GB2239455A

GB2239455A - Lipolytic enzyme inhibitors

Info

Publication number: GB2239455A
Application number: GB9027268A
Authority: GB
Inventors: Toshiyuki Miyazaki; Hirofumi Motoi; Toshiaki Kodama; Taturo Maeda; Takahiro Tsujita; Hiromichi Okuda
Original assignee: Nisshin Seifun Group Inc
Current assignee: Nisshin Seifun Group Inc
Priority date: 1989-12-25
Filing date: 1990-12-17
Publication date: 1991-07-03
Anticipated expiration: 2010-12-17
Also published as: GB9027268D0; GB2239455B; FR2656311B1; FR2656311A1; DE4040874A1

Abstract

A lipolytic enzyme inhibitor is disclosed which comprises a basic protein, a basic polypeptide or a salt thereof. The inhibitor is useful as a dieting agent for the prevention of obesity and lipemia and as an additive for food and feed. The inhibitor is preferably a protamine, a histone, a purothionin, a polylysine or a polyarginine.

Description

LIPOLYTIC ENZYME INHIBITORS

BACKGROUND OF THE INVENTION Field of the Invention

This invention relates to inhibitors of enzymes participating in lipolysis. More particularly, the invention relates to lipolytic enzyme inhibitors which comprises basic proteins and/or basic polypeptides. Description of the Prior Art

It has been reported in a number of references that proteins such as serum albumin, 8-1actoglobulin and certain soybean proteins inhibit some kinds of lipases (see, for example, Journal of Lipid Research, Vol. 25, 1984, pages 1214-1221). In the presence of bile acids, however, these proteins lose their lipase inhibitory activity and do not function as a lipase inhibitor in vivo.

SUMMARY OF THE INVENTION our continuing study on the proteinous lipase inhibitors has revealed that basic proteins, basic peptides and the salts thereof inhibit or suppress the activity of lipolytic enzymes in the presence of bile acids.

Thus the present invention provides a lipolytic enzyme inhibitor which comprises as an active ingredient at least one of a basic protein, a basic peptide and salts thereof.

- 2 DETAILED DESCRIPTION OF THE INVENTION

The term "lipolytic enzyme inhibitor" as used herein refers to an agent having a function of inhibiting or suppressing the activity of lipolytic enzymes such as lipases, thereby inhibiting or suppressing hydrolysis of lipids which results in inhibiting or suppressing the intestinal absorption of lipids.

The lipolytic enzyme inhibitors of the present invention are effective in the condition wherein lipids are emulsified in the presence of bile acids, thus effectively acting in vivo.

The basic proteins, basic polypeptides and salts thereof which can be used in the invention include purothionins contained in wheat; purothionin analogues contained in other cereals than wheat (including barley and rye) such as purothionin-analogous polypeptides widely distributed in barley as disclosed in Japanese Patent Publication No. 57840/1986 and purothionin-analogous polypeptides occurring in rye as disclosed in J. Agric. Food Chem., Vol. 26, No. 4, pages 794-796 (1978); protamine; histone; polylysine; polyarginine and salts thereof.

Three kinds of purothionin, ai-, (X 2_# 2purothionin have some differences in amino acid sequence, each having the following amino acid sequences. Any purothionins can be used in the present invention.

1 cl 1 1 5 10 15 20 25 ui-Purothionin H, N-Lys-Ser-Cys-Cys -A r g -S c r-Thr-Leu-G I y-Arg-Asn- Cys-Ty r-Asn-Leu-Cys-Arg-A I a-Arg-Gly-A I a-G 1 n-Lys-Leu-Cys a2-Purothionin H 2N-Lys-Ser-Cys-Cys -A r g -Thr-Thr-Leu-G I y-Arg-Asn-Cys- Tyr-Asn-Leu-Cys-Arg-Ser-Arg-Gly-A l a-G 1 n-Lys-Leu-Cys Purothionin H N-Lys-Ser-Cys-Cys-Lys -S er -Thr-Leu-G l y-Arg-Asn-Cys- Tyr -As n-Leu-Cys-Arg-,k 1 a-Arg-Gly-A 1 a-G 1 n-Lys-Leu-Gys- 1 W 1 Ala-GIY-Val-Cys-Arg-Cys-Lys-1 le-Ser-Ser-Gly-Leu-Ser-Cys-Pro-Lys-Gly-PhePro-Lys-COOR -S er -Thr-Va 1 -Cys- A rg-Cys-Lys-Leu-Thr-Ser-G l y-Leu-Ser-Cys-Pro-Lys - Cl y-Ph'e-Pro-Lys-COOH -Ata-Asn-Val-Cys-Arg-Cys-Lys-Leu-Thr-Ser-Gly-Leu-Ser-Cys-Pro-Lys-Asp-PhePro-Lys-COOR - 4 There are known three types of protamines including mono-, di- and tri- protamines and five types of histones including H1, H2A, H2B, H3, H4 and H5. Any types of those can be used in the present invention.

Polylysine is distinguished by the site of peptide bond and composed of cpolylysine represented by formula (I) H-(CH 2)4_ CH-CO1 1 IN& 12 n (1) wherein n represents the degree of polymerization of lysine and apolylysine represented by formula (II) --NH-CH-CO-- 2)4 2) n (II) wherein n is as defined above. Any polylysines and salts thereof may be used in the invention. C-Polylysine and its salts are preferred because they can maintain in vivo the function of suppressing or inhibiting the lipolytic enzymes over a longer period of time, thus leading to more reduced total lipid absorption as compared with a-polylysine, other basic proteins and polypeptides and salts thereof. Polylysines of formulas (I) and (II) wherein n is 4 or more, particularly 5 or more are more effective because of their higher activities of inhibiting lipolytic enzyme. E:- C QZ 1%; Polylysines wherein n is less than 9 have low antimicrobial activity. Thus c-polylysines wherein n is 5-9 will have an effect on inhibition of lipid absorption without damage to intestinal flora.

The amino acids composing basic proteins and peptides include two kinds of optical isomers, L- and Dforms. The basic proteins and peptides derived from natural products are known to be usually composed of L-amino acids.

The basic proteins and polypeptides as well as salts thereof which are used in the invention may be composed of either or both of L- and D-amino acids.

The lipolytic enzyme inhibitors of the present invention may contain a salts thereof alone or The lipolytic basic protein, a basic peptide and in combination therewith. enzyme inhibitors of the invention can be administered to human being and various including livestock and chicken as well as pet poultry such as cattle, animals such as dog and effective dose will vary depending on the type, physical conditions, etc. of the subjects to be administered. Preferably, they may be given at dose for individual subjects.

animals horse and cat. The age and any suitable

The lipolytic enzyme inhibitors of the invention is formulated into a preparation for oral administration. They may be administered either alone or in admixture with carrier conventionally used in pharmaceutical industry or in is combination with other drugs. Furthermore, they can be used in any form of preparations such as tablets, granules, capsules, powders or the like.

In addition, the lipolytic enzyme inhibitors of the invention may also be administered as an additive for food and feed. Thus the inhibitors of the invention are useful as an additive for food and feed.

Administration of the lipolytic enzyme inhibitors of the invention to human being and other animals can inhibit lipolytic enzymes to suppress or inhibit the hydrolysis of lipids so that rapid intestinal absorption of ingested lipids can be inhibited and total fat absorption can also be controlled at a low level, thereby achieving a variety of effects such as prevention of lipemia and obesity. Thus the inhibitors of the invention are useful as a dieting agent for the prevention of obesity and lipemia.

Of the lipolytic enzyme inhibitors of the invention, especially Cpolylysines maintain their lipid absorption-inhibitory actions in the living body over a long period of time, which result in largely reduced total fat absorption in vitro.

The invention is further illustrated by the following non-limitative examples.

EXAMPLE 1 (Preparation of purothionin) A crude purothionin mixture containing a 1 -, U 2_ 7 - and 8-purothionins was obtained starting from weak wheat flour according to the method described in Agr. Biol. Chem., Vol. 34, No. 7, pages 1089- 1094 (1970). The resulting crude mixture was purified to isolate a 2- purothionin and 8purothionin, respectively. (Preparation of olive oil emulsion) 250 mg of an olive oil, 21.5 mg of sodium cholate as a bile acid component and 30 mg of phosphatidylcholine were added to 5 ml of a 300 mM potassium phosphate buffer solution at pH 6.8 (called hereafter "potassium phosphate buffer").

The mixture was ultrasonicated to prepare an olive oil emulsion. (Preparation of protein (polypeptide)-containing solution) One ml of the potassium phosphate buffer was added to each 4 mg of the crude purothionin mixture, the purified ot 2-purothionin and the purified 3-purothionin, bovine serum albumin (called hereafter "BSA", A7030, Sigma Co., Ltd., U.S.A.), 8-1actoglobulin (L0130, Sigma Co., Ltd.) and ovalbumin (A5503, Sigma Co., Ltd.) to prepare six kinds of protein (polypeptide)-containing solutions. (Preparation of lipase solution) Porcine pancreatic lipase (manufactured by Sigma Co., Ltd.) was added to the potassium buffer solution to prepare a lipase solution containing 100 units porcine pancreatic lipase per ml.

8 (Assay of lipase inhibitory activity of proteins) The control and six test samples were prepared in the following manner.

pl of an olive oil emul.sion as prepared above were used as a substrate.

pl of potassium phosphate buffer was added to the substrate to prepare an emulsion for control sample. Each 50 pl of the protein(polypeptide)containing solutions as prepared above was added to 100 pl of the substrate to prepare six emulsions for test sample. All of the emulsions were incubated for 5 min. To each of those emulsions was added 50 pl of the lipase solution as prepared above. The resulting mixture was incubated at 37'C for one hour, subsequently to which were added 3 ml of an extraction solvent (chloroform: methanol: n-heptane = 49: 1: 49). After being shaken for 5 min., the mixture was centrifuged at 3000 rpm for 5 min. The upper layer was removed with an aspirator and to the remaining solution was added 1 ml of a copper reagent (0.45 M triethanol amine, 0.05 N acetic acid, 3.4% copper sulfate pentahydrate and 20% sodium chloride). The mixture was shaken for 5 min. and centrifuged at 3000 rpm for 5 min. 0.5 ml of a solution was taken from the upper layer and to the solution was added 0.5 ml of a color developing agent (a solution of 0.1% bathocuproine and 0.05% butyl hydroxyanisole in the above extraction solvent).

The resulting solutions for control and test - 9 samples were measured for the absorbance at 480 nm (A480) using photometer to determine the amount of the free fatty acids as formed, i.e. , the activity of the porcine pancreatic lipase.

The results are shown in Table 1 in which the activity is expressed in terms of the percentage of the Abs480 in the case where control sample was hydrolyzed by the pocine pancreatic lipase.

Table 1

Protein (polypeptide) Activity of porcine contained in samples Isoelectric pH pancreatic lipase (%) Control 100 Crude purothionins Ca. 10 6.0 a 2Purothionin Ca. 10 0 8-Purothionin Ca. 10 0 Ovalbumin Ca. 4-5 107.0 13-Lactoglobulin Ca. 5 103.9 BSA Ca. 4-5 105.4 It is seen from the results shown in Table 1 that the lipase activity is much inhibited when the inhibitor of the invention comprising crude purothionins or a purified (X 2- or B-prothionin is added in the presence of bile acid, whereas ovalbumin, 8-1actoglobulin and BSA, not belonging to basic proteins do not have lipase inhibitory activity under the specified condition in this example. EXAMPLE 2 Activity of porcine pancreatic lipase was investigated in the same way as in Example 1 except that the crude purothionin- or B-purothionin- containing solution was diluted to a protein concentration of.0.2 mg/ml. Lipase activity of the crude purothionin-containing sample was 97.5%, thereby indicating that crude purothionin in such concentration has little inhibitory activity against porcine pancreatic lipase. On the contrary, the lipase activity of the 3-purothionin-containing sample was 0%, thereby indicating that 8-purothionin was capable of greatly inhibiting the activity of porcine pancreatic lipase even in such concentration.

Furthermore, in a separate experiment carried out in a similar manner as given above, B-purothionin was able to reduce the activity of porcine pancreatic lipase to 5.2% even in its concentration of 0.04 mg/ml representing its great inhibitory activity of porcine pancreatic lipase. EXAMPLE 3 (Preparation of cholesterol oleate emulsion) 32.5 mg of cholesterol oleate and 25 mg of phosphatidylcholine and 21.5 mg of sodium cholate were added to 5 ml of the potassium phosphate buffer. The solution was ultrasonicated to prepare a cholesterol oleate emulsion. (Preparation of protein-containing solution) Following the same procedure as in Example 1, four protein-containing solutions were prepared, each of which h contains the crude purothionins, BSA, B-1actoglobulin or ovalbumin. (Preparation of cholesterol esterase solution) A cholesterol esterase isolated from porcine pancreas was added to a 300 mM potassium phosphate buffer solution to prepare an enzyme solution containing 40 pg/ml of cholesterol esterase. (Assay of cholesterol esterase inhibitory activity of protein) The control and four test samples were prepared in the following manner.

pl of an olive oil emulsion as prepared above were used as a substrate.

pl of potassium phosphate buffer was added to the substrate to prepare an emulsion for control sample. Each 50 V1 of the protein(polypeptide)containing solutions as prepared above was added to 100 pl of the substrate to prepare four emulsions for test sample. All of the emulsions were incubated for 5 min. To each of those emulsions was added 50 pl of the lipase solution as prepared above. The resulting mixture was incubated at 37'C for one hour, subsequently to which were added 3 ml of the same extraction solvent as used in Example 1. After being shaken for 5 min., the mixture was centrifuged at 3000 rpm for 5 min. The upper layer was removed with an aspirator and to the remaining solution was added 1 ml of the same copper 12 - reagent as used in Example 1. The mixture was shaken for 5 min. and centrifuged at 3000 rpm for 5 min. 0.5 ml of a solution was taken from the upper layer and to the solution was added 0.5 ml of the same color developing agent as used in Example 1.

The resulting solutions for control and test samples were measured for the absorbance at 480 nm (A480) using photometer to determine the amount of the free fatty acids as formed, i.e., the activity of the cholesterol esterase.

The results are shown in Table 2 in which the activity is expressed in terms of the percentage of the Abs480 in the case where control sample was hydrolyzed by cholesterol esterase.

Table 2

Protein (polypeptide) contained in samples Activity of cholesterol esterase Control Crude purothionins Ovalbumin B-Lactoglobulin BSA 9.8 93.6 89.9 120.0 From the results shown in Table 2, it is seen that the cholesterol esterase activity is much inhibited when the inhibitor of the invention comprising crude purothionins, i.e., basic protein was added in the presence of a bile 13 - acid, whereas ovalbumin, 8-lactoglobulin and BAS, not belonging to basic proteins have no or a little, if any, cholesterol esterase inhibitory activity under this condition.

EXAMPLE 4 (Preparation of olive oil emulsion) 250 mg of an olive oil, 21.5 mg of sodium cholate as a bile acid component and 30 mg of phosphatidylcholine were added to 5 ml of a 200 mM Tris buffer solution at pH 6.8 (called hereafter "Tris buffer").

The mixture was ultrasonicated to prepare an olive oil emulsion. (Preparation of protein- or peptide-containing solution) ml of Tris buffer was added to each 1 mg of the same purified 8- purothionin as used in Example 1, protamine (P4005, Sigma Co., Ltd., U.S. A.), histone H2A (H6881, Sigma Co., Ltd.), histone H3 (H4380, Sigma Co., Ltd.), (x-poly-Llysine (3075, Peptide Research Co., Ltd.) and poly- Larginine (P3892, Sigma Co., Ltd.) to prepare six kinds of protein- or peptide-containing solutions. (Preparation of lipase solution) Porcine pancreatic lipase (manufactured by Sigma Co., Ltd.) was added to Tris buffer to prepare an enzyme solution containing 100 units porcine pancreatic lipase per M1. (Assay of lipase inhibitory activity of proteins) - 14 The control and six test samples were prepared in the following manner.

pl of an olive oil emulsion as prepared above were used as a substrate.

pl of Tris buffer was added to the substrate to prepare an emulsion for control sample. Each 50 U1 of the protein(polypeptide)-containing solutions as prepared above was added to 100 pl of the substrate to prepare six emulsions for test sample. All of the emulsions were incubated for 5 min. To each of those emulsions was added 50 pl of the lipase solution as prepared above. The resulting mixture was incubated at 370C for one hour, subsequently to which were added 3 ml of the same extraction solvent as used in Example 1. After being shaken for 5 min., the mixture was centrifuged at 3000 rpm for 5 min. The upper layer was removed with an aspirator and to the remaining solution was added 1 ml of the same copper reagent as used in Example 1. The mixture was shaken for 5 min. and centrifuged at 3000 rpm for 5 min. 0.5 ml of a solution was taken from the upper layer and to the solution was added 0.5 ml of the same color developing agent as used in Example 1.

The resulting solutions for control and test samples were measured for the absorbance at 480 nm (A480) using photometer to determine the amount of the free fatty acids as formed, i.e., the activity of the porcine pancreatic lipase.

h The results are shown in Table 1 in which the activity is expressed in terms of the percentage of the Abs480 in the case where control sample was hydrolyzed by the pocine pancreatic lipase.

Table 3

Protein (polypeptide) Activity of porcine contained in samples Isoelectric pH pancreatic lipase (%) Control 8-Purothionin Protamine Histone H2A Histone H3 aPoly-L-lysine Poly-L-arginine is 1.3 1.3 3.7 2.3 6.9 16.5 From the results shown in Table 3 it is seen that the activity of lipase is much inhibited when the inhibitor of the present invention comprising purified 8-purothionin, protamine, histone, a-poly-L-lysine or poly-Larginine, a kind of basic protein or peptide was added under the specified condition in this example. EXAMPLE 5 Two groups of 10 SD male rats (9 weeks age, 200 9 average bodyweight) were prepared.

g of corn oil, 6 g of yolk lecithine and 12.5 g of glycerol were added to distilled water to make up to 100 M1. The mixture was ultrasonicated to prepare an emulsion.

16 - The rats of first group were each given 2 ml of the emulsion and 100 mg of E-poly-L-lysine manufactured by Chisso Corp. (containing dextrin and E- poly-L-lysine at a weight ratio of 1:1 and having formula (I) wherein n is ca. 30), orally using a gastric probe. Blood was drawn at predetermined intervals from the tail vein, and serum t.riglyceride concentration was measured using Kyowa Medics enzyme Kit TG, determined for mean value (mg/dl) per animal and calculated on the basis of serum triglyceride concentration prior to administration (taken as 0 mg/dl)(According to the invention).

The rats of second group were orally given 2 ml of the emulsion and 50 mg of dextrin. Serum triglyceride concentration was measured at predetermined intervals, determined for the average value and calculated in the same way as above (Comparative example).

The results are shown in Table 4 below.

Table 4

Elapsed time after administration (hrs.) Inventive 0 (before administration) 0 Blood concentration of neutral fats (ml/dl) Comparative 0 1 53.3+2.5 a 176.8+16.3 2 32.4+4.9a 124.1+25.2 3 16.7+3.2 a 79.6+11.0 4 11.1+3.0 a 62.0+10.5 7 4.2+1.0 a 17.3+3.0 a: Significantly different at K0.01 As seen from the results in Table 4, in the case where the rats are given a test sample containing c-poly-Llysine (a basic peptide of the invention), the intestinal lipid absorption is inhibited and serum triglyceride concentration can be controlled at a low level over a period of time from the beginning to as far as 7 hours after administration, resulting in largely reduced total fat absorption. These results indicate that E-polylysine has an inhibitory activity in vivo against lypolytic enzyme over a long period of time.

EXAMPLE 6 (Preparation of peptide-containing solutions) Two peptide-containing solutions were prepared by adding 1 mg of distilled water to each 1 mg of a-poly-Llysine hydrochloride (3075, Peptide Research Co., Ltd.) and a-poly-D-lysine hydrobromide (P7886, Sigma Co., Ltd.). (Assay of lipase inhibitory activity of polylysine) The control and two test samples were prepared in the following manner.

M1 of an olive oil emulsion prepared in the same way as in Example 4 were used as a substrate.

pl of Tris buffer was added to the substrate to prepare an emulsion for control sample. Each 50 gl of the polypeptide-containing solutions as prepared above was added to 100 pl of the substrate to prepare two emulsions for test sample. After incubating the emulsions for 5 min., there was added 50 pl of the enzyme solution prepared in the same way as in Example 4. The resulting mixture was incubated at 370C for one hour.

Further, the incubated mixture was treated and measured for the activity of porcine pancreatic lipase in the same way as in Example 4.

The results are shown in Table 5 below. Table 5 Polylysine contained Activity of porcine in samples pancreatic lipase Control a-Poly-L-lysine hydrochloride a-Poly-D-lysine hydrobromide 1.6 1.1 It is seen from the above data that both L and D polylysines can greatly inhibit the lipase activity under the specified condition in this example. EXAMPLE 7 (Preparation of peptide-containing solutions) Lysine oligomers having a degree of polymerization of 2-5 as shown in Table 6 below were prepared using a peptide synthesizer (Biolynx 4170, LKB-Pharmacia Co., Ltd.). Each of the oligomers was purified by high performance liquid chromatography. Each of the purified oligomers was added to distilled water to make up to 1 pmol/ml. Seven z - 19 is lysine oligomer-containing solutions were thus prepared. (Assay of lipase inhibitory activity of lysine oligomers) The control and six test samples were prepared in the following manner.

pl. of an olive oil emulsion prepared in the same way as in Example 4 were used as a substrate.

pl of Tris buffer was added to the substrate to prepare an emulsion for control sample. Each 50 gl of the lysine oligomer-containing solutions as prepared above was added to 100 pl. of the substrate to prepare six emulsions for test sample. After incubating the emulsions for 5 min., there was added 50 pl. of a lipase solution containing 100 units of porcine pancreatic lipase per ml as prepared in the same way as in Example 4. The resulting mixture was incubated at 37C for one hour.

The results are shown in Table 6.

- Table 6

Lysine oligomer contained in samples Control a- (L-lysine) 2 a-(L-lysine) 4 (x-(L-lysine) 5 E-(L-lysine) 3 E-(L-lysine) 4 E-(L-lysine) 5 Activity of porcine pancreaticlipase 103.6 103.8 2.1 89.0 53.8 3.1 As seen from the above data, lysine oligomers of a type with the polymerization degree of 5 or more strongly inhibit lipase activity, whereas the oligomers with the polymerization degree of 4 or below have no lipase inhibitory activity at all under the specified in this example. Further, lysine oligomers of c type with the polymerization degree of 4 has a lipase inhibitory activity but not high enough, whereas the oligomer with the polymerization degree of 5 has very high lipase inhibitory activity. It is understood from Table 6 that lysine oligomer with higher degree of polymerization has greater lipase inhibitory activity than lower polymerized lysine.

It is reported that E-lysine oligomers with the polymerization degree of about 10 or higher possess strong antimicrobial property. From such report and the results shown in Table 6 it is anticipated that E-lysine oligomers 1 h having the polymerization degree of 5-9 which will produce no adverse effect upon intestinal flora can be used more effectively as a lipolytic enzyme inhibitor.

Claims

1. A lipolytic enyme inhibitor which comprises as an active ingredient a basiceprotein, abasic polypeptide or a salt thereof.

2. An inhibitor according to claim 1 comprising a basic protein which is histone or protamine.

3. An inhibitor according to claim 2 wherein the basic protein is histone HI, H11A, H2B, H3, H4 or H5, or mono,di-or triprotamine.

4. An inhibitor according to claim 1 comprising a basic polypeptide which is a purothionin, a purothionin analogue, a polylysine or a polyarginine.

5. An inhibitor according to claim 4 wherein the basic polypeptide is a,-, a2- or B-purothionin.

6. An inhibitor according to claim 4 whrein the basic polypeptide is aand B-polylysine.

7. An inhibitor according to claim 1 whrein the basic polypeptide is alysine oligomer having a polymeriation degree of 5 or more, c-lysine oligoner having a polymerization degree of 5 or more or a mixture thereof.

8. An inhibitor according to claim 1 comprising a salt of a basic polypeptide which is a-poly-L-lysine hydrochloride or a-poly-D-lysine hydrobr6mide.

9. A dieting agent comprising a lipolytic enzyme inhibitor according to any one of claims 1 to 8 protein.

10. An additive for food and feed comprising a lipolytic enzyme inhibitor according to any one of claims 1 to 8.

11. An inhibitor according to any one of claims 1 to 8 for use in delaying or preventing the enzymatic hydrolysis of lipids.

12. Use of an inhibitor according to any one of claims 1 to 8 in the preparation of a medicament for delaying or preventing the enzymatic hydrolysis of lipids.

is

13. An inhibitor according to claim 1 and substantially as hereinbefore described with reference to any one of the Examples.

Publ shed 1991 atThe Patent Office. State House. 66171 High Holborn. London WC1 R 4TP. Further copies may be obtained from 11 Sales Branch. UnAt 6. Nine Mile Point, cwmfelinfach. Cross Keys, Newport NPI 7HZ. Printed by Muluplex techniques lid. St Mary Cray. Kent.