11 7~9~t 1
-- 2 --
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()~
-- 4 --
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
-- 8 --
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.
EXAMPLE 1
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.
EXAMPLE 2
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
-- 10 --
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
- 12 -
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
16 -
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
- 17 -
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
- 19 -
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~.
- 20 -
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~
- 23 -
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~
- 26 -
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
- 29 -
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
- 30 -
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.
