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WO1991007439A1 - Regulation de la secretion de lait - Google Patents

Regulation de la secretion de lait Download PDF

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Publication number
WO1991007439A1
WO1991007439A1 PCT/GB1990/001743 GB9001743W WO9107439A1 WO 1991007439 A1 WO1991007439 A1 WO 1991007439A1 GB 9001743 W GB9001743 W GB 9001743W WO 9107439 A1 WO9107439 A1 WO 9107439A1
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WO
WIPO (PCT)
Prior art keywords
protein
milk
protein according
peak
kda
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/GB1990/001743
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English (en)
Inventor
Caroline Victoria Pauline Addey
Malcolm Peaker
Colin James Wilde
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BTG International Ltd
National Research Development Corp UK
Original Assignee
National Research Development Corp UK
British Technology Group Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by National Research Development Corp UK, British Technology Group Ltd filed Critical National Research Development Corp UK
Priority to JP90515417A priority Critical patent/JPH05506639A/ja
Publication of WO1991007439A1 publication Critical patent/WO1991007439A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/14—Drugs for genital or sexual disorders; Contraceptives for lactation disorders, e.g. galactorrhoea
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to a newly isolated protein from cow's milk and the use of the protein or antibodies thereto for the control of milk secretion in lactating animals.
  • the rate of milk secretion by a lactating animal is regulated by the frequency of milk removal.
  • studies by workers at the Hannah Research Institute, Ayr, Scotland on lactating goats have shown that another factor is involved. This is an inhibitor which decreases milk secretion at a local level, i.e. at the individual gland of an udder.
  • inhibitory activity is present in a 10-30 KDa fraction of whey from cow's milk, that this fraction can be separated by anion exchange FPLC (Fast Protein Liquid Chromatography) into a number of peaks and that the inhibitory activity is concentrated mainly in two particular peaks out of eight.
  • anion exchange FPLC Fast Protein Liquid Chromatography
  • One currently preferred definition is a protein which inhibits milk secretion by lactating cows and which is present in the second significant peak (labelled "2A" in Figure 1) when a nominally 10-30 KDa fraction of the whey proteins of the milk is resolved on an anion exchange column containing particles of monodisperse hydrophilic polymers having pendent -CH 2 N(CH 3 ) 3 + groups, the particle diameter being 10 ⁇ 0.5 ⁇ m, especially a "Mono Q" column, using 10 mM imidazole buffer, pH 7.0 and a sodium chloride elution gradient.
  • the molecular weight of the peak 2A protein as determined by gel filtration chromatography is about 7 KDa.
  • the peak 2A protein is further resolved, by gel filtration chromatography on a cross-linked agarose gel having a particle size of 10 ⁇ 2 ⁇ m, such as "Superose", the major component, which is that which appears second, comprises the inhibitory protein.
  • peak 2A (or its equivalent in which the whey is not fractionated before anion exchange chromatography) is resolved on a chromatofocussing column containing particles of monodisperse hydrophilic polymers having pendant tertiary (-N + HR 2 ) and quaternary (-N + R 3 ) amine groups where R represents an organic group, the particle diameter being 10 ⁇ 0.5 ⁇ m, especially a "Mono "P column, using 10 mM imidazole. pH 6.5 and amphoteric buffer of pH 4.0 to create a pH gradient of 6.5-4.0, the protein is present in the second significant peak ("2A.2").
  • any combination of one or more of the above features, together with the inhibitory action of the protein, might be sufficient to define the protein uniquely and accordingly applicant does not wish to be limited unnecessarily to specific combinations, in case one of them or some aspect of one of them might later be re-determined and found not sufficiently to approximate to their definition given above, while the remaining features are confirmed, and leave no doubt as to the identity of the protein.
  • Precisely which features are the most meaningful and the most reliable are, in any case, a matter of judgement, the preferred definitions given above reflecting applicant's current judgement. It will be appreciated, therefore, that the protein defined by other combinations of features herein set forth is to be considered as encompassed by the invention.
  • a property which might be useful for defining the protein is the isoelectric point (pl). It has been found that peak 2A gives a pl in the range 4.8 to 5.0 when determined in a tube of polyacrylamide gel, while peak 2A.2 gives a pi of 4.9-5.0 determined by isoelectric focussing.
  • the protein can be in glycosylated or unglycosylated form.
  • Antibodies to the protein, whether polyclonal, monoclonal or engineered, are within the scope of this invention.
  • the administration of the inhibitor to decrease milk yield or an antibody thereto to suppress at least partly the action of the inhibitor, to cows or other animals is within the invention.
  • Figure 1 shows the resolution of the 10-30 KDa fraction by anion-exchange chromatography:
  • Figure 2 and 3 show the further resolution of two of the peaks of the Figure 1 anion exchange chromatography by gel filtration chromatography
  • Figure 4 shows the further resolution of one of the peaks obtained from anion-exchange chromatography by chromatofocussing. Description of the preferred embodiments
  • the protein of the invention exists in cow's milk, probably in glycosylated form. It is believed that the effect of glycosylation is simply for attachment of the protein to the appropriate cells within the mammary gland. It would be expected, therefore, that the protein could be administered locally to the gland in an unglycosylated form.
  • Antibodies can be raised against the protein of the invention by any conventional methods, e.g. as polyclonal antisera, mouse monoclonal antibodies, cow-mouse hybrid monoclonal antibodies or as engineered antibodies, by any of the currently available methods. Passive immunisation methods can then be used to generate a reduction in the effect of the natural inhibitor, when this is desired in order to increase milk yield. Frequently, however, there will be a need to reduce milk yield in order to meet milk quotas, in which event the inhibitor Itself is administered. Conventional carriers and adjuvants known in vaccination can be used.
  • the invention is applicable to any animal responsive to the inhibitor defined herein. Since the 10-30 KDa goat's milk fraction has been successfully found to reduce milk accumulation and relevant enzyme activities when injected into the mammary gland of rabbits, it is likely that the cow's milk inhibitor will be effective in some other lactating animals.
  • a dose in the range of 1 to 50 ⁇ g, especially 5 to 20 ⁇ g of inhibitor is likely to be effective and should be repeated as required, e.g. daily, and possibly reduced when given over long periods.
  • the protein of the invention can be obtained from cow's milk by the method described in the Example or some variant thereon. It can be recovered in pure form from an eluate by extensive dialysis against water (using an appropriate membrane for retention of the protein, e.g. with a nominal molecular weight cut-off of about 6 KDa) and freeze-drying. However, it is expected that it would be synthesised by protein synthesis or by a recombinant DNA method.
  • This Example describes the preparation and properties of the inhibitor of the invention.
  • the whey fraction was subjected to ultrafiltration using a filter with a nominal cut-off value of molecular weight 30,000 Dal tons (Da).
  • the filtrate obtained with the 30,000 Da filter was concentrated by ultrafiltration with a 10,000 Da filter.
  • the 10,000-30,000 Da fraction was dialysed extensively against water, sterilized by filter sterilization and concentrated by freeze-drying for anion exchange chromatography.
  • the 10-30 KDa whey fraction was resolved on a "MonoQ HR 10/10" anion exchange column (Pharmacia) using an "FPLC” chromatography system (Pharmacia).
  • the whey fraction was dissolved in 10 mM imidazole at four times its concentration in the original milk and the pH adjusted to 7.0. Before chromatography, the sample and buffer (degassed) were filtered through 0.2 ⁇ m filters. 1 ml of the 4 x concentrated whey fraction was loaded for each separation; the flow rate was 4.0 ml/min. A sodium chloride elution gradient was used.
  • Fractions containing protein peaks eluted from the column were dialysed extensively against distilled water, freeze-dried and stored at -20°C, before use in the next stage.
  • Figure 1 of the drawings shows the elution of protein from the chromatography column. Protein concentration, as absorption of light at 280 nm, on the left-hand ordinate is plotted against cumulative volume of eluted material on the abscissa. The plot is shown as lines. The right-hand ordinate is calibrated to show the sodium chloride gradient, from 0 to 1.0M. used in the eluant. The broken line is a plot of the sodium chloride concentration.
  • Mammary tissue was cultured as explants, small pieces of parenchymal tissue approximately lcm 3 and weighing 0.5-0.7 mg. Explants were prepared from mammary tissue of mid-pregnant New Zealand White rabbits as described by R. Dils & I.A. Forsyth in Methods in Enzymology 72 , 724-742 (1981). The explants were cultured in a defined culture medium (Medium 199; Gibco Europe Ltd., Paisley, UK) on stainless steel grids each holding 30 explants, so that the explants were in contact with the medium but not completely submerged in it.
  • a defined culture medium Medium 199; Gibco Europe Ltd., Paisley, UK
  • the medium was supplemented throughout with insulin (5 ⁇ g/ml), cortisol (100ng/ml) and prolactin (l ⁇ g/ml).
  • Explants were cultured in this medium under an atmosphere of air/CO 2 (19:1 v/v) for 42 h, with replenishment of medium after 24 h.
  • groups of explants (3 or 4 groups per treatment) were transferred into fresh medium containing hormones and one of the fractions of cow milk under test.
  • the milk fractions tested in this experiment were obtained from cow's milk whey which had not been ultrafiltered (as distinct from the 10-30 KDa fraction referred to above), but which had been fractionated by anion exchange chromatography as described above.
  • Explants were homogenized at 4°C in 1.0 ml of 10mM Tris/ Hll, pH 7.0, containing 5mM ethyl eneglycol-bis-(2-aminoethyl ether) N,N,N' ,N'-tetraacetic acid (EGTA) and 2mM phenylmethane- sulphonyl fluoride by 10 strokes with a glass/PTFE homogenizer, followed by soni cation for 30s (Kontes ultrasonic cell disruptor, 30% maximum power), and a particle-free supernatant was prepared by centrifugatlon at 10,000g for 5 min.
  • EGTA ethyl eneglycol-bis-(2-aminoethyl ether) N,N,N' ,N'-tetraacetic acid
  • EGTA ethylmethane- sulphonyl fluoride
  • casein was prepared from the particle-free supernatant by precipitation at its isoelectric point, and the precipitate was subjected to SDS-polyacryl amide gel electrophoresis, as described by C.J. Wilde et al., Exp. Cell Res. 151, 519-532 (1984). Bands corresponding to casein polypeptides were visualized by staining with Coomassie brilliant blue, and were excised and counted for [ 3 H] radioactivity as described by S.M. Russell et al., Biochim. Biophys. Acta 714, 34-45 (1982).
  • [ 14 C] lactose was selectively precipitated from explant homogenates and culture medium using ethanol/diethyl ether (3:1, v/v), H.3. Kuhn & A. White, Biochem. J . 148, 77-84 (1975) and the radioactivity of the precipitate counted. Results were corrected for carry-through of [ 14 C] glucose from culture medium (usually ⁇ 0.08%), by measuring [ 14 C] radioactivity after extraction of uncultured medium. The addition of milk fractions did not affect the distribution of secreted products between the extracellular space of the explants and the medium.
  • the amount of radioactive material (casein and lactose) was expressed as a percentage of that produced by the explants to which no milk fraction had been added. The results are shown in Table 1. The figures in parenthesis are the numbers of experiments performed on various peaks.
  • peak 2 was the more active than the others in inhibiting lactose synthesis, a major determinant of milk yield, and casein synthesis.
  • Results are the mean ⁇ s.e.m. for 3 experiments. It will be seen that both fractions were inhibitory to some extent, although lack of clear separation of the inhibitor component between peaks 2A and 2B accounts for some of these effects.
  • peak 2A is resolved into three separated peaks and a shoulder downstream of the first main peak.
  • the components of m.w. 7 and 4 KDa were assigned to the first main peak and its shoulder respectively.
  • the 7 KDa component is clearly the "main one of peak 2A.
  • Peak 2B was resolved into three separated peaks and a shoulder upstream of the main peak, the components of m.w. 7 and 4 KDa being assigned to the shoulder and main peak respectively.
  • the molecular weights of the principal components were determined to be about 7 and 4 KDa. (An attempt at m.w. determination by SDS-PAGE gave anomalous results: it appears that high molecular weight aggregates form).
  • the unexpectedly low mol ecul ar weights can probably be explained by clogging of the nominally 10,000 Dalton filter during ultrafiltration, allowing smaller molecules to be retained.
  • Isoelectric focussing was performed in tube gels (diameter, 4mm; length 11.5 cm).
  • 4% polyacryl amide gels were prepared essentially as described by P.H. 0'Farrell, 3. Biol. Chem. 250. 4007-4021 (1975) using a mixture of ampholines (4% v/v pH range 5-8; 1% v/v pH range 3.5-10; BioRad), which gave a linear gradient in the range 4.0-9.0.
  • Samples 25 ⁇ g of the peak 2 protein
  • Samples (25 ⁇ g of the peak 2 protein) were dissolved in a solution containing 9.5M urea, 2% (w/v) NP40, 1.6% (v/v) pH 5-8 ampholines and 0.4% (v/v) pH 3.5-10 ampholines.
  • the anodic and cathodic solutions were 10 mM H 3 PO 4 and 20 mM NaOH respectively. Electrophoresis was at 300 V for 18 h, followed by 400 V for 4 h. Gels were extruded and fixed first in 25% (v/v) isopropanol/10% (v/v) acetic acid, then in 5% (w/v) TCA/5% (w/v) sulphosalicylic acid/1% (v/v) methanol, and were stained 25% (v/v) isopropanol/10% (v/v) acetic acid containing 0.1% (w/v) Coomassie Blue. Destaining was In isopropanol/acetic acid.
  • the major band has an isoelectric point of 4.84; the minor band focusses at an isoelectric point of 4.92.
  • Chromatofocussing separates proteins on the basis of their isoelectric point (pl). Resolution with the Pharmacia "Mono P HR 5/20" column is such that molecules differing in pi by only 0.02 pH units can be separated.
  • “Mono P” is a weak anion exchanger, based on monobeads, i.e. monodisperse hydrophilic polymer particles (10 ⁇ 0.5 ⁇ m diameter) into which various tertiary (-N + HR 2 ) and quaternary (-N + R 3 ) amine groups are introduced.
  • “Mono P” has a buffering capacity and the amount of charge it carries will vary with pH. Consequently, its ionic capacity will also vary with pH.
  • a pH gradient is formed on the column by equilibrating it with start buffer and eluting with another buffer which is added in increasing amounts, thereby adjusting the solution progressively to a lower pH. Proteins bound to the column at the starting pH are eluted at different points on the pH gradient according to their pl.
  • a "Mono P" column was pre-washed with 1 ml of 1 M NaOH, and the equilibrated in .10 mM imidazole, pH 6.5.
  • the sample 50 ⁇ g of ultrafiltered bovine whey protein, peak 2A, obtained by anion-exchange chromatography as described in section 2 of this Example, was applied once the pH of the column eluant had returned to 6.5.
  • the column was eluted with a 1/10 v/v dilution of "Polybuffer” 74, pH 4.0 in distilled water.
  • "Polybuffer” contains numerous amphoteric buffering substances of different pKa. Buffers were filtered through 0.2 ⁇ m filters and degassed before use. Elution was carried out at a flow rate of 0.5 ml/min, until the pH of the eluant was 4.5. Protein eluting from the column was detected by monitoring absorbance of the eluant at 280 nm.
  • Peak 2A eluted as three major (2A.1, 2A.2 and 2A.6) and three minor peaks (2A.3, 2A.4 and 2A.5) as shown in Figure 4.
  • the relative abundance of these components varies between separations. However, the separation is qualitatively reproducible in terms of the relative positions of the peaks.
  • the second major peak Peak 2A.2 which elutes at pH 5.0-4.9 is that which predominantly contains the inhibitor (see section 7 below).
  • the bioassays were carried out as described in section 3.
  • the samples tested in this experiment had been fractionated by chromatofocussing of ion-exchange peak 2A as described in section 5. They were dissolved in 10mM Hepes. pH 7.4, at twice their concentration in the original milk and added to an equal volume of twice concentrated culture medium so as to be at 100% of their original milk concentration in normal strength culture medium. Control cultures containing only the milk fraction diluent were included in each experiment. Average rates of lactose and caesin synthesis were measured as described previously (section 3).
  • the amount of radioactive material was expressed as a percentage of that produced by the explants to which no milk fraction was added.
  • the results are shown in Table 3 below.
  • the figures in parenthesis are the numbers of experiments performed on the various peaks. Results are the mean ⁇ s.e.m. where three experiments were carried out.
  • Peak 2A.2 (section 7) was carried out using an FPLC chromatography system and a Superose 12 HR10/30" column (Pharmacia), as described in section 4 for the anion-exchange peaks.
  • Peak 2A.2 eluted as the sole protein-containing peak.
  • the other peaks contained no detectable protein and were of low molecular weight ( ⁇ 1kDa).
  • the molecular weight of Peak 2A.2 was calculated to be about 7.0 kDa which was the molecular weight estimated for anion-exchange peak 2A in section 4.
  • PhaseGel IEF PhaseGel IEF 4-6.5.
  • PhaseGel IEF media are homogeneous polyacryl amide gels containing "Pharmalyte” carrier ampholytes.
  • “Pharmalyte” generates stable, linear pH gradients in the gels during electrophoresis, in this case in the pH range 4 to 6.5. Proteins migrate under an electric field, essentially unhindered by the porous gel, to a point in the pH gradient that corresponds to their pi (isoelectric point).
  • Peak 2A.2 (section 6) which had been extensively dialysed against distilled water was applied to one well of the gel, "Pharmacia pi Calibration Kit” proteins were applied to wells on both sides of the sample well.
  • the resulting protein bands were visualised by staining with Coomassie blue after electrophoresis. Peak 2A.2 gave rise to a single protein band corresponding to pi 4.9-5.0.

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Abstract

Protéines inhibant la sécrétion de lait par des vaches allaitant, et présente dans la seconde crête significative (2A) lorsqu'une fraction nominale de 10 à 30 kDa des protéines de lactosérum dulait est dissoute dans une colonne d'échange anionique ''mono Q'', utilisant un tampon d'imidazole de 10 mM, un pH de 7,0 et un gradient d'élution de chlorure de sodium.
PCT/GB1990/001743 1989-11-13 1990-11-13 Regulation de la secretion de lait Ceased WO1991007439A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP90515417A JPH05506639A (ja) 1989-11-13 1990-11-13 乳の分泌の調整

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB8925595.4 1989-11-13
GB898925595A GB8925595D0 (en) 1989-11-13 1989-11-13 Control of secretion of milk

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WO1991007439A1 true WO1991007439A1 (fr) 1991-05-30

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PCT/GB1990/001743 Ceased WO1991007439A1 (fr) 1989-11-13 1990-11-13 Regulation de la secretion de lait

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EP (1) EP0575311A1 (fr)
JP (1) JPH05506639A (fr)
AU (1) AU645272B2 (fr)
CA (1) CA2068533A1 (fr)
GB (2) GB8925595D0 (fr)
IE (1) IE64841B1 (fr)
WO (1) WO1991007439A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992020714A1 (fr) * 1991-05-10 1992-11-26 British Technology Group Ltd. Controle de la secretion de lait chez les vaches laitieres
US5496802A (en) * 1991-05-10 1996-03-05 British Technology Group Ltd Control of milk secretion
WO1998023667A1 (fr) * 1996-11-26 1998-06-04 Dr. Beck & Co. Ag Procede de preparation de polyester-imides contenant des groupes carboxyle et hydroxyle et leur utilisation dans des vernis pour fils de fer

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2255562B (en) * 1991-05-10 1994-11-09 Nat Res Dev Bovine protein inhibitor for control of milk secretion

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB8925594D0 (en) * 1989-11-13 1990-01-04 Nat Res Dev Control of milk secretion

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
Dialog Information Services, File 15 S: Medline, NLM accession number 89234827, Butler WR et al: "J Dairy Sci (UNITED STATES) Mar 1989, 72 (3) p767-83 *
Dialog Information Services, File 155: Medline NLM accession number 88289973, Wilde CJ et al: "Feed- backinhibition of milk secretion: the effect of a fraction of goat milk on milk yield and composition"& Q J Exp Physiol (ENGLAND) May 1988, 73 (3) p391-7 Cited in the application *
Dialog Information Services, File 155: Medline NLM accession number 89375505, Wilde CJ et al: "Regulation of intracellular casein degradation by secreted milk proteins", & Biochim Biophys Acta (NETHERLANDS) Sep 15 1989, 992 (3) p315-9 Cited in the application *
Dialog Information Services, file 155: Medline, NLM accession number 86141121, Manji B et al: "Rapid separation of milk whey proteins by anion exchange chromatography", & J Dairy Sci Dec 1985, 68 (12) p3176-9 *
Dialog Information Services. File 10: Agricola, Dialog accession number 87047695, Hill, A.R. et al: "Separation and quantification of whey proteins by size exclusion chromatography", & Canadian Instituteof Food Science and Technology journal. Dec 1986. v.19 (5) p. 227-230 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992020714A1 (fr) * 1991-05-10 1992-11-26 British Technology Group Ltd. Controle de la secretion de lait chez les vaches laitieres
US5496802A (en) * 1991-05-10 1996-03-05 British Technology Group Ltd Control of milk secretion
WO1998023667A1 (fr) * 1996-11-26 1998-06-04 Dr. Beck & Co. Ag Procede de preparation de polyester-imides contenant des groupes carboxyle et hydroxyle et leur utilisation dans des vernis pour fils de fer
US6211326B1 (en) 1996-11-26 2001-04-03 Schenectady International, Inc. Method for the production of polyester imides containing carboxyl- and hydroxyl groups and their usage in wire enamels

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AU6645390A (en) 1991-06-13
IE904087A1 (en) 1991-05-22
JPH05506639A (ja) 1993-09-30
IE64841B1 (en) 1995-09-06
GB9024649D0 (en) 1991-01-02
GB2238052A (en) 1991-05-22
CA2068533A1 (fr) 1991-05-14
AU645272B2 (en) 1994-01-13
GB2238052B (en) 1993-09-29
GB8925595D0 (en) 1990-01-04
EP0575311A1 (fr) 1993-12-29

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