CA1170991A - Products prepared from the serum thymic factor or its analogs and derivatives, a process for preparing these products, the use of these products as medicaments, tracers obtained from these products and the use of these tracers in detection processes - Google Patents
Products prepared from the serum thymic factor or its analogs and derivatives, a process for preparing these products, the use of these products as medicaments, tracers obtained from these products and the use of these tracers in detection processesInfo
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- CA1170991A CA1170991A CA000378037A CA378037A CA1170991A CA 1170991 A CA1170991 A CA 1170991A CA 000378037 A CA000378037 A CA 000378037A CA 378037 A CA378037 A CA 378037A CA 1170991 A CA1170991 A CA 1170991A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
- C07K7/062—Serum thymic factor
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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Abstract
ABSTRACT
The invention relates to new products prepared from serum thymic factor (Facteur Thymique Serique-FTS) or from its analogs and derivatives, to the use of these products as medicaments and to the use of these products for the preparation of tracers for radio-immunological detection processes.
These new products are prepared by treating FTS or its analogs and derivatives with a metal or a metal-containing protein substance.
The products thus obtained show increased biological activity in relation to the products from which they are derived.
These products may be used as medicaments for the treatment of illnesses attributable to an unbalance of T lymphocytea.
The invention relates to new products prepared from serum thymic factor (Facteur Thymique Serique-FTS) or from its analogs and derivatives, to the use of these products as medicaments and to the use of these products for the preparation of tracers for radio-immunological detection processes.
These new products are prepared by treating FTS or its analogs and derivatives with a metal or a metal-containing protein substance.
The products thus obtained show increased biological activity in relation to the products from which they are derived.
These products may be used as medicaments for the treatment of illnesses attributable to an unbalance of T lymphocytea.
Description
11 7~9~t 1 1 l1ais invention reLate~ ~o ne~/ products prepared from the serulll th~llic factor (l`TS) or its an.llogs and derivatives, to a process for their preparation and to their use as medicaments.
Tne invention also relates to radioactive tracers obtained frorn tllese ncw products and ~o the use of these tracers in a pro~ess for the radio-immunoJogical detection of lTS in a sample.
Fl`S is a nonapeptide isolated from pig's ser~n and present in the thymus and tlle nonnal serum of various species.
The thymic origin of FTS, suggested by the disappear<mce of ITS from the ser~n after thyrnectomy, ~as recently directly demonstrated by the localisation by immunofluorescence in the thymic epithelium of purified antibodies produced against synthetic I:TS.
FTS llas numerous in vitro and in vivo biological activities, including in particular the induction of the tlleta antigen characteristic of the T lympllocytes in mice in theta-negative cells of bone marrow and spieen from thymectomised mice wllich fonn rosettes.
FTS has been characterised ~y a ~iological test, commonly kno~ as the "rosette test", wllich is based on induction of the sensitivity to azathioprine of rosette-forming cells from the spleen of thymectomised mice. The rosette test was described by J.F. ~CII .~nd I~I. DA2~ENNE in ~ OLOGY, 25, 353 (1973).
This test is based on the ability of l`TS to render theta-positive and azathioprine-sensitive tlle theta-negative rosette-formillg cells obtained frorl- the spleen of adult th~nectomised mice. Azathioprine is a purine analog whicll, like anti-tlleta ser~n, inhibits tlle formation of tll~nus-~ependent rosette-fonning ce]ls. In normal rnice, rosette forlllation is inhibited by 1 ~g/ml of azatllioprine wllereas, ~.
1:~.7(3~1 in adult thymectomised mice, from 50 to 100 ~g/ml of this compound are necessary to inhibit rosette formation. FTS as well as the thymic extracts imparts to the rosette-forming cells of adult thymectomised mice, after incubation for 90 minutes at 37C, a sensitivity to azathioprine identical with that of the rosette-forming cells of normal mice, which may be used as a basis for a reproducible and quantitative in vitro and ln vivo biological test.
In the rosette test, the thymic activity of a sample (FTS or thymic extract or a serum sample) is determined by incubation in a haemolysis tube with 3 x 106 cells from the spleen of adult C 57/B1 6 mice (supplied by the Centre d'Elevage des Animaux de Laboratoire du C.N.R.S. 45 Orleans, La Source) which had been thymectomised ten to twenty days previously.
The thymectomy method is described by M. DARDENNE and J.F. BACH
in Immunology 25, 343 (1973) on page 344.
Incubation is carried out for 90 minutes at 37C in the presence of azathioprine (Az) in a concentration of 10 ~g/ml.
This concentration is intermediate between the minimal Az concentration which inhibits 50% of the rosette-forming cells (RFC) from the spleen of normal mice (1 ~g/ml) and that which inhibits the rosette-forming cells of thymectomised animals (50 ~g/ml). After incubation, 12 x 106 sheep red blood cells (SRBC) are added to the tubes containing the cells and the sampleO The cells and the sample are centrifuged for six minutes at 1400 revolutions per minute and are resuspended gently and care-fully by rotation on a rotary agitator (10 cm in diameter) at low speed (10 revolutions per minute).
117()~
1 ~he l~C are counted i.n a hae[natocytolneter. In the absence of th~nic activity, the nurnber oL Rl;C is 1210/10 cells - 120 (standard deviation, Sv). In the presence of thylnic activity, tlle guantity oE RFC is reduced to a level of 200 to 400/10 cells. In the absence of Az, the lTS
does not inhibit the l~IC.
In vitro rosette test: The th~-nic activity is defined as the lot~7est concentration of the sample ~hich induces inhibition o.~ more than 50~10 of the rosette-forming cells from the spleen of adult thymectomised mice in the presence of 10 ~-g/ml of aæathioprine.
In vivo: a) The activity of the serurn is expressed as the thymic factor level (determined by the rosette test described a~ove) in the serum of adult th~nectomised mice collected two or four hours after the injection of 0. 1 Tlg or 1 ng of sc~ple ~bsorbed on carboxymethyl cel].ulose or injected alone: the activity of the scr~n is expressed as tlle highest dilution of the serum allo~7ing the inhibition of 5~/o of the rosette-forming cells.
b) The activity of the spleen cells is expressed as the sensitivity to azathioprine of these cells of adult thymectomised mice isolated 24 hours after the injection of 0.1 ng to 1 ng of sample absorbed on carboxymethyl cellulose or injected alone.
1.7e have no~? found that there is no correlation bet~een the activities observed in the in vitro and in vivo tests and tha~ it is possible to obtain products ~.7hich are more active t'nan those obtained hitllerto particularly in vivo.
I~le have sllown that, according to the methods employed, the chelnical synthesis of lTS can possibly result in a product having an in vitro and itl vivo 7~iological activity t~7hich is ~luantitatively inferior (up to 100 and 117~
1 1000 times) to tllat o~served wi~ll I`TS o~ natural origin.
0ll tlle o~her hand, ~e have Eound that thc treatrnent of the syn~lletic lrS l~y an agent adapted to cllelate metals, sucll as a chelating resin, rcsults in a product ~hich has completely lost its in vitro and in vivo biological activity.
We have been able to show, by means of atomic absorption spectrometry analysis o the different batches of FTS, that the active batches contained smal.l quantities of heavy metals, in particular zinc, whereas the inactive batches were completely free from such materials.
The essenti.al object of the invention is the preparation of new products from ~TS or its analogs and derivatives in order to obtain products of constant quality ~hich may be used either as a medicament or in the preparation of tracers or radio-imllluIlological detention processes.
l`he invention relates more particularly to net~
products characterised in that ~TS or one of its analogs or derivativcs is combined ~ith a metal t~hich is capable of forming a IV co-ordination complex having a tetrahedral shape.
The metal which is involved may be a diva].ent metal, sucl- as zinc or copper, or a trivalent metal such as aluminium, but zinc is the preferred rnetal.
The quantity of metal which is in the complexes o the invention, as determined ~y atomic spectrometry, must be such that the weigllt ratio of metal to the complex is greater than 1/200.
l`he products of the invention have an in vivo ~h~-ni.c activity in the rosette test mentioned above wllicll is multiplied by a factor of at least ten or even, ~'7~991 1 usually of c~t least 100 witl~ respect to the activity of ~llc rcference l'TS (l~`'l`S of natural or symthetic origin as outained previously).
~`.ccording to the invention, the product as described herein, in wllicll the mctal is zinc, injec~ed in a dose of 0.1 ng per mouse, has an in vivo cactivity in tlne rosette test whicll is ten times greater than that obtained for the reference ~''l`S injected in a dose of 1 ng per mouse.
'l'he invention also relates to a process for the preparation of new products prepared from FTS or its analogs or derivatives, which comprises treating FTS
or one of its analogs or derivatives with a metal or a metal-containing protein.
In a pr~ferred embodiment of the invention, the FTS
lS or one of its analogs or derivatives is brough~ into contact with the mctal or one of its salts at a pil of between 7 and 9 for ten to thirty minutes at ambient temperature, for example from 20 to 37OC.
In this cmbodiment, it is of advantage to use a proportion of 1 nole of FTS or an analog or derivative of FTS for 0.1 to 10 moles of the metal or of one of its salts, preferably 1 mole of the Metal or of one of its salts.
In another embodiment of the process according to the invention, the new products are prepared by treating l`TS or one or its analogs or derivatives with a metal-containing protein substance.
This metal-containing protein substance may be for example a ser~un fraction or a fraction of a thymic extract.
For e~ample, the metal-containing protein substance used may be a frac~ion of the Thymosine iraction 5 l~licll will be referred hereinatcr as thc 'lactivating fraction" by 9~
1 virtue oE its activating properties with respect to lTS
or its ana]ogs and derivatives.
In 1965, ~.L. GOL~ST~IN et. a]. isolated from calf thylllus a more or less impure preparation ~hich they called Thyrnosirle lraction 5 and ~ iCh iS a mixture of n~nerous polypeptides having various biological activities.
This preparation has been used in humans in pllase 1 and 2 clinical ~rials ~ith as yet inconclusive, but promising results.
i~ore recently, A.L. GOLDSTEII~ pu~lished the se~luence of certain pel~tides contained in the Fraction 5, na!nely 1' ~1' ~ but none of these peptides has been ound to show any structural similarity to l`TS.
Our o~n prelirninary studies l~ave shown that tlle Thymosine lraction 5 contained one or more substances active in the above-mentioned rosette test and having physical or cnernical ~roperties comparable ~ith tllose of FTS (molecular weigllt, electrical charge, aminoacid composition).
The above-mentioned activating fraction is prepared by fractionating Thymosine lraction 5 in a colurnn of G-100 (Pharmacia 0.9 crn x 100 cm) in a pl-l 7.3 0.2 l~
phospllate buffer to eliminate the molecule(s) slmilar to FTS, only the eluted fractions ~ith elution volurnes of from 15 to 22 ml being retained.
This fraction contains molecules having a rnolecular ~eight in the range from 7500 to 12500 (tracers used:
IGG, bovine albumin, Cytochrome C and tritiated ~rs).
This fraction is sensitive to heat: its activity is destroyed by heating to 60OC in less than 15 minutes and to 100oC in less than 5 minutes.
l`his fr~ction, whicll itself has no activity in 9~1 the rosette test,shows activating properties with respect to FTS or its analogs and derivatives insofar as it enables their biological activity to be increased by a factor of approximately 103.
, The process for preparing the new products from the activating fraction emanating from the Thymosine Fraction 5 is characterised in that FTS or one of its analogs or derivatives is incubated with this activating fraction, the mixture obtained after incubation is fractionated and the fraction showing the greatest activity in the rosette test is selected.
In this process, the incubation temperature is in the range from 30 to 40C, the incubation time is between 10 minutes and 1 hour and incubation is carried out at a pH-value in the range from 6 to 8.
The particularly preferred conditions comprise carry-ing out incubation for 20 minutes at a temperature of approx-imately 37 C and at a pH value of 7.3.
Irrespective of the process used (treatment with a metal or metal salt or treatment with a metal-containing protein substance), it is preferable to eliminate at the out-set any trac~ of metal which may be present in the preparation of FTS or one of its analogs or derivatives by means of a chelating agent capable of chelating metals, for example by chromatography in a column of chelating resin of the "Chelex*
100" type. This allows a chemically homogeneous product to be obtained which is biologically inactive and which, after being treated with the metal or one of its salts, produces a homogeneous product with reproducible results, these products thus being acceptable for pharmaceutical application.
The FTS or the FTS analogs or derivatives which are *A Trade Mark 1~7~ 9~
used in the ~rocess according to the invention are those which have been synthesised in the liquid phase by the method ,l described by BRICAS, E., MARTINEZ J., BLANOT, D., AUGER, G., DARDENNE, M., PLEAU, J.M., and BACH, J.F., 5th Intern. Peptide Symposium, La Jolla, California, U.S.A., June 1977, and those claimed in French Patent Application No. 77 15963 filed 25th May, 1977 and published December 15, 1978 as French Patent No.
Tne invention also relates to radioactive tracers obtained frorn tllese ncw products and ~o the use of these tracers in a pro~ess for the radio-immunoJogical detection of lTS in a sample.
Fl`S is a nonapeptide isolated from pig's ser~n and present in the thymus and tlle nonnal serum of various species.
The thymic origin of FTS, suggested by the disappear<mce of ITS from the ser~n after thyrnectomy, ~as recently directly demonstrated by the localisation by immunofluorescence in the thymic epithelium of purified antibodies produced against synthetic I:TS.
FTS llas numerous in vitro and in vivo biological activities, including in particular the induction of the tlleta antigen characteristic of the T lympllocytes in mice in theta-negative cells of bone marrow and spieen from thymectomised mice wllich fonn rosettes.
FTS has been characterised ~y a ~iological test, commonly kno~ as the "rosette test", wllich is based on induction of the sensitivity to azathioprine of rosette-forming cells from the spleen of thymectomised mice. The rosette test was described by J.F. ~CII .~nd I~I. DA2~ENNE in ~ OLOGY, 25, 353 (1973).
This test is based on the ability of l`TS to render theta-positive and azathioprine-sensitive tlle theta-negative rosette-formillg cells obtained frorl- the spleen of adult th~nectomised mice. Azathioprine is a purine analog whicll, like anti-tlleta ser~n, inhibits tlle formation of tll~nus-~ependent rosette-fonning ce]ls. In normal rnice, rosette forlllation is inhibited by 1 ~g/ml of azatllioprine wllereas, ~.
1:~.7(3~1 in adult thymectomised mice, from 50 to 100 ~g/ml of this compound are necessary to inhibit rosette formation. FTS as well as the thymic extracts imparts to the rosette-forming cells of adult thymectomised mice, after incubation for 90 minutes at 37C, a sensitivity to azathioprine identical with that of the rosette-forming cells of normal mice, which may be used as a basis for a reproducible and quantitative in vitro and ln vivo biological test.
In the rosette test, the thymic activity of a sample (FTS or thymic extract or a serum sample) is determined by incubation in a haemolysis tube with 3 x 106 cells from the spleen of adult C 57/B1 6 mice (supplied by the Centre d'Elevage des Animaux de Laboratoire du C.N.R.S. 45 Orleans, La Source) which had been thymectomised ten to twenty days previously.
The thymectomy method is described by M. DARDENNE and J.F. BACH
in Immunology 25, 343 (1973) on page 344.
Incubation is carried out for 90 minutes at 37C in the presence of azathioprine (Az) in a concentration of 10 ~g/ml.
This concentration is intermediate between the minimal Az concentration which inhibits 50% of the rosette-forming cells (RFC) from the spleen of normal mice (1 ~g/ml) and that which inhibits the rosette-forming cells of thymectomised animals (50 ~g/ml). After incubation, 12 x 106 sheep red blood cells (SRBC) are added to the tubes containing the cells and the sampleO The cells and the sample are centrifuged for six minutes at 1400 revolutions per minute and are resuspended gently and care-fully by rotation on a rotary agitator (10 cm in diameter) at low speed (10 revolutions per minute).
117()~
1 ~he l~C are counted i.n a hae[natocytolneter. In the absence of th~nic activity, the nurnber oL Rl;C is 1210/10 cells - 120 (standard deviation, Sv). In the presence of thylnic activity, tlle guantity oE RFC is reduced to a level of 200 to 400/10 cells. In the absence of Az, the lTS
does not inhibit the l~IC.
In vitro rosette test: The th~-nic activity is defined as the lot~7est concentration of the sample ~hich induces inhibition o.~ more than 50~10 of the rosette-forming cells from the spleen of adult thymectomised mice in the presence of 10 ~-g/ml of aæathioprine.
In vivo: a) The activity of the serurn is expressed as the thymic factor level (determined by the rosette test described a~ove) in the serum of adult th~nectomised mice collected two or four hours after the injection of 0. 1 Tlg or 1 ng of sc~ple ~bsorbed on carboxymethyl cel].ulose or injected alone: the activity of the scr~n is expressed as tlle highest dilution of the serum allo~7ing the inhibition of 5~/o of the rosette-forming cells.
b) The activity of the spleen cells is expressed as the sensitivity to azathioprine of these cells of adult thymectomised mice isolated 24 hours after the injection of 0.1 ng to 1 ng of sample absorbed on carboxymethyl cellulose or injected alone.
1.7e have no~? found that there is no correlation bet~een the activities observed in the in vitro and in vivo tests and tha~ it is possible to obtain products ~.7hich are more active t'nan those obtained hitllerto particularly in vivo.
I~le have sllown that, according to the methods employed, the chelnical synthesis of lTS can possibly result in a product having an in vitro and itl vivo 7~iological activity t~7hich is ~luantitatively inferior (up to 100 and 117~
1 1000 times) to tllat o~served wi~ll I`TS o~ natural origin.
0ll tlle o~her hand, ~e have Eound that thc treatrnent of the syn~lletic lrS l~y an agent adapted to cllelate metals, sucll as a chelating resin, rcsults in a product ~hich has completely lost its in vitro and in vivo biological activity.
We have been able to show, by means of atomic absorption spectrometry analysis o the different batches of FTS, that the active batches contained smal.l quantities of heavy metals, in particular zinc, whereas the inactive batches were completely free from such materials.
The essenti.al object of the invention is the preparation of new products from ~TS or its analogs and derivatives in order to obtain products of constant quality ~hich may be used either as a medicament or in the preparation of tracers or radio-imllluIlological detention processes.
l`he invention relates more particularly to net~
products characterised in that ~TS or one of its analogs or derivativcs is combined ~ith a metal t~hich is capable of forming a IV co-ordination complex having a tetrahedral shape.
The metal which is involved may be a diva].ent metal, sucl- as zinc or copper, or a trivalent metal such as aluminium, but zinc is the preferred rnetal.
The quantity of metal which is in the complexes o the invention, as determined ~y atomic spectrometry, must be such that the weigllt ratio of metal to the complex is greater than 1/200.
l`he products of the invention have an in vivo ~h~-ni.c activity in the rosette test mentioned above wllicll is multiplied by a factor of at least ten or even, ~'7~991 1 usually of c~t least 100 witl~ respect to the activity of ~llc rcference l'TS (l~`'l`S of natural or symthetic origin as outained previously).
~`.ccording to the invention, the product as described herein, in wllicll the mctal is zinc, injec~ed in a dose of 0.1 ng per mouse, has an in vivo cactivity in tlne rosette test whicll is ten times greater than that obtained for the reference ~''l`S injected in a dose of 1 ng per mouse.
'l'he invention also relates to a process for the preparation of new products prepared from FTS or its analogs or derivatives, which comprises treating FTS
or one of its analogs or derivatives with a metal or a metal-containing protein.
In a pr~ferred embodiment of the invention, the FTS
lS or one of its analogs or derivatives is brough~ into contact with the mctal or one of its salts at a pil of between 7 and 9 for ten to thirty minutes at ambient temperature, for example from 20 to 37OC.
In this cmbodiment, it is of advantage to use a proportion of 1 nole of FTS or an analog or derivative of FTS for 0.1 to 10 moles of the metal or of one of its salts, preferably 1 mole of the Metal or of one of its salts.
In another embodiment of the process according to the invention, the new products are prepared by treating l`TS or one or its analogs or derivatives with a metal-containing protein substance.
This metal-containing protein substance may be for example a ser~un fraction or a fraction of a thymic extract.
For e~ample, the metal-containing protein substance used may be a frac~ion of the Thymosine iraction 5 l~licll will be referred hereinatcr as thc 'lactivating fraction" by 9~
1 virtue oE its activating properties with respect to lTS
or its ana]ogs and derivatives.
In 1965, ~.L. GOL~ST~IN et. a]. isolated from calf thylllus a more or less impure preparation ~hich they called Thyrnosirle lraction 5 and ~ iCh iS a mixture of n~nerous polypeptides having various biological activities.
This preparation has been used in humans in pllase 1 and 2 clinical ~rials ~ith as yet inconclusive, but promising results.
i~ore recently, A.L. GOLDSTEII~ pu~lished the se~luence of certain pel~tides contained in the Fraction 5, na!nely 1' ~1' ~ but none of these peptides has been ound to show any structural similarity to l`TS.
Our o~n prelirninary studies l~ave shown that tlle Thymosine lraction 5 contained one or more substances active in the above-mentioned rosette test and having physical or cnernical ~roperties comparable ~ith tllose of FTS (molecular weigllt, electrical charge, aminoacid composition).
The above-mentioned activating fraction is prepared by fractionating Thymosine lraction 5 in a colurnn of G-100 (Pharmacia 0.9 crn x 100 cm) in a pl-l 7.3 0.2 l~
phospllate buffer to eliminate the molecule(s) slmilar to FTS, only the eluted fractions ~ith elution volurnes of from 15 to 22 ml being retained.
This fraction contains molecules having a rnolecular ~eight in the range from 7500 to 12500 (tracers used:
IGG, bovine albumin, Cytochrome C and tritiated ~rs).
This fraction is sensitive to heat: its activity is destroyed by heating to 60OC in less than 15 minutes and to 100oC in less than 5 minutes.
l`his fr~ction, whicll itself has no activity in 9~1 the rosette test,shows activating properties with respect to FTS or its analogs and derivatives insofar as it enables their biological activity to be increased by a factor of approximately 103.
, The process for preparing the new products from the activating fraction emanating from the Thymosine Fraction 5 is characterised in that FTS or one of its analogs or derivatives is incubated with this activating fraction, the mixture obtained after incubation is fractionated and the fraction showing the greatest activity in the rosette test is selected.
In this process, the incubation temperature is in the range from 30 to 40C, the incubation time is between 10 minutes and 1 hour and incubation is carried out at a pH-value in the range from 6 to 8.
The particularly preferred conditions comprise carry-ing out incubation for 20 minutes at a temperature of approx-imately 37 C and at a pH value of 7.3.
Irrespective of the process used (treatment with a metal or metal salt or treatment with a metal-containing protein substance), it is preferable to eliminate at the out-set any trac~ of metal which may be present in the preparation of FTS or one of its analogs or derivatives by means of a chelating agent capable of chelating metals, for example by chromatography in a column of chelating resin of the "Chelex*
100" type. This allows a chemically homogeneous product to be obtained which is biologically inactive and which, after being treated with the metal or one of its salts, produces a homogeneous product with reproducible results, these products thus being acceptable for pharmaceutical application.
The FTS or the FTS analogs or derivatives which are *A Trade Mark 1~7~ 9~
used in the ~rocess according to the invention are those which have been synthesised in the liquid phase by the method ,l described by BRICAS, E., MARTINEZ J., BLANOT, D., AUGER, G., DARDENNE, M., PLEAU, J.M., and BACH, J.F., 5th Intern. Peptide Symposium, La Jolla, California, U.S.A., June 1977, and those claimed in French Patent Application No. 77 15963 filed 25th May, 1977 and published December 15, 1978 as French Patent No.
2,391,994 and in its Addition No. 78 11870 filed 21st April, 1978 and published November 16, 1979 as French Patent No.
2,423,481~
FTS is a nonapeptide of the sequence pGlu - ALa -Lys - Ser - Gln - Gly - Gly - Ser - Asn and the FTS analogs and derivatives referred to herein are derived from this sequence in the manner described in the French Patent Appli-cation and its Addition mentioned above.
The Thymosine Fraction 5 used for preparing the activating fraction according to the invention was obtained by A.L. GOLDSTEIN's method tGOLDSTEIN A.L., SALTER, F.D. and WHITE, A., 1966, Proc. Nat. Acad. Sci. USA, 56, 1010-1017, and GOLDSTEIN, A.L., GUHA, A., ZATZ, M.M., HARDY, M., WHITE, A., Proc. Nat. Acad. Sci. USA, 69, 1800-1803~.
Other features of the invention will now be described in the following examples.
Preparation of the activating fraction:
The Thymosine Fraction 5 prepared by the above-mentioned method was fractionated in a first stage in a column of G-100 tPharmacia 0.9 cm x 100 cm) in a 0.2 M pH 7.3 phos-phate buffer to eliminate the above-mentioned molecule(s~
similar to or identical with FTS.
Only the eluted fractions with elution volumes of from 15 to 22 ml are retained and used for the described tests.
These fractions, which will be referred to hereinafter as _ g _ "activating -fractions", contain molecules having molecular weights in the range from 7500 to 12,500 and do not show any activity in the rosette test.
The quant.ities of activating fractions used will be expressed in the following in equivalents of Fraction 5 (EqF 5), i.e. in equivalents of the quantity of Fraction 5 used for its preparation.
Preparation of an activated protein:
1 mg of FTS corresponding to the formula p Glu - Ala -Lys - Ser - Gln - Gly - Gly - Ser - Asn synthesised in the liquid phase by the method described above is incubated with 50 mg (EqF 5) of the activating fraction of Example 1 in a tube of plastics material for 20 minutes at 37C in a 0.2 M
pH 7.3 phosphate buffer.
After incubation, the mixture is applied to a column of Sephadex* G-25 and the active fraction detected by the rosette test is recovered in the fractions having an elution volume of from 1.8 to 2.2 Vo.
This fraction is freed from salts by dry filtration on an Amicon* UM 2 membrane and is then taken up to 0.02 M pH
6.1 phosphate buffer and applied to a column of carboxy methyl cellulose (Whatman) balanced by the same phosphate buffer.
An NaCl gradient is applied after a passage of 50 ml of the initial buffer. The fractions showing activity in the rosette test are recovered in ~e fractions containing 0.12 M
of NaCl.
The fractions obtained after incubationl~with the acti-vating fraction are from 102 to 103 times more active than the initial preparation of FTS.
These active fractions are then freed from salts by * Trade Marks 1~'7~91 1 dry iltration on a~ unicon ~l 2 membrane and subse~luently subjected to an c~ninoacid ..nalysis.
e results of this an.llysis show that thc fraction thus recovered has a high biological activity and an ~ninoacid colnposition comp.ltible t7ith that of tl-e initial l`TS.
It follows that incubation of the ~TS tJith the activating fraction according to the invention emanating from a thymic ext~act, but having no biological activity of its own in the rosette test, increascs the ~iological activity of the synthetic l;`TS by a ~actor of 103.
The aminoacid analysis and the behaviour on Sephadex G-25 and car~oxyrmetllyl cellulose of the "activated"
preparation confi~n-l that the ~TS is the synthetic ~lS
itself in activated rorm.
In the presence of the activating fraction (as distinct from the lTS), the lrS is transfonned after 20 minutes at 37C and this transformation increases its biological activity very significantly.
As in the case of treatment with a metal or met:al salt, this transfo~nation is indicative of the fixing of a metal to the starting product.
It has been possible to shot~ that this metal does in act emanate ~rom the activating fraction bccause, after passage over an agent capa~le of chelating metals (for example Chelcx 100), these activating fractions lose their ability to activate ~TS or its analogs and derivatives.
L,~IPL~ 3 ,~ctivation by a metal ~iaterials and liethods 1. i'eactants:
.
The synthetic llS t~as prepared ~y the rnethod described 1~'7~39~1 above. The ~tritium-labelled FTS was obtained by the tritation of an FTS analog containing a lysine having an acetylenic bond (SASAKI A.W., MORGAT J.L. and E. BRIGAS, 16th European Sympo-sium on Peptides, Elsinger, Denmark, September, 1980, K.
BRUNGELDT, page 224).
The zinc was used in the form of its optionally radial-labelled chloride (65Zn Cl~).
2. Chromatography:
The treatment with Chelex 100 (Biorad Lab.) was carried out by mixing 0.2 ml of the solution containing the product under investigation with 0.2 ml of a 50 mg/ml suspension of Chelex. Chromatography on Biogel* P-2 was carried out using plastic columns (60 x 0.9 cm).
2,423,481~
FTS is a nonapeptide of the sequence pGlu - ALa -Lys - Ser - Gln - Gly - Gly - Ser - Asn and the FTS analogs and derivatives referred to herein are derived from this sequence in the manner described in the French Patent Appli-cation and its Addition mentioned above.
The Thymosine Fraction 5 used for preparing the activating fraction according to the invention was obtained by A.L. GOLDSTEIN's method tGOLDSTEIN A.L., SALTER, F.D. and WHITE, A., 1966, Proc. Nat. Acad. Sci. USA, 56, 1010-1017, and GOLDSTEIN, A.L., GUHA, A., ZATZ, M.M., HARDY, M., WHITE, A., Proc. Nat. Acad. Sci. USA, 69, 1800-1803~.
Other features of the invention will now be described in the following examples.
Preparation of the activating fraction:
The Thymosine Fraction 5 prepared by the above-mentioned method was fractionated in a first stage in a column of G-100 tPharmacia 0.9 cm x 100 cm) in a 0.2 M pH 7.3 phos-phate buffer to eliminate the above-mentioned molecule(s~
similar to or identical with FTS.
Only the eluted fractions with elution volumes of from 15 to 22 ml are retained and used for the described tests.
These fractions, which will be referred to hereinafter as _ g _ "activating -fractions", contain molecules having molecular weights in the range from 7500 to 12,500 and do not show any activity in the rosette test.
The quant.ities of activating fractions used will be expressed in the following in equivalents of Fraction 5 (EqF 5), i.e. in equivalents of the quantity of Fraction 5 used for its preparation.
Preparation of an activated protein:
1 mg of FTS corresponding to the formula p Glu - Ala -Lys - Ser - Gln - Gly - Gly - Ser - Asn synthesised in the liquid phase by the method described above is incubated with 50 mg (EqF 5) of the activating fraction of Example 1 in a tube of plastics material for 20 minutes at 37C in a 0.2 M
pH 7.3 phosphate buffer.
After incubation, the mixture is applied to a column of Sephadex* G-25 and the active fraction detected by the rosette test is recovered in the fractions having an elution volume of from 1.8 to 2.2 Vo.
This fraction is freed from salts by dry filtration on an Amicon* UM 2 membrane and is then taken up to 0.02 M pH
6.1 phosphate buffer and applied to a column of carboxy methyl cellulose (Whatman) balanced by the same phosphate buffer.
An NaCl gradient is applied after a passage of 50 ml of the initial buffer. The fractions showing activity in the rosette test are recovered in ~e fractions containing 0.12 M
of NaCl.
The fractions obtained after incubationl~with the acti-vating fraction are from 102 to 103 times more active than the initial preparation of FTS.
These active fractions are then freed from salts by * Trade Marks 1~'7~91 1 dry iltration on a~ unicon ~l 2 membrane and subse~luently subjected to an c~ninoacid ..nalysis.
e results of this an.llysis show that thc fraction thus recovered has a high biological activity and an ~ninoacid colnposition comp.ltible t7ith that of tl-e initial l`TS.
It follows that incubation of the ~TS tJith the activating fraction according to the invention emanating from a thymic ext~act, but having no biological activity of its own in the rosette test, increascs the ~iological activity of the synthetic l;`TS by a ~actor of 103.
The aminoacid analysis and the behaviour on Sephadex G-25 and car~oxyrmetllyl cellulose of the "activated"
preparation confi~n-l that the ~TS is the synthetic ~lS
itself in activated rorm.
In the presence of the activating fraction (as distinct from the lTS), the lrS is transfonned after 20 minutes at 37C and this transformation increases its biological activity very significantly.
As in the case of treatment with a metal or met:al salt, this transfo~nation is indicative of the fixing of a metal to the starting product.
It has been possible to shot~ that this metal does in act emanate ~rom the activating fraction bccause, after passage over an agent capa~le of chelating metals (for example Chelcx 100), these activating fractions lose their ability to activate ~TS or its analogs and derivatives.
L,~IPL~ 3 ,~ctivation by a metal ~iaterials and liethods 1. i'eactants:
.
The synthetic llS t~as prepared ~y the rnethod described 1~'7~39~1 above. The ~tritium-labelled FTS was obtained by the tritation of an FTS analog containing a lysine having an acetylenic bond (SASAKI A.W., MORGAT J.L. and E. BRIGAS, 16th European Sympo-sium on Peptides, Elsinger, Denmark, September, 1980, K.
BRUNGELDT, page 224).
The zinc was used in the form of its optionally radial-labelled chloride (65Zn Cl~).
2. Chromatography:
The treatment with Chelex 100 (Biorad Lab.) was carried out by mixing 0.2 ml of the solution containing the product under investigation with 0.2 ml of a 50 mg/ml suspension of Chelex. Chromatography on Biogel* P-2 was carried out using plastic columns (60 x 0.9 cm).
3. Rosette Test:
The spleen cells of C57BL/6 mice thymectomised at the age of 6 weeks are resistant to azathioprine (10 g/ml). The thymic extracts or the FTS or the serums containing it provide the cells with their sensitivity to azathioprine. The test results are expressed by the minimum inhibiting concentration of FTS or the maximum inhibiting dilution of serum.
II RESULTS
l. Loss of biological activity of FTS in the presence of a metal-chelating agent An aqueous solution of synthetic FTS is contacted with Chelex 100 resin. The FTS which is not retained by the resin is tested in the rosette test. Table I below shows that the FTS has lost most of its _ vitro and in vivo activity_ after this treatment. Similar results are observed with natural FTS (normal serum ultrafiltrate of mice) treated under the same conditions.
* A Trade Mark 117(:~991 2. i~ei.lpl)earallce of the l~iolo~ical activity of FTS prctreated passage over Chelex 100 aEter thc: addition of zinc c;r other InetaLs Lhe addition of lO ng of ZnCL2 to 100 ng of F LS
( sy~ etic or natural ) pretreatecl l~y passage over Chelex 100 followed by incul~ation f:or 10 minutes at 200C brings about not only complete restoration o the in vivo and in vi tro biologi cal activity, but also a significant increase in this activity over the activity initially observed before any treatrnent: (l`able I). Zinc chloride is active on its own in the in vitro and in vivo rosette tests.
Activation o the FTS is optimal ~t a neutral (or slightly alkaline) pll. The need for contact between the zinc and the ~TS is demonstrated by the absence of any reappearance of the l~iological activity observed in the in vivo tests when the FTS passed over Chelex 100 and the zinc chloride are inj ected at an interval of 1 minute .
Metals other than zinc are capable of activating FTS:
aluminium (as active as zinc~, copper, manganese, chromium, iron and nickel (distinctly less active than zinc), whereas indium, thallium, cadmium, lead, col)alt and tin have no ef f ect .
3. I)irect dcmo.ns~tral:ion of tl~e fixing of zinc to_ the lTS
molecule The tritiated FTS is eluted with distilled ~ater from a column of 13iogel P-2 in a singLe peak (ractions 10-14, V /V : 1. 3) which recovers the biological activity of which the level is higher than in the very first fractiolls (10-12). Applied to the same column, the ~nCl2 is eluted with a V /V of 2.2 (fractions 19-22).
I~hell a mixture of 0.1 ,ug of tritiated FTS (previously passed over Chelex 100) and 10 to 30 ng of ZnC12 is appliecl to the 1~7(~9~1.
1 col~nn o~ ~iogel P-2, tlle radioactivity corresponding to Zn appears in two peaks: the first, whicll is the larger of the two, corresponds very precisely to the biological activity alld ~o the first h~lf of the radioactivity peak of thc tritiated I`TS (fractions ~ to 12), whilst the second corresponds to that of the free zinc (fractions 19 to 23).
This result shows that a percentage of the order of 1 ~/O of zinc is fixed to the FTS.
III ~ISCUSSI~
Irhese tests sho~ that the lTS loses its activity after passage over a metal-cllelating resin and recovers its activity completely after thc addition of various metal salts, such as those of zinc or aluminium, and partly after the addition of certain others. The zinc is fixed to the FTS with an affinity constant sufficient to allow separation from the free zinc on Biogel P-2. Thus, the FTS is only active after certain Metals, particularly zinc, have been arrested.
The presence of biological activity and the fixing of zinc in the very first fractions of tlle FTS peak eluted on Biogel P-2 indicates the existence of two forms of ~TS. Only the first of these two forms to which the zinc is fixed is biologically active in the rosette test.
Other results indicate that these two forms also differ in their antigenicity.
Table I below sho~s the results of the in vivo and invitro tests and ~emonstrates the increase in the biological activity of the activated products according to th~ invention obtained by treating FTS either with a metal or ~7ith the activating fraction cmanating from the Thymosine fraction 5.
1~'7U~9 1 'l`~Lr I
In vitro activity: In vivo activiLy:
minimum inhibiting inhibiting concentration dilution of the serl~n 30 minutes after the administration of l ng of FTS
.
Synthetic l~`S5.10 6ng/ml 1/80 000 Synthetic ~ rs passed over Chelex 100 1 ng/n-l 1/256 Synthetic ITS passed over Chelex 1Q0 ~- zinc chloride 2.10 ng/ml 1/1o6 Zinc chloride aloneno activity 1/4 Synthetic FTS 5.10 ng/ml 1/80 000 Synthetic F'l`S passed over Chelex 100 ~- activating fraction 3.10 ng/ml 1/10 Svnthetic FTS passed over Che~x 100 + activating fraction passed over Chelex 100 lng/ml 1/100 Normal mouse serum (ultrafiltrate) 1/128 Normal serum passed over Chelex 100 1/4 Normal serurn passed over 1~128 Chelex 100 + zinc chloride Normal ser~lm passed over 1/128 Chelex 100 ~ activating fraction Normal serum passed over 1/4 Chelex 100 + activating fraction passed over Chelex 100 Serum from th~-nectornised mice 1/4 Serum from th~lectomised mice 1/4 -~ zinc cilloride Ser~ll from th~nectomised mice 1/4 ~- activating fraction 11'7~3991 1 Table I shows tllat tlle ~`TS pretreated ~y passage over Chelex 100 subsequell~Ly returns to a substantially increased in vitro biological activity level and to an even higher in vivo biological activity leveL after treatment with a metal or witll the activating fractioll, this metal and this activating fraction themselv2s sho~.7ing no biological activity in the rosette test.
l`able I also shows th.lt certain products according to the invention are active in concentratiolis below 10 ng/ml.
lor the in vivo tests, the activated derivatives of the invention were injected into thymectomised mice ~hich were six weeks old and used eight weeks after thymectomy.
After 30 minutes in the serum they induce the appearance of a biologiccll activity which is from 10 to 100 times greater than that obtained using natural ETS, imparting to the rosette-forming spleen cells a sensitivity to azathioprine .
This increase in biological activity is clearly shown in Figure 1 which compares the biological activity of the lTS, passed over a chelatin~ resin, then treated witll zinc sulfate at pll 7 and injected in a dose of 0.1 ng per mouse (curve I) with the activity of the '~TS ~7hich has merely been passed over the resin, then injected in a dose of 1 ng per mouse (curve II), and with the activity of the reference FTS which was injected in a dose of 1 ng per mouse (curve III).
Tlle curves of ligure 1, as well as those of ligures 2 to 6, express the variation in the activity of the product injected in tlle serum, expressed by the active serum clilution as a function of the time elapsed after the injection. The curves of Figure 1 compare the activity of the reference ~TS, injected in ~17~991 1 the mollse~, the activity of the FTS treated witll a chelating resin ~nd injected into the mouse, and the activity of a product according to the invention whicll was injected in the mouse, the latter being much more active.
Figure 2 shows the activation which was obtained ]n vivo by ~TS passed over a chelating resin, then treated with zinc sulfate using various weight ratios of ~n/I;TS
(O.Ol(curve Ia);O.l(curve Ib)and l(curve Ic)) and injected into the mouse, in cornparison with the FTS which ~as merely passed over a chelating resin and injected into the mouse (curve II).
The best activation is obtained for weight ratios of from 0.1 to 10.
Figures 3 and 4 compare the activities obtained in vlvo by tlle FTS passed over a chelating resin, then treated with different metals, that is the foLlowing metal salts:
Z-inc sulfate (curve Id) Copper sulfate (curve Ie) Ferrous sulfate (curve If) Cobalt chloride (curve Ig) ~ickel chloride (curve Ih) Chromium chloride (curve Ii) ~langanese chloride (curve Ij) Aluminium nitrate (curve Il~) clnd injected into the mouse, with the activity obtained using the FTS which was merely passed over a chelating resin and injected into the mouse (curve II).
Figure 5 shows the activity obtained in vivo by l:TS passed over a chelating resin~ then treated with zinc sulfate either for ten minutes at 200C (curve Il) or ten minutes at 370C (curve Im), and injected into the mouse, 99~
l~ -1 in co;lparison with t~-le ITS whicll was merely passed over a cllelating resin and i.njected into the mouse. This ligure sho~s tha~ there are no significant differences in activation between the treatment effected at 200C and that effected at 370C.
r`igure 6 shows tl~e ac~ivity obtained in vivo by the FTS, WhiCil ~as passed over a chelating resin then treated with zinc, for different injection doses into the mouse:
0.001 ng (curve In), 0.01 ng (curve Io) 0.1 ng (curve Ip) and 1 ng (curve Iq).
~t the s~ne time, the _ vivo treatlllent of thymectomised mice with the produc~s of the invention completely restores the sensitivity to azathioprine and to the antitheta serum of the rosette-forming cells from tl~e spleen of these mice, as is shown by the results in the Table II below:
L~L~ II
Sensitivity of the s~leen of thymectomised mice (Tx) to azathioprine, the mice being injected 24 hours before the test with 0.1 or 1 ng of reference ITS or of products of the inveTItion.
.. . ..
Treatment :Sensitivity of spleen to azathloprine Untreated nomlal spleen . 0.7 y Untreated Tx spleen 25 y 25 Tx spleen treated with 1 ng of l~TS passed over Chelex 100 : 25 y Tx spleen treated with 1 ng of reference . 0.7 y Tx spleen treatcd with 0.1 ng of FTS passed over Chelex 100 and treated with 0.1 ng of ZnS04 0.7 y Lx spleen treated witll 0.01 ng of ~TS passed over Chelex 100 and ~reated witll 0.1 ng of ZnS0~. 0.7 y 117~991 T~l~Ll` Il (continuation) Treatlnent : Sensitivity of spleen to azatllioprine . ~
Tx sl)leen treated witl1 O.Ol ng :
of ll`S passe~ over Chelex 100 -~ activ~ting fraction : 0-7 r Tx spleen treated Witl1 O.Ol ng of l:TS passed over Chelex 100 ~- activa~ing rraction passed over Chelex 100 . 25 l rl`hese e~periments were repeated using differcnt doses of the proclucts: rom l to lO0 p~ hich allowed a demonstration of the relationship between the dose and effect and showed the absence of acute toxicity, w}1ether the product is injec ed alone (in physiological serurn) or c~bso-rbed on carboxymethyl cellulose.
Tl1ese results sl1ow that in vivo a considerable activation is obtained by the zinc and, to a lesser e~tent, by other metals.
The products of tlle invention have the same phanrlacologic~l properties and the same therapeutic applications as lTS or its analogs or derivatives.
It has already been pointed out in the publications cited earlier on relating to lTS analogs and derivatives tl1at ~l~ese compoul1ds rnay l1ave eitl1er the s~ne thymic activity as l`TS or an inl1il)iti11g or antagonistic e~fect on this thymic activity.
Tl1e compounds which are active in the rosette test all have the s~ne th~1lic activity as E`TS wl~ereas certain cornpouncls Whi.CIl are in<active in the rosette test have an inhibiting or antagonistic e'ciect on the tl1~nic activity.
117~g~.
1 In every case, these deri.vati.ves rnay be used as rnedicaments for treating illncsses attributable to an unbalance o T lymphocytes, ~articularly auto~i~nune illnesses corresponding to a collapse of tlle suppressor lyrn~ ocytes, viral illnesses correspondin~ to a reduction in the"llelper" lympllocytes and tumoral illnesses caused by a deficit of "Kil3.er" ly~npllocytes.
lable III below sllo~7s the effect of the trea~rnent ~7itll ~inc on tlle biological activity of certain I`TS analogs and derlvatives.
1:~7~)9~1 -- ~1 --. - ~ t, G' G~ G\ ~ G` Ul ~r- ~ -1~1~ C` ~) ~) 1~) ~ ~, ~, ~J ~ G\ ~ ~ r~ O ~ ~ ~;
fD ~
. __ H
,~
1'- (D O
rr rt o~
It rr 1-.
O tD
O rr t l ~ J t ~ 0~
. rr I I I ~ I ~
U~ !'' 1'' ~ ~
O ~) ~r O ~-+ + -1- + I ~ N ~
H
I I I I I ~ I I I + I I I C
rr _ .
~ ~ 0~
-' -!- 1-~ -~ I + +
o ~
C~ ~ p, ~ ~D rr U) 0~
_~
I_,L I I ' _~_ I _,L -1- 1-ll- -t- -1- 1 ~ , , (~
It 1~7~9~
~ ~J ,_j O'~ CO 0~ 00 -J
O O O O ~I C~ ~ ~ O ~ CO ~ _n ~ ~ ~ ~D rr n I'' C. H
__ _ , . .. _ H
I _~L ~ _~L _,L ~ ~ It ~. --O O O
cn ~ O
D O
0 ~
l.
O ~D ~:
rt ~ ~
Q.
+~ ' t- ~ I I ~-- rt ~
IJ.
O ~
_ .
1 ~ ~ 7~ 7. ~ 7.' ~. 7'~ 7- ~ .
.
r~
.__.
I I I I I I + I I I I I I I _L I I I H ';~
~ S.
. ~
O ~`C
O ~
-t :j: ~ H
0~' ~
__ . H
, , , , , , , +~ ,~, , , , ~ I I I I -1-IJ
It .---o , _,L ,L + _,L + ~. , , , + , , I + -,L + ~L -1- -',- I 3 o ~
~ (D
__ tn o ' + I + -5- -1- + -,' -~- -,L -,' -,L ~ , + , , , , ,,, . ~:' ____ . _ 117~9g~
IL is il~lL)ortant to note tll.~t cert<lin pel~tides (iios.
37, 38, 47, 45, GO, 63, 84 and 96) ~ icll are c~mpletely or suL)s~antially inacl~ive in the rosette t:est l~ecome active afccr trcatlnent t~ith zinc.
In lal;le III, the letters ~D stand for not-detc~rrnined ~hilst tl~e letter R me.ms tl~at the compound corresponds to a delayed activity, i.e. to a prolonged activity.
On the otller hand, tlle l~epticle nurnbers correspond to the i~ollo~ing peptides:
Ser-Gln-Gly-Gly-Ser-Asn (36) Lys-Ser-Gln-Gly-Gly-Ser-Asn (37) Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn (38) PyroGlu Ala-Lys-Ser-Gln-Gly-Gly-Ser (42) Gln-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn (18) PyroGlu-D-Ala-Lys-Ser-Gln-Gly-Gly-Ser-l~sn (47) D-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn (45) PyroGlu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-~ (53) PyroGlu-Ala-Lys-Ser-Gln-Gly-Gly-Ala-Asn (59) PyroGlu-Ala-Lys-Ala-Gln-Gly-Gly-Ser-Asn (GO) ~ Gln-~la-Lys-Ser-Gln-Gly-Gly-Ser-Asn (34) YyroGlu-Ala-l)-Lys-Ser-Gln-Gly-Gly-Ser-Asn (61) PyroGlu-Ala-Lys(l~6-acetyl)-Ser-GLn-Gly-Gly-Ser-Asn (G2) PyroGlu-Ala-Orn-Ser-Gln-Gly-Gly-Ser-Asn (63) PyroGlu-Ala-Lys-(N-methyl)Ser-Glll-Gly-Ser-Asn (64) PyroGlu-Ala-Lys-D-Ser-Gln-Gly-Gly-Ser-Asn (66) PyroGlu-Ala-I.ys-Ser-l)-Gln-Gly-Gly-Ser-Asn (71) PyroGlu-Ala-(2,G-di~ninollexynoyl)-Ser-Gln-Gly-Gly-Scr-Asn (77) PyroGlu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Gln (79) Lys(ii acetyl)-Ser-Gln-Gly-Gly-Ser-Asn (82) 30 L)-Lys-Ser-Gln-Gly-Gly-Ser-Asn (83) Orn-Ser-Gln-Gly-Gly-Ser-Asn (84) .Lys-Ser-GLn-Gly-Gly-Ser-Asn (85) 1~'7~9g~
- ~4 -1 PyroGI.u-Ala-I.ys-~er-G:LIl-Gly-Gl.y-Ser-~3-Ala-i~ll2 (~6) Lys-Ser-Gln-l)-Ala-Gly-Ser-Asn (~) PyroGlu-A]a-Lys-Ser-Gln-~-Ala[Gly-Ser-Asn (89) PyroGlu-AI.a-Lys-Ser-Gln-Gly-Gly-Ser-~-Asn (~0) 5 l'yroGlu-Ala-ilep-Ser-Gln-Gly-GlyOSer-Asn (91) ~yroGlu-Ala-Lys-Ser-Gln-Gly-Gly-SeL^-Asn-i~l~2 (93) PyroGiu-AIa-Lys-Ser-Asn-G].y-Gly-Ser-Asn (9~) PyroG].u-Ala Lys-Ser-~va-Gly-Gly-Ser-Asn (97) Z-~la-I.ys-Ser-Gln-Gly-Ser-Asn (lOS) 10 ~-PyroGlu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn (106) D-Gln-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn (107~
Pro-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn (109) PyroGlu-Ala-l~rg-Ser-Gln-Gly-Gly-Ser-Asn (111) I'yroGlu-Ala-liar-Ser-Gln-Gly-Gly-Ser-Asn (112) PyroGlu-Ala-Lys-(i~ -acetyl)-Ser-Gln-~ la-Gly-Ser-~sn(113) In the above peptides, the aminoacids are des.ignated by their stcmdard abbreviations. In addition, thc figure 2 denotes the benzyl oxycarbollyl group.
As mentioned above, the activated products according to the invention may be used in the treatment of illnesses attributable to an ullbalance of T lympllocytes, particularly corresponding to:
- a collapse of the suppressor lymphocytes: auto-immune illnesses, such as lupus, particul2rly lupus erythematosus, disseminated sclerosis, rhumatoid polyar~hritis;
- a reduction in the "helper" ly~npllocytes: bacterial and viral infections, particul2rly herpes recurrens zona, varicella;
- a deficit of "kil].er" lymphocytes: tumoral illnesses, in particular nasopharyngeal cancers, epidermoid cancer of the lung, infiltrating cancer of tlle bladder, mel~momas, ncoplasllls .
1~ 7(~9~
1`ILC? products according to the present inventio11 may admillistered intravenuous]y or int:rallluseularly, in tlle forr~
of suppositories, aerosols or orally in protected form.
Suitable vehic] es ~.7hicll may be used in the eornposition S includc for ~x~ r)le st:erile ' iguicls, sucll as ~ai:er, or a pllysiologica] so]ution or subs~anees cap.lble of imparting a clel.lyed effect or evell preparations eapable of l)eirlg adlninistere(l Ln the fonn of suppositories, aerosols or sublingu:llly. In addition to a vehiele, the eompositions according to the invention may also eomprise otller ingredients, sueh as stabilisers, oxiclation inhibitors, suspendin~ agents or preservatives, such as pheno l or ehlorobut~mol and agents of the same type. The ~inal solution may readily be s terilised l)y eonventional f il t: rat iOII teel-mi~lues .
Tlle eomposition used in aecordarlce ~`.ith the invention contains in aclueous so]ution a suffieient quantity o the agent aceording to the invention to be tllerapeutieally effeetive. The doses to be administered depend to a large extent upon the eondition and ~eight of the patient to be treated.
~or ex~unple, it is possible to administer a dose of 100 to 5000 ~L~ per inj ection and preferably 500 llg per inj eetion repeated three times per ~Jeek. This eorresponds to an approximate dose of 10 ~/~g in hum~ns.
l`he activated products according to the invention maj~ also be used in the preparation of radioaetive traeers suitable for use in radio-immunologieal deteetion proeesses for detecting the level of l TS presellt` in the serum.
The ro s e t te t e s t has sho~ n that the l eve l o f ~TS
in the serum is ~nodi fied in certain irmmuno-pathologieal situations, sueh as lupus and rnyasthenie eonditions(J.l~.
1~7~)~9~
1 ~ACIi ~t coll., ~ m. i~.Y. ~cad. Sci. 249 (1975), pa~e 18G).
i;o~7ever, since tl-le rosette test is a semi-qucmtitative test, tllere is a neecl to develop a quantitative detection process ~71~icll is sufficiently sellsitive to ~110~7 tlle direct detection oL llS in tlle blood.
Tlle process according to the inve-ltion ~or cletecting E`TS in a sample is cllarclcterised in tilat a predetenilined quantity of said sample is contacted with a predetermined quantity o~ tracer and witll a predetermined quantity of anti-llS antibody so as to bring about a competitive reaction between the tracer and tlle ILS of tlle sample in their combination witll the anti-FrS Lultibody, the quantity of Eree tracer or tracer combined witll tl~e .mtibody is determined after tlle reaction llaS reaCIIed a state of equilibriu:-n .md tlle value obtained is compared witll the values obtained for l;no~l and increasing quantities o~ l"rS.
l`lle anti-L;TS antibody is prepared by ~ixing ti~e ~`TS or one of its analogs or derivatives to a support to obtain an immunogen, injecting the i~mnunooen into an animal c~nd subsequently collecting the serum of tlle animal.
rlle tracer is obtained by labelling ~7itll a radioactive isotope and preferably by introducing an iodisable molecule into the activated product according to the invention and subsequently iodising tlle iodisable derivative thus obtained.
To carry out the detection process, it is possible to use a box of reactants optionally containing tlle tracer and tlle ~mtibody required for detection.
l`lle detection process is illustrated by the following ~;xalop 1 c .
1~7V~l II'LL 4 iateria1s an(1I~ietllods c ~ t s ~ e syn~l~etic FTS t~JaS prepared by the method described above.
2. I'adioactive traccr A synthetic analog of ITS, for cxamplc ~-aminobenzoyl-G]nl-I`TS, prepared by E. ~RICAS (Universitc de Paris Sud Orsay), is laI~eLled i~itll iodine 125 in the presence of ch]or~unine ~` (the P-aminobenzoyl-Glnl-lTS is only one ex&-lple amongst o~hers of ~TS analogs or derivativcs wlllch may be used for preparing the radioactive tracer). Tlle lTS- 5I is scparated from ~he free iodine b~- filtration over Sephadex G-25 gel eluted with O.l ~I pil 7.6 tris-ElCl buffer. ~ uot fractions of ~rs- I (approximately S x 106 cpm) are mixed ~ith a suspension of Chele~ lOO
(SO mg/ml), follo~?ed by incubation for 30 mlnutes in tlle presence of lOO ng of zinc chloride. l`l1e mixture is then chromatographed on Biogel P2, eluted ~ith O.l i~I pll 7.6 tris-ilCl buffer. lhe peak correspondin~ to tlle 1`TS-Zn is recovered and used for radio-immunological detection.
3. Anti-~TS ant _odv TI1e ~TS is rendered immunogenic by fixing it to bovine albumin serum in the presence of glutaraldehyde (6). The immunogen thus obtained, incorporated in ~reund's complete adjuvc-Lnt, is injected into rabbits (l mg per rabbit). ~ booster injection (also of l mg) is administered l month after the first injection and the animals are bled 15 days after the booster injection.
The spleen cells of C57BL/6 mice thymectomised at the age of 6 weeks are resistant to azathioprine (10 g/ml). The thymic extracts or the FTS or the serums containing it provide the cells with their sensitivity to azathioprine. The test results are expressed by the minimum inhibiting concentration of FTS or the maximum inhibiting dilution of serum.
II RESULTS
l. Loss of biological activity of FTS in the presence of a metal-chelating agent An aqueous solution of synthetic FTS is contacted with Chelex 100 resin. The FTS which is not retained by the resin is tested in the rosette test. Table I below shows that the FTS has lost most of its _ vitro and in vivo activity_ after this treatment. Similar results are observed with natural FTS (normal serum ultrafiltrate of mice) treated under the same conditions.
* A Trade Mark 117(:~991 2. i~ei.lpl)earallce of the l~iolo~ical activity of FTS prctreated passage over Chelex 100 aEter thc: addition of zinc c;r other InetaLs Lhe addition of lO ng of ZnCL2 to 100 ng of F LS
( sy~ etic or natural ) pretreatecl l~y passage over Chelex 100 followed by incul~ation f:or 10 minutes at 200C brings about not only complete restoration o the in vivo and in vi tro biologi cal activity, but also a significant increase in this activity over the activity initially observed before any treatrnent: (l`able I). Zinc chloride is active on its own in the in vitro and in vivo rosette tests.
Activation o the FTS is optimal ~t a neutral (or slightly alkaline) pll. The need for contact between the zinc and the ~TS is demonstrated by the absence of any reappearance of the l~iological activity observed in the in vivo tests when the FTS passed over Chelex 100 and the zinc chloride are inj ected at an interval of 1 minute .
Metals other than zinc are capable of activating FTS:
aluminium (as active as zinc~, copper, manganese, chromium, iron and nickel (distinctly less active than zinc), whereas indium, thallium, cadmium, lead, col)alt and tin have no ef f ect .
3. I)irect dcmo.ns~tral:ion of tl~e fixing of zinc to_ the lTS
molecule The tritiated FTS is eluted with distilled ~ater from a column of 13iogel P-2 in a singLe peak (ractions 10-14, V /V : 1. 3) which recovers the biological activity of which the level is higher than in the very first fractiolls (10-12). Applied to the same column, the ~nCl2 is eluted with a V /V of 2.2 (fractions 19-22).
I~hell a mixture of 0.1 ,ug of tritiated FTS (previously passed over Chelex 100) and 10 to 30 ng of ZnC12 is appliecl to the 1~7(~9~1.
1 col~nn o~ ~iogel P-2, tlle radioactivity corresponding to Zn appears in two peaks: the first, whicll is the larger of the two, corresponds very precisely to the biological activity alld ~o the first h~lf of the radioactivity peak of thc tritiated I`TS (fractions ~ to 12), whilst the second corresponds to that of the free zinc (fractions 19 to 23).
This result shows that a percentage of the order of 1 ~/O of zinc is fixed to the FTS.
III ~ISCUSSI~
Irhese tests sho~ that the lTS loses its activity after passage over a metal-cllelating resin and recovers its activity completely after thc addition of various metal salts, such as those of zinc or aluminium, and partly after the addition of certain others. The zinc is fixed to the FTS with an affinity constant sufficient to allow separation from the free zinc on Biogel P-2. Thus, the FTS is only active after certain Metals, particularly zinc, have been arrested.
The presence of biological activity and the fixing of zinc in the very first fractions of tlle FTS peak eluted on Biogel P-2 indicates the existence of two forms of ~TS. Only the first of these two forms to which the zinc is fixed is biologically active in the rosette test.
Other results indicate that these two forms also differ in their antigenicity.
Table I below sho~s the results of the in vivo and invitro tests and ~emonstrates the increase in the biological activity of the activated products according to th~ invention obtained by treating FTS either with a metal or ~7ith the activating fraction cmanating from the Thymosine fraction 5.
1~'7U~9 1 'l`~Lr I
In vitro activity: In vivo activiLy:
minimum inhibiting inhibiting concentration dilution of the serl~n 30 minutes after the administration of l ng of FTS
.
Synthetic l~`S5.10 6ng/ml 1/80 000 Synthetic ~ rs passed over Chelex 100 1 ng/n-l 1/256 Synthetic ITS passed over Chelex 1Q0 ~- zinc chloride 2.10 ng/ml 1/1o6 Zinc chloride aloneno activity 1/4 Synthetic FTS 5.10 ng/ml 1/80 000 Synthetic F'l`S passed over Chelex 100 ~- activating fraction 3.10 ng/ml 1/10 Svnthetic FTS passed over Che~x 100 + activating fraction passed over Chelex 100 lng/ml 1/100 Normal mouse serum (ultrafiltrate) 1/128 Normal serum passed over Chelex 100 1/4 Normal serurn passed over 1~128 Chelex 100 + zinc chloride Normal ser~lm passed over 1/128 Chelex 100 ~ activating fraction Normal serum passed over 1/4 Chelex 100 + activating fraction passed over Chelex 100 Serum from th~-nectornised mice 1/4 Serum from th~lectomised mice 1/4 -~ zinc cilloride Ser~ll from th~nectomised mice 1/4 ~- activating fraction 11'7~3991 1 Table I shows tllat tlle ~`TS pretreated ~y passage over Chelex 100 subsequell~Ly returns to a substantially increased in vitro biological activity level and to an even higher in vivo biological activity leveL after treatment with a metal or witll the activating fractioll, this metal and this activating fraction themselv2s sho~.7ing no biological activity in the rosette test.
l`able I also shows th.lt certain products according to the invention are active in concentratiolis below 10 ng/ml.
lor the in vivo tests, the activated derivatives of the invention were injected into thymectomised mice ~hich were six weeks old and used eight weeks after thymectomy.
After 30 minutes in the serum they induce the appearance of a biologiccll activity which is from 10 to 100 times greater than that obtained using natural ETS, imparting to the rosette-forming spleen cells a sensitivity to azathioprine .
This increase in biological activity is clearly shown in Figure 1 which compares the biological activity of the lTS, passed over a chelatin~ resin, then treated witll zinc sulfate at pll 7 and injected in a dose of 0.1 ng per mouse (curve I) with the activity of the '~TS ~7hich has merely been passed over the resin, then injected in a dose of 1 ng per mouse (curve II), and with the activity of the reference FTS which was injected in a dose of 1 ng per mouse (curve III).
Tlle curves of ligure 1, as well as those of ligures 2 to 6, express the variation in the activity of the product injected in tlle serum, expressed by the active serum clilution as a function of the time elapsed after the injection. The curves of Figure 1 compare the activity of the reference ~TS, injected in ~17~991 1 the mollse~, the activity of the FTS treated witll a chelating resin ~nd injected into the mouse, and the activity of a product according to the invention whicll was injected in the mouse, the latter being much more active.
Figure 2 shows the activation which was obtained ]n vivo by ~TS passed over a chelating resin, then treated with zinc sulfate using various weight ratios of ~n/I;TS
(O.Ol(curve Ia);O.l(curve Ib)and l(curve Ic)) and injected into the mouse, in cornparison with the FTS which ~as merely passed over a chelating resin and injected into the mouse (curve II).
The best activation is obtained for weight ratios of from 0.1 to 10.
Figures 3 and 4 compare the activities obtained in vlvo by tlle FTS passed over a chelating resin, then treated with different metals, that is the foLlowing metal salts:
Z-inc sulfate (curve Id) Copper sulfate (curve Ie) Ferrous sulfate (curve If) Cobalt chloride (curve Ig) ~ickel chloride (curve Ih) Chromium chloride (curve Ii) ~langanese chloride (curve Ij) Aluminium nitrate (curve Il~) clnd injected into the mouse, with the activity obtained using the FTS which was merely passed over a chelating resin and injected into the mouse (curve II).
Figure 5 shows the activity obtained in vivo by l:TS passed over a chelating resin~ then treated with zinc sulfate either for ten minutes at 200C (curve Il) or ten minutes at 370C (curve Im), and injected into the mouse, 99~
l~ -1 in co;lparison with t~-le ITS whicll was merely passed over a cllelating resin and i.njected into the mouse. This ligure sho~s tha~ there are no significant differences in activation between the treatment effected at 200C and that effected at 370C.
r`igure 6 shows tl~e ac~ivity obtained in vivo by the FTS, WhiCil ~as passed over a chelating resin then treated with zinc, for different injection doses into the mouse:
0.001 ng (curve In), 0.01 ng (curve Io) 0.1 ng (curve Ip) and 1 ng (curve Iq).
~t the s~ne time, the _ vivo treatlllent of thymectomised mice with the produc~s of the invention completely restores the sensitivity to azathioprine and to the antitheta serum of the rosette-forming cells from tl~e spleen of these mice, as is shown by the results in the Table II below:
L~L~ II
Sensitivity of the s~leen of thymectomised mice (Tx) to azathioprine, the mice being injected 24 hours before the test with 0.1 or 1 ng of reference ITS or of products of the inveTItion.
.. . ..
Treatment :Sensitivity of spleen to azathloprine Untreated nomlal spleen . 0.7 y Untreated Tx spleen 25 y 25 Tx spleen treated with 1 ng of l~TS passed over Chelex 100 : 25 y Tx spleen treated with 1 ng of reference . 0.7 y Tx spleen treatcd with 0.1 ng of FTS passed over Chelex 100 and treated with 0.1 ng of ZnS04 0.7 y Lx spleen treated witll 0.01 ng of ~TS passed over Chelex 100 and ~reated witll 0.1 ng of ZnS0~. 0.7 y 117~991 T~l~Ll` Il (continuation) Treatlnent : Sensitivity of spleen to azatllioprine . ~
Tx sl)leen treated witl1 O.Ol ng :
of ll`S passe~ over Chelex 100 -~ activ~ting fraction : 0-7 r Tx spleen treated Witl1 O.Ol ng of l:TS passed over Chelex 100 ~- activa~ing rraction passed over Chelex 100 . 25 l rl`hese e~periments were repeated using differcnt doses of the proclucts: rom l to lO0 p~ hich allowed a demonstration of the relationship between the dose and effect and showed the absence of acute toxicity, w}1ether the product is injec ed alone (in physiological serurn) or c~bso-rbed on carboxymethyl cellulose.
Tl1ese results sl1ow that in vivo a considerable activation is obtained by the zinc and, to a lesser e~tent, by other metals.
The products of tlle invention have the same phanrlacologic~l properties and the same therapeutic applications as lTS or its analogs or derivatives.
It has already been pointed out in the publications cited earlier on relating to lTS analogs and derivatives tl1at ~l~ese compoul1ds rnay l1ave eitl1er the s~ne thymic activity as l`TS or an inl1il)iti11g or antagonistic e~fect on this thymic activity.
Tl1e compounds which are active in the rosette test all have the s~ne th~1lic activity as E`TS wl~ereas certain cornpouncls Whi.CIl are in<active in the rosette test have an inhibiting or antagonistic e'ciect on the tl1~nic activity.
117~g~.
1 In every case, these deri.vati.ves rnay be used as rnedicaments for treating illncsses attributable to an unbalance o T lymphocytes, ~articularly auto~i~nune illnesses corresponding to a collapse of tlle suppressor lyrn~ ocytes, viral illnesses correspondin~ to a reduction in the"llelper" lympllocytes and tumoral illnesses caused by a deficit of "Kil3.er" ly~npllocytes.
lable III below sllo~7s the effect of the trea~rnent ~7itll ~inc on tlle biological activity of certain I`TS analogs and derlvatives.
1:~7~)9~1 -- ~1 --. - ~ t, G' G~ G\ ~ G` Ul ~r- ~ -1~1~ C` ~) ~) 1~) ~ ~, ~, ~J ~ G\ ~ ~ r~ O ~ ~ ~;
fD ~
. __ H
,~
1'- (D O
rr rt o~
It rr 1-.
O tD
O rr t l ~ J t ~ 0~
. rr I I I ~ I ~
U~ !'' 1'' ~ ~
O ~) ~r O ~-+ + -1- + I ~ N ~
H
I I I I I ~ I I I + I I I C
rr _ .
~ ~ 0~
-' -!- 1-~ -~ I + +
o ~
C~ ~ p, ~ ~D rr U) 0~
_~
I_,L I I ' _~_ I _,L -1- 1-ll- -t- -1- 1 ~ , , (~
It 1~7~9~
~ ~J ,_j O'~ CO 0~ 00 -J
O O O O ~I C~ ~ ~ O ~ CO ~ _n ~ ~ ~ ~D rr n I'' C. H
__ _ , . .. _ H
I _~L ~ _~L _,L ~ ~ It ~. --O O O
cn ~ O
D O
0 ~
l.
O ~D ~:
rt ~ ~
Q.
+~ ' t- ~ I I ~-- rt ~
IJ.
O ~
_ .
1 ~ ~ 7~ 7. ~ 7.' ~. 7'~ 7- ~ .
.
r~
.__.
I I I I I I + I I I I I I I _L I I I H ';~
~ S.
. ~
O ~`C
O ~
-t :j: ~ H
0~' ~
__ . H
, , , , , , , +~ ,~, , , , ~ I I I I -1-IJ
It .---o , _,L ,L + _,L + ~. , , , + , , I + -,L + ~L -1- -',- I 3 o ~
~ (D
__ tn o ' + I + -5- -1- + -,' -~- -,L -,' -,L ~ , + , , , , ,,, . ~:' ____ . _ 117~9g~
IL is il~lL)ortant to note tll.~t cert<lin pel~tides (iios.
37, 38, 47, 45, GO, 63, 84 and 96) ~ icll are c~mpletely or suL)s~antially inacl~ive in the rosette t:est l~ecome active afccr trcatlnent t~ith zinc.
In lal;le III, the letters ~D stand for not-detc~rrnined ~hilst tl~e letter R me.ms tl~at the compound corresponds to a delayed activity, i.e. to a prolonged activity.
On the otller hand, tlle l~epticle nurnbers correspond to the i~ollo~ing peptides:
Ser-Gln-Gly-Gly-Ser-Asn (36) Lys-Ser-Gln-Gly-Gly-Ser-Asn (37) Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn (38) PyroGlu Ala-Lys-Ser-Gln-Gly-Gly-Ser (42) Gln-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn (18) PyroGlu-D-Ala-Lys-Ser-Gln-Gly-Gly-Ser-l~sn (47) D-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn (45) PyroGlu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-~ (53) PyroGlu-Ala-Lys-Ser-Gln-Gly-Gly-Ala-Asn (59) PyroGlu-Ala-Lys-Ala-Gln-Gly-Gly-Ser-Asn (GO) ~ Gln-~la-Lys-Ser-Gln-Gly-Gly-Ser-Asn (34) YyroGlu-Ala-l)-Lys-Ser-Gln-Gly-Gly-Ser-Asn (61) PyroGlu-Ala-Lys(l~6-acetyl)-Ser-GLn-Gly-Gly-Ser-Asn (G2) PyroGlu-Ala-Orn-Ser-Gln-Gly-Gly-Ser-Asn (63) PyroGlu-Ala-Lys-(N-methyl)Ser-Glll-Gly-Ser-Asn (64) PyroGlu-Ala-Lys-D-Ser-Gln-Gly-Gly-Ser-Asn (66) PyroGlu-Ala-I.ys-Ser-l)-Gln-Gly-Gly-Ser-Asn (71) PyroGlu-Ala-(2,G-di~ninollexynoyl)-Ser-Gln-Gly-Gly-Scr-Asn (77) PyroGlu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Gln (79) Lys(ii acetyl)-Ser-Gln-Gly-Gly-Ser-Asn (82) 30 L)-Lys-Ser-Gln-Gly-Gly-Ser-Asn (83) Orn-Ser-Gln-Gly-Gly-Ser-Asn (84) .Lys-Ser-GLn-Gly-Gly-Ser-Asn (85) 1~'7~9g~
- ~4 -1 PyroGI.u-Ala-I.ys-~er-G:LIl-Gly-Gl.y-Ser-~3-Ala-i~ll2 (~6) Lys-Ser-Gln-l)-Ala-Gly-Ser-Asn (~) PyroGlu-A]a-Lys-Ser-Gln-~-Ala[Gly-Ser-Asn (89) PyroGlu-AI.a-Lys-Ser-Gln-Gly-Gly-Ser-~-Asn (~0) 5 l'yroGlu-Ala-ilep-Ser-Gln-Gly-GlyOSer-Asn (91) ~yroGlu-Ala-Lys-Ser-Gln-Gly-Gly-SeL^-Asn-i~l~2 (93) PyroGiu-AIa-Lys-Ser-Asn-G].y-Gly-Ser-Asn (9~) PyroG].u-Ala Lys-Ser-~va-Gly-Gly-Ser-Asn (97) Z-~la-I.ys-Ser-Gln-Gly-Ser-Asn (lOS) 10 ~-PyroGlu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn (106) D-Gln-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn (107~
Pro-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn (109) PyroGlu-Ala-l~rg-Ser-Gln-Gly-Gly-Ser-Asn (111) I'yroGlu-Ala-liar-Ser-Gln-Gly-Gly-Ser-Asn (112) PyroGlu-Ala-Lys-(i~ -acetyl)-Ser-Gln-~ la-Gly-Ser-~sn(113) In the above peptides, the aminoacids are des.ignated by their stcmdard abbreviations. In addition, thc figure 2 denotes the benzyl oxycarbollyl group.
As mentioned above, the activated products according to the invention may be used in the treatment of illnesses attributable to an ullbalance of T lympllocytes, particularly corresponding to:
- a collapse of the suppressor lymphocytes: auto-immune illnesses, such as lupus, particul2rly lupus erythematosus, disseminated sclerosis, rhumatoid polyar~hritis;
- a reduction in the "helper" ly~npllocytes: bacterial and viral infections, particul2rly herpes recurrens zona, varicella;
- a deficit of "kil].er" lymphocytes: tumoral illnesses, in particular nasopharyngeal cancers, epidermoid cancer of the lung, infiltrating cancer of tlle bladder, mel~momas, ncoplasllls .
1~ 7(~9~
1`ILC? products according to the present inventio11 may admillistered intravenuous]y or int:rallluseularly, in tlle forr~
of suppositories, aerosols or orally in protected form.
Suitable vehic] es ~.7hicll may be used in the eornposition S includc for ~x~ r)le st:erile ' iguicls, sucll as ~ai:er, or a pllysiologica] so]ution or subs~anees cap.lble of imparting a clel.lyed effect or evell preparations eapable of l)eirlg adlninistere(l Ln the fonn of suppositories, aerosols or sublingu:llly. In addition to a vehiele, the eompositions according to the invention may also eomprise otller ingredients, sueh as stabilisers, oxiclation inhibitors, suspendin~ agents or preservatives, such as pheno l or ehlorobut~mol and agents of the same type. The ~inal solution may readily be s terilised l)y eonventional f il t: rat iOII teel-mi~lues .
Tlle eomposition used in aecordarlce ~`.ith the invention contains in aclueous so]ution a suffieient quantity o the agent aceording to the invention to be tllerapeutieally effeetive. The doses to be administered depend to a large extent upon the eondition and ~eight of the patient to be treated.
~or ex~unple, it is possible to administer a dose of 100 to 5000 ~L~ per inj ection and preferably 500 llg per inj eetion repeated three times per ~Jeek. This eorresponds to an approximate dose of 10 ~/~g in hum~ns.
l`he activated products according to the invention maj~ also be used in the preparation of radioaetive traeers suitable for use in radio-immunologieal deteetion proeesses for detecting the level of l TS presellt` in the serum.
The ro s e t te t e s t has sho~ n that the l eve l o f ~TS
in the serum is ~nodi fied in certain irmmuno-pathologieal situations, sueh as lupus and rnyasthenie eonditions(J.l~.
1~7~)~9~
1 ~ACIi ~t coll., ~ m. i~.Y. ~cad. Sci. 249 (1975), pa~e 18G).
i;o~7ever, since tl-le rosette test is a semi-qucmtitative test, tllere is a neecl to develop a quantitative detection process ~71~icll is sufficiently sellsitive to ~110~7 tlle direct detection oL llS in tlle blood.
Tlle process according to the inve-ltion ~or cletecting E`TS in a sample is cllarclcterised in tilat a predetenilined quantity of said sample is contacted with a predetermined quantity o~ tracer and witll a predetermined quantity of anti-llS antibody so as to bring about a competitive reaction between the tracer and tlle ILS of tlle sample in their combination witll the anti-FrS Lultibody, the quantity of Eree tracer or tracer combined witll tl~e .mtibody is determined after tlle reaction llaS reaCIIed a state of equilibriu:-n .md tlle value obtained is compared witll the values obtained for l;no~l and increasing quantities o~ l"rS.
l`lle anti-L;TS antibody is prepared by ~ixing ti~e ~`TS or one of its analogs or derivatives to a support to obtain an immunogen, injecting the i~mnunooen into an animal c~nd subsequently collecting the serum of tlle animal.
rlle tracer is obtained by labelling ~7itll a radioactive isotope and preferably by introducing an iodisable molecule into the activated product according to the invention and subsequently iodising tlle iodisable derivative thus obtained.
To carry out the detection process, it is possible to use a box of reactants optionally containing tlle tracer and tlle ~mtibody required for detection.
l`lle detection process is illustrated by the following ~;xalop 1 c .
1~7V~l II'LL 4 iateria1s an(1I~ietllods c ~ t s ~ e syn~l~etic FTS t~JaS prepared by the method described above.
2. I'adioactive traccr A synthetic analog of ITS, for cxamplc ~-aminobenzoyl-G]nl-I`TS, prepared by E. ~RICAS (Universitc de Paris Sud Orsay), is laI~eLled i~itll iodine 125 in the presence of ch]or~unine ~` (the P-aminobenzoyl-Glnl-lTS is only one ex&-lple amongst o~hers of ~TS analogs or derivativcs wlllch may be used for preparing the radioactive tracer). Tlle lTS- 5I is scparated from ~he free iodine b~- filtration over Sephadex G-25 gel eluted with O.l ~I pil 7.6 tris-ElCl buffer. ~ uot fractions of ~rs- I (approximately S x 106 cpm) are mixed ~ith a suspension of Chele~ lOO
(SO mg/ml), follo~?ed by incubation for 30 mlnutes in tlle presence of lOO ng of zinc chloride. l`l1e mixture is then chromatographed on Biogel P2, eluted ~ith O.l i~I pll 7.6 tris-ilCl buffer. lhe peak correspondin~ to tlle 1`TS-Zn is recovered and used for radio-immunological detection.
3. Anti-~TS ant _odv TI1e ~TS is rendered immunogenic by fixing it to bovine albumin serum in the presence of glutaraldehyde (6). The immunogen thus obtained, incorporated in ~reund's complete adjuvc-Lnt, is injected into rabbits (l mg per rabbit). ~ booster injection (also of l mg) is administered l month after the first injection and the animals are bled 15 days after the booster injection.
4. Pre~ ation o~ the biological sunr~les lresh serurns from normal and th~nectomised mice are u1trafiltered OII ~nicon C150 and used extemporaneously.
117(~991 - ~$ -.SUI.'I7S
'1. Corn,7,~.lrison oE th~ anti~-,c-licity of tlle t~o tracers 'I`lle antiserum obtained by immunisation against the synt,letic I~"rS combines tlle t~,70 tr~ccrs under investigation (I~TS I alld l~TS-i,n I). `~efined as the maximuri-dilution of the ~ntiser~n ~Jhicll produces a 40~,' combination of the radioactive tracer, ~lle strengtll of tlle antiserum is 1/50 000 ~or LTS I and 1/20 000 for FTS-Zn I.
2. 7~isplace~nent of the combinatioll by ~'TS and by the serum of r.orlllal or th~lectornised ~ice 'l'he syn~lletic I~TS inllibits the combination of tlle t~70 tracers ~ith tlle antibody, the inllibition level reaching 80^/o at tlle higllest concentrations of the peptide.
The sensitivity of detection as expressed by tlle hormone dose ~7hicll inllibits 50~,~ of tl-le coln7~ination is 10 pg for lTS I and 60-~0 pg for rTS-Zn I.
'Len sarnples of serurn froml normal mice and tell sarnples of serum from thyrnectomised mice ~7ere comparcd for their ability to displace thc com7~ination of tlle two tracers described above with tlle rabbit antiserum. No significant difference is observed ~litll FTS I bet~een tlle serurns emanating frotn nonnal and tllylrlectornised anirnals.
On the otller hand, altllough thc ten s~mples from nonnal mice all displace tlle com7~ination of tlle lTS Zn I, none o~ the serutns from the thymectomised mice (even llen used in undiluted forrn) affects tlle combination.
The displacenlent obtained ~:ith the norrnal serums (diluted to 1/2) varies frorn 40 to 607~o according to tlle s~lr,lples.
The displacement is still significant ~7hen tlle serums are diluted to 1/8th.
III ~ISCUSSIOi~
The Eoregoing results sllo~.7 that the antibodies o~tained 117~991 in ral~bits ater immullisation against syntlletic l TS
combitle l~oth the l;'TS an(l the lTS-~n tracers, albeit to a lcsser extent in the case of l`'l'S-;~n. The synthet:ic ~TS cli spl.accs th2 colllbi.natioll oL the t~`70 tracers with the
117(~991 - ~$ -.SUI.'I7S
'1. Corn,7,~.lrison oE th~ anti~-,c-licity of tlle t~o tracers 'I`lle antiserum obtained by immunisation against the synt,letic I~"rS combines tlle t~,70 tr~ccrs under investigation (I~TS I alld l~TS-i,n I). `~efined as the maximuri-dilution of the ~ntiser~n ~Jhicll produces a 40~,' combination of the radioactive tracer, ~lle strengtll of tlle antiserum is 1/50 000 ~or LTS I and 1/20 000 for FTS-Zn I.
2. 7~isplace~nent of the combinatioll by ~'TS and by the serum of r.orlllal or th~lectornised ~ice 'l'he syn~lletic I~TS inllibits the combination of tlle t~70 tracers ~ith tlle antibody, the inllibition level reaching 80^/o at tlle higllest concentrations of the peptide.
The sensitivity of detection as expressed by tlle hormone dose ~7hicll inllibits 50~,~ of tl-le coln7~ination is 10 pg for lTS I and 60-~0 pg for rTS-Zn I.
'Len sarnples of serurn froml normal mice and tell sarnples of serum from thyrnectomised mice ~7ere comparcd for their ability to displace thc com7~ination of tlle two tracers described above with tlle rabbit antiserum. No significant difference is observed ~litll FTS I bet~een tlle serurns emanating frotn nonnal and tllylrlectornised anirnals.
On the otller hand, altllough thc ten s~mples from nonnal mice all displace tlle com7~ination of tlle lTS Zn I, none o~ the serutns from the thymectomised mice (even llen used in undiluted forrn) affects tlle combination.
The displacenlent obtained ~:ith the norrnal serums (diluted to 1/2) varies frorn 40 to 607~o according to tlle s~lr,lples.
The displacement is still significant ~7hen tlle serums are diluted to 1/8th.
III ~ISCUSSIOi~
The Eoregoing results sllo~.7 that the antibodies o~tained 117~991 in ral~bits ater immullisation against syntlletic l TS
combitle l~oth the l;'TS an(l the lTS-~n tracers, albeit to a lcsser extent in the case of l`'l'S-;~n. The synthet:ic ~TS cli spl.accs th2 colllbi.natioll oL the t~`70 tracers with the
5 ant.i.serurr~ y contrast, lmdcr l:l~e conditions oE these tesl s, the l`'.l'S of b;.ologica1 orig~in prcsent in the serum of norrnal rnice only -inl~ibits the cotnl~ination of the FTS-~n tracer, tlle speci;Eicity of inhibi~ ion being demonstrated by tlle conlplel:e absellce of displaccment ol)served ~7ith the 10 seruin of ~hylllectomised micc.
These rcsull:s confLL n tlle e~ istence Or l;-`70 fonns OL
E'TS, one having picl~ed up a metal, in thc present case zinc, and being bio1Ogica1 actlve and .mtigeni.cal1y close to natura1 honnone and the other, free froln Inetal, being 15 bio1Ogi.cal1y inactive and anti~;enically ciif ferent rom tllyl,lic hormone. Tlle s-yntl-etic FTS wllicll is not passed over Chele~ 100 and ~ ich is probab].y bioloe,ic~l:l.ly activc by virtue of the traces of zinc present in il; (~), delnonstrably includes l~TS free rom zinc. Its content 20 of FTS-Zn or of l TS complex witll other metals was not le termined .
It i.s still difficult to tell wlletller the rabl)its iinm~mised witll syntlletic ~rs produccd t~;/o families of antibodies each recognising one fonn of the peptide 25 (witll or without zinc) or instead only one antibody g iving rise to a crossed reaction between the l~TS ancl the l'TS-Zn (but recognisin the l'TS ~7ith greater a.ffinity t:han the ~TS-~:n). This hypotllesis is compatible ~7ith the significant (rnore tllan 50^~) displacement of the 30 colnbin~tion of the.l;`TS-~n tr~c.er observed both wit:ll sy~ hetic l'l`S and witll natural ~'l`S. It implies that natul;ll l`TS only displaces the l`'l`S-~n tracer to ~7hicil it 1~7(399 1 is sl-ructur.llly closer ~ilan the zinc ~ree l:TS and il~ls no ercct on tlle cornbination o~ the zinc-free-~'rS tr~icer :f:OL
~hlcl~ the ra~it antil~ody llas a greater affinity.
These rcsull:s confLL n tlle e~ istence Or l;-`70 fonns OL
E'TS, one having picl~ed up a metal, in thc present case zinc, and being bio1Ogica1 actlve and .mtigeni.cal1y close to natura1 honnone and the other, free froln Inetal, being 15 bio1Ogi.cal1y inactive and anti~;enically ciif ferent rom tllyl,lic hormone. Tlle s-yntl-etic FTS wllicll is not passed over Chele~ 100 and ~ ich is probab].y bioloe,ic~l:l.ly activc by virtue of the traces of zinc present in il; (~), delnonstrably includes l~TS free rom zinc. Its content 20 of FTS-Zn or of l TS complex witll other metals was not le termined .
It i.s still difficult to tell wlletller the rabl)its iinm~mised witll syntlletic ~rs produccd t~;/o families of antibodies each recognising one fonn of the peptide 25 (witll or without zinc) or instead only one antibody g iving rise to a crossed reaction between the l~TS ancl the l'TS-Zn (but recognisin the l'TS ~7ith greater a.ffinity t:han the ~TS-~:n). This hypotllesis is compatible ~7ith the significant (rnore tllan 50^~) displacement of the 30 colnbin~tion of the.l;`TS-~n tr~c.er observed both wit:ll sy~ hetic l'l`S and witll natural ~'l`S. It implies that natul;ll l`TS only displaces the l`'l`S-~n tracer to ~7hicil it 1~7(399 1 is sl-ructur.llly closer ~ilan the zinc ~ree l:TS and il~ls no ercct on tlle cornbination o~ the zinc-free-~'rS tr~icer :f:OL
~hlcl~ the ra~it antil~ody llas a greater affinity.
Claims (27)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for the preparation of new products from the serum thymic factor (FTS) or its analogs or derivatives, char-acterised in that FTS or one of its analogs or derivatives is treated with a metal or metal-containing protein substance, wherein the said metal is capable of forming a IV coordination complex having a tetrahedral configuration.
2. A process as claimed in claim 1, characterised in that FTS or its analog or derivative is brought into contact with a metal or one of its salts for 10 to 30 minutes at a pH-value of from 7 to 9 and at ambient temperature.
3. A process as claimed in claim 2, characterised in that FTS or its analog or derivative is used in a proportion of 1 mole for 0.1 to 10 moles of metal or metal salt.
4. A process as claimed in claim 2, characterised in that the metal is a divalent or trivalent metal.
5. A process as claimed in claim 4, characterised in that the metal is zinc.
6. A process as claimed in claim 1, characterised in that the metal-containing protein substance is a serum fraction.
7. A process as claimed in claim 1, characterised in that the metal-containing protein substance is a fraction of a thymic extract.
8. A process as claimed in claim 7, characterised in that the metal-containing protein substance is a fraction of the Thymosine Fraction 5 prepared by fractionating the Thymosine Fraction 5 in a column of G-100 (Pharmacia 0.9 cm x 100 cm) in pH 7.3 0.2M phosphate buffer to eliminate the molecule(s) similar to FTS whilst retaining only the eluted fractions with elution volumes of from 15 to 22 ml.
9. A process as claimed in claim 8, characterised in that the FTS or one of its analogs or derivatives is incubated with said fraction, the mixture obtained after incubation is frac-tionated and the fraction with the greatest activity in the rosette test is selected.
10. A process as claimed in claim 9, characterised in that incubation is carried out for 10 minutes to 1 hour at a pH-value of from 6 to 8 and at a temperature of from 30 to 40°C.
11. A process as claimed in claim 1, characterised in that, before it is treated with the metal or metal-containing protein substance, the FTS or one of its analogs or derivatives is treated with a chelating agent capable of chelating metals.
12. A product obtained by the process claimed in claim 1, 2 or 3.
13. A product obtained by the process claimed in claim 4, 5 or 6.
14. A product obtained by the process claimed in claim 7, 8 or 9.
15. A product obtained by the process claimed in claim 10 or 11.
16. A product made in accordance with the process of claim 1, 2 or 3 characterised in that the metal is a divalent or trivalent metal.
17. A product made in accordance with the process of claim 4, 5 or 6 characterised in that the metal is a divalent or trivalent metal.
18. A product made in accordance with the process of claim 7, 8 or 9 characterised in that the metal is a divalent or trivalent metal.
19. A product made in accordance with the process of claim 10 or 11 characterised in that the metal is a divalent or trivalent metal.
20. A product made in accordance with the process of claim 1, characterised in that the metal is zinc.
21. A product made in accordance with the process of claim 1, 2 or 3 characterised in that the ratio by weight of metal to the complex is greater than 1:200.
22. A product made in accordance with the process of claim 4, 5 or 6 characterised in that the ratio by weight of metal to the complex is greater than 1:200.
23. A product made in accordance with the process of claim 7, 8 or 9 characterised in that the ratio by weight of metal to the complex is greater than 1:200.
24. A product made in accordance with the process of claim 10 or 11 characterised in that the ratio by weight of metal to the complex is greater than 1:200.
25. A tracer comprising the product made in accordance with the process of claim 1 labelled by a radioactive isotope.
26. A tracer as claimed in claim 25, characterised in that an iodisable molecule is introduced into the product beforehand and the iodisable derivative thus obtained is subsequently iodised.
27. A process for detecting FTS contained in a sample, characterised in that a predetermined quantity of said sample is contacted with a predetermined quantity of the tracer claimed in claim 25 or 26 and a predetermined quantity of anti-FTS antibody so as to bring about a competitive reaction between the tracer and the FTS of the sample in their associ-ation with the anti-FTS antibody, the quantity of free tracer or tracer associated with the antibody is determined after the reaction has reached a state of equilibrium and the value obtained is compared with the values obtained for known and increasing quantities of FTS.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB8017027 | 1980-05-22 | ||
| GB8017027 | 1980-05-22 | ||
| GB8030507 | 1980-09-22 | ||
| GB8030507 | 1980-09-22 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1170991A true CA1170991A (en) | 1984-07-17 |
Family
ID=26275622
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000378037A Expired CA1170991A (en) | 1980-05-22 | 1981-05-21 | Products prepared from the serum thymic factor or its analogs and derivatives, a process for preparing these products, the use of these products as medicaments, tracers obtained from these products and the use of these tracers in detection processes |
Country Status (8)
| Country | Link |
|---|---|
| EP (1) | EP0041019B1 (en) |
| AU (1) | AU553640B2 (en) |
| CA (1) | CA1170991A (en) |
| DE (1) | DE3166925D1 (en) |
| DK (1) | DK221681A (en) |
| ES (2) | ES8306022A1 (en) |
| GR (1) | GR75636B (en) |
| IE (1) | IE51744B1 (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4634682A (en) * | 1982-11-30 | 1987-01-06 | Sloan-Kettering Institute For Cancer | Radioimmunoassays for the serum thymic factor (FTS) |
| CS256897B1 (en) * | 1984-12-17 | 1988-04-15 | Evzen Kasafirek | Series thymic factor's analogue-type peptides |
| IT1188550B (en) * | 1986-02-07 | 1988-01-14 | Sclavo Spa | SYNTHETIC PEPTIDE WITH INTERLEUKINA 1 HUMAN ACTIVITY |
| FR2830451B1 (en) * | 2001-10-09 | 2004-04-30 | Inst Nat Sante Rech Med | USE OF THYMULIN-LIKE PEPTIDES (PAT) FOR THE MANUFACTURE OF PAIN MEDICATIONS |
| CN101400322B (en) | 2006-03-20 | 2013-05-29 | 史密夫和内修有限公司 | Acetabular cup assembly for multiple bearing materials |
| FR2930156B1 (en) * | 2008-04-21 | 2011-10-28 | Cll Pharma | USE OF NONAPEPTIDE PAT IN THE TREATMENT OF AUTOIMMUNE DISEASES. |
| WO2021030689A2 (en) * | 2019-08-15 | 2021-02-18 | Cytolyf Therapeutics Llc | Zinc activated thymulin and methods of preparation and administration |
-
1981
- 1981-05-20 GR GR65000A patent/GR75636B/el unknown
- 1981-05-20 DK DK221681A patent/DK221681A/en not_active Application Discontinuation
- 1981-05-21 EP EP19810400807 patent/EP0041019B1/en not_active Expired
- 1981-05-21 CA CA000378037A patent/CA1170991A/en not_active Expired
- 1981-05-21 AU AU70905/81A patent/AU553640B2/en not_active Ceased
- 1981-05-21 IE IE113581A patent/IE51744B1/en not_active IP Right Cessation
- 1981-05-21 DE DE8181400807T patent/DE3166925D1/en not_active Expired
- 1981-05-22 ES ES502911A patent/ES8306022A1/en not_active Expired
-
1982
- 1982-12-16 ES ES518298A patent/ES8501628A1/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| GR75636B (en) | 1984-08-02 |
| DE3166925D1 (en) | 1984-12-06 |
| ES518298A0 (en) | 1984-12-16 |
| ES502911A0 (en) | 1983-05-01 |
| ES8501628A1 (en) | 1984-12-16 |
| ES8306022A1 (en) | 1983-05-01 |
| EP0041019A3 (en) | 1982-03-10 |
| AU553640B2 (en) | 1986-07-24 |
| AU7090581A (en) | 1981-11-26 |
| DK221681A (en) | 1981-11-23 |
| EP0041019B1 (en) | 1984-10-31 |
| EP0041019A2 (en) | 1981-12-02 |
| IE811135L (en) | 1981-11-22 |
| IE51744B1 (en) | 1987-03-18 |
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