chicks when incorporated into the diet at a level of 0.25%, but not when added at 0.1%. As with rats, a dose-related inhibition of growth was observed. Activity in rabbits Tetronic 701, when added at 0.25% to the diet of cholesterol-fed rabbits, showed considerable hypocholesterolaemic activity (Table 6). Again, as in rats and chicks, a significant depression in body weight gain was caused by the comTABLE EFFECT CELLES

8 OF =

TETRONICS

Substance

OF

[4--l

4C]CHOLESTEROL

FROM

MIXED

MI-

Cholesterol precipitated (% initial cholesterol added)

None Neomycin Tetronic Tetronic Tetronic Tetronic Tetronic Tetronic Tetronic Tetronic Tetronic Tetronic Tetronic

ON PRECIPITATION

sulphate 304 504 701 702 704 904 1502 1504 1508 1704 1707

0 99 0 0

65 39 4 0 0 0 0 0 0

a Each substance was added to 5 ml miceIlar solution to give a final concentration of 3 m&z/ml. Eacn mcubation contained 0.5 @f cholesterol and each result is the mean of duplicate assays.

pound. However, hypocholesterolaemic activity was observed at 5 weeks, when body weight gain was not significantly different from that of control animals. Cholesterol absorption in rats The uptake of a single dose of radioactive cholesterol into serum and liver of rats given Tetronic 701 in the diet for 21 days was significantly lower than in control rats (Table 7), apart from the figure for liver in rats given 0.1% Tetronic. This indicates that Tetronic 701 may exert its hypocholesterolaemic activity by inhibiting the absorption or re-absorption of cholesterol from the gastro-intestinal tract, although it is possible that the same results could have been obtained by a shift of the absorption curve to the right, without any change in net absorption. Micelle stability in vitro In an attempt further to clarify the mode of action of the Tetronics, their effect on the stability of simple micelles was studied by a method [6] that measures the capacity of a substance to remove cholesterol from mixed solutions. The results are given in Table 9. Neomycin sulphate, a polybasic antibiotic, which was used as a positive control, completely precipitated cholesterol from the micelle (as was shown by Thompson et al. [6]), in accord with the suggested mode of hypocholesterolaemic activity for this substance. Of the Tetronics tested, only 701 and 702 were effective in destabilising the micelle, the latter being less active than the former and neither being as nearly as effective as neomycin sulphate. It should be noted that the Tetronics showing activity in this test were the only ones showing hypocholesterolaemic activity in vivo (see Table 2). Discussion We have examined a wide range of compounds from the Tetronic series of non-ionic block polymers. Only two of the compounds tested (Tetronics 701 and 702) showed hypocholesterolaemic activity in a primary screen and the former was chosen for further investigation. The activity of 701 and 702 does not seem to correlate with any obvious physical property of the compounds. Both compounds are defoaming agents, but two other commercial defoaming agents (with differing structures) showed no hypocholesterolaemic activity (J. Green, this laboratory, unpublished results). Tetronic 701 has shown significant hypocholesterolaemic activity in a variety of animal models, namely: the cholesterol-fed chick, the cholesterol-fed rabbit, the rat fed on diets containing cholesterol and bile acids and the rat fed on a semi-synthetic cholesterol-free diet. This spectrum of activity suggests that the mode of action of Tetronic 701 is associated with an inhibition of cholesterol absorption. The reduced uptake of radiolabelled cholesterol into liver and serum of rats given Tetronic 701 supports this hypothesis. Such an inhibition is clearly not specific for cholesterol, for hypocholesterolaemic activity was nearly always associated with a depression in growth and efficiency of foodstuff utilisation. It has been shown (J. Bunyan, this laboratory, private communica-

558

tion) that Tetronic 701 and 702 both increase the output of faecal fat in normal rats. However, at a critical level of dietary Tetronic (0.5% in our hypercholesterolaemic rats), it appears possible to achieve an effective lowering of serum cholesterol without marked growth depression. Certain esters of Tetronic 701 were shown to have hypocholesterolaemic activity. Two of these active esters (phenyl carbamate and benzoate) had a significantly smaller effect on growth than did Tetronic 701 itself. In a study in vitro of the effect of Tetronics on micellar solutions, it was shown that only Tetronics 701 and 702 had destabilising activity, and this is in accord with their hypocholesterolaemic effect in vivo. However, their activity was weak compared to that of neomycin sulphate, which completely disrupts the micelle. It could be concluded that the hypocholesterolaemic activity of Tetronic 701 can only be partly explained by its disruptive effect on bile acid micelles. The properties of the compounds described in this paper suggest that either Tetronic 701 or one of its esters could prove to be useful for the treatment of hypercholesterolaemia in man. Thus, the tetrabenzoate ester of Tetronic 701 has been found to have a remarkably low order of toxicity in the rat and the baboon. However, when given to beagle dogs at a level of 2 g/kg/d for 14 days, the compound produced vacuolation in the lymphoid tissue. The exact nature of the phenomenon requires further investigation. Acknowledgements We are grateful to Dr. H.H. LeVeen (Veterans Administration Hospital, New York) for his interest and useful discussions during the course of this work. We thank Mr. D. Hiley and his staff for care of the animals.

References Hernandez. H.H., Peterson, D.W.. Chaikoff. I.L. and Dauben. W.G.. Absorption of cholesterol-4-I 4C in rats fed mixed soybean sterols and sitosterol. Proc. Sot. Exp. Biol. Med., 83 (1953) 498. Huff. J.W.. Gilfillan. J.L. and Hunt, V.M., Effect of cholestyramine, a bile acid binding polymer, on plasma cholesterol and faecal bile acid excretion in the rat. Proc. Sot. Exp. Biol. Med.. 114 (1963) 352. Morgan, B., Heald. M., Brooks. S.G., Tee. J.L. and Green, J., The interactions between dietary saponin. cholesterol and related sterols in the chick, Poultry Sci.. 51 (1972) 677. Folch. J., Lees. M. and Stanley, G.H.S., A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem.. 226 (1957) 497. from the Atkin, S.D.. Morgan, B., Baggaley, K.H. and Green. J., The isolation of 2,3-oxidosqualene liver of rats treated with 1-dodecylimidazole, a novel hypocholesterolaemic agent, Biochem. J., 130 (1972) 153. Thompson, G.R.. Macmahon, M. and Claes, P., Precipitation by neomycin compounds of fatty acid and cholesterol from mixed micellar solutions, Eur. J. Clln. Invest., 1 (1970) 40. Technicon Instruments Corporation, Technical Bulletin N-24a. 1967. British Patent No. 1371507. Diplock, A.T., Green. J., Bunyan, J., McHale, D. and Muthy, I.R., Vitamin E and stress. Part 3 (The metabolism of D-a-tocopherolin the rat under dietary stress with silver). Brit. J. Nutr., 21 (1967) 115,