Note: Descriptions are shown in the official language in which they were submitted.
3~7
M~DICAMEl`TT CO~i~l?RISII~TG T~E I'~ODIJCT OF T~TE ~E~CT10?~
, . . . ~
OF ~ C1 TO C6 ~ ~
The invention relates to a medicamen-t comprising
a5 active substa.nce the produc-t of the reaction - hereinafter
called "product" - of a C1 -to C6 carboxylic acid, par-ticularly
bu-tyric, propionic, hexanoic and ace-tic a.cids, on a. ba.sic
amino acid of -the group constituted by arginine and lysine;
the produc-ts so obtained are called below bu-tyrate7 propionate,
hexanoa-te and a.cetate.
It is directed also at the medicamen-t comprising
the above-sa.id product and interferon.
It is also direc-ted at the medicament comprising
-the a.bove-said product and a.n immunostimulant agent.
It is directed la.s-tly at m.edicaments compri3in~
the above-said product, interferon and a.n im~unos-timulant
agentO
Among the above~said produc-ts~ the butyrate i5
particularly preferred.
The sodium salt of butyric acid has been described
as increasing the antitumoral effect and the antiviral effect
of interferon.
Admi~istered alone, sodium butyrate doesno-t hnve
any of these two effects.
It was, consequently, impossible -to foresee that
butyrates, propionates, hexonoates and acetates of the above-
said b~sic amino acids, which had never been used hitherto in
medicine, would have, applied alone, an antiviral and anti-
tumoral action~
,,~
~ 0~7
It is however this that the inventors have had -the
merit of demo~strating at the end of extensive research.
It follows that the medicamen-t according to the in-
vention is characterized by -the fac-t that it comp:ris~s~ as
acti.ve substance, at least olle of -the products of the reaction
of a. ~1 to C6 carboxylic acid on a.rginine or lysine, prefer-
ably arginine or lysine butyrate.
~ pplicants have also shown tha-t the product con-
cerned and par-ticularly said basic amino acid butyra-tes
stimulated the antitumoral action of interferon and of immuno-
stimulant agents~
It follows that ~he medicarnent according to the in-
vention is also characterized by the fact that it comprises,
as active substance, at leas-t one of the products concerrled
and, preferably, arginine or lysine butyra-te as well as int.er-
feron and/or an immunostimulant agentO
~ he medicament according -to the inven-tion is also
characterized by the fact -that in a.ddition to -the active sub~
stance it comprises a no ~ oxic and pharmacologically accept-
able excipient.
The medicarnent according to the inverltion i5 ad.~in-
istered parenterally, intravenously or orally.
It is a.dvantageously presented in pharrnaceutical
forms constituted by isotonic solutions of the product (admin-
isterable, for example, intravenously) or by pow~ers or drink-
able ampoules for oral adrninistration, comprising the bu-tyrate
in freeze-dried form in the case of powders a.nd in aqueous
solution form in -the case of drinkable ampoules~ In the case
3~7
of arginine butyrate, the iso-tonic solution contains 178
millimoles o~ produc-t per litre of solution. It is interes-ting
to note tha-t the butyrate-based medicament does no-t have the
unpleasant smell of butyric acid.
V~len the medicament co~nprises also interferon and/or
certain immunostimulant agents, the parenteral route is
possible~ In -this case, -the medicamen-t comprises the two or
even three ac-tive principles if necessary in separate packagings~
The possibility of having one in the presence of the other
particularly the bu-tyrate and interferon7 also exists.
The ef~ec-tive amount
- of product, preferably basic ami~o acid butyrate and,
possibly,
- interferon and~or immunostimulant agent
is that which~ administered to the mouse, causes;
- an an-tiviral effec-t de-tectable by the survival o~
mice which have been infec-ted with 100 -times the LD50 o~--the
virus of encephalomyocarditis as regards the product, par-
ticularly the basic amino acid butyrate~ taken alone (the
presence of interferon and/or immunos-timulan-t agent being able
to further imProve the results),
- an anti-tumoral effect detectable by the increase
in the average survival of mice to which an asci-tes tumor has
been grafted, as regards the product, particularly the basic
amino acid butyrate, w~ether used alone or conjointly with
interferon andjor wi-th the immunostimulan-t agen-t.
The daily dose may be, as regards -the product, par-
ticularly the basic amino acid butyra-te, used alone, comprised
between 0~2~ mg and 0.56 mg per g of mouse (which corresponds
to the amount a.dministered to a mouse of 25 g by one demi-
millilitre of solution containing 50 millimoles per litre
as reg~rds the lower limi-t and con-tainin~ loO millimoles per
litre as re~a.rds the upper limi-t).
0-ther tcsts have shown that -this dose could be
further not~bl.y lowered, and this -to a range of about 0.07 mg
to 0.009 mg o:f product per gram of mouse (which corresponds
to -the administration to a mouse of 25 g of a demi-milliliter
Of solution with 12.5 mmoles per litre as regards the upper
limit and a solu-tion of 1 6 mmoles per litre as regards the
lower limit).
In the ca~e of administration to men, it is possible
a.lso -to expect that the probable dose of 10 -to 20 g of active
subs-tance per day could be loweredg par-ticularly -to 0.5 g
daily.
When conjointly the product, notably the bu-tyrate
and interferon and/or the immunostimulant agent are used,
- -I;he dose o~ produc-t, notably of butyra.te 7 iS
20unchan~ed,
- the dose of interferon administerable to man is
comprised between 1 and 5~106 in-terferon units,
- the dose of immunostimulan-t agent is a function
of the nature of the la-tter.
25For example, in the case of corynebacterium parvum
-taken as immunostimulant agent, -the single dose of the treat~
ment (applied 18 hours before the experimental cell graft, at
the time of the latter or three days later~ is 200 microgra~s
~L2~03~7
~or a 25 g mouse. In -the ca.se of adminis-tration to man, this
dose is that ~enerally used for this produc-t in -the applica.-
tions already }mown.
The product, ~articula~ly the ba.sic am:ino.acid
r~ butyrate, is pre~erably admi.n:istered several times, and this
is the s~e for interferon~
The dosage is ada.pted as a func-tion o~ -the subject
and of the disease -trea-ted.
The immunostimulant agen-t may be:
- cor~nebacterium parvum,
: ~ isoprinosine,`
~ s-taphylococci extract,
- cimctidine,
- levamisole,.
1~ ~ particularly preferred medica.rnen-t accordin~ -to
the invent;ion is based on:
- ei-ther the product of the rcaction of a C1 to C6
carboxylic acid, preferably hexanoic acid and, more preferably
still, butyric acid on arginine and comprlses possibly inter-
feron and/or an immunostimulant agen-t,
- or the produc-t o~ the reaction of a C1 to C6
carboxylic acid, preferably hexanoic and, more preferably
still3 butyric acid on lysine and comprises possibly intcr~
feron and/or a.n immunostimulan-t agent.
~5 The absence o~ ~toxicity of the procluct, particularly
of the basic amino acid butyrate, at the above-said dose~ ha.s
been demonstrated by the experiments carried out on -the mouse.
In fact, administered a.t the dose of 5 to 10 ~/kg
of body weigh-t daily to a group o~ 15 mice, no death was
3b7
observed a-t the end of 100 da.ys.
The therapeutic coefficients of interferon and of
i~m~lnostimulan-t agents, particularly those mentioned abov~e,
are wcll Icnovm and th~re is no need, consequen-tly, to discuss
~he mat~er a~aill hereO
The excipien-t en-tering into ~he consti-tution of
tho medic~ment according to thc invention may be dis-tilled
water or a~a.in a buffered solu-tion comprisin~ if nece3sa.ry
the usual preservative agents.
Prepara.-tion of products of the reaction of a C1 -to C6
.carboxylic acid, particularly of basic amino acid bu-tyra.tes
entering in to the constitu-tion of -the ac-tive subs-tance of
-the mcdicamen-ts according -to the invention, ma.y be carried
ou-t by nQu-traliz~-tion of the acid, particularly oI butyric
acid, by the basic amino acid -to a pII 7~ or even to p~I 7.30
By way of example, there a.re indicated-the prepara~
tion of ar~ininc and lysi~e butyra-tes.
The two bu-tyrates concerned are prepared by addi.n~
drop by drop the basic amino acid to the bu-tyric acid and by
fol].owin~ the development of the plI wi-th a pH meter. Meutrali-ty
is obta.ined, -that is to say the butyrate is prepared by the
addition of one mole of bu-tyric acid to one mole of basic
amino a.cid, The salt obtained does not have the unpleasan-t
smell of bu-tyric acid.
The physical and/or chemical constants of arginine
butyrate a.re~
- empirical formula : C10~224N4 2
- molecular weight : 28003
3'7
- melting point : 145C
~ rotatory power : 16~6
- pil at 1~o ~ 7~3
The experiments which ha.v~ ~na.bled ~pplican-ts to
dcmonstratc the antiviral activity of the p:roduc-t and pa.r-
ticularly o~ -the basic amino acid b-utyrate taken alone and
the an-titumoral activi-ty of -this product alone or when it is
applied conjoin-tly with interferon and/or an immunostimulant
agent, will now be described~
First of a.ll, the experiments showing -the antiviral
activity of the basic acid butyrate taken alone are described.
In -these experiments, the antiviral a.ctivl-ty aga.inst
the le-thal effect of 100 ~D50 of thc virus of encephalomyo-
cc~ditis is shown,
The laboratory animals used were mice of the "Swlss"
type comin~ from a breeding unit belonging to l~pplicants
The virus employed was that of encephalomyocarditis
of the mouse (EMC) of the Mengo strain cul-tivated on ~ 929
murine cells.
20 The titer of -the virus suspensions was determ1.ned
by the "pla~e method" again using ~ 929 murine cells. It is
assumed -that 5 pla~e-forl~ing units (PFU)~ correspona -to a
lethal dose LD50
The virus is innoculated intrap~ritoneally.
. The experimen-tal process consists of innocula-ting
to groups o~ 15 a.nima.ls:
- in a f.irst stage, arginine butyrate (0~56 mg per
gram of mouse) to one group and lysine butyrate
(0.47 mg per gram of mouse) -to another group,
~2~ 93~7
- in a second stage, after about 18 hours, 100 ~50
of virus.
The following experiments are also carried out, by
way of comparison:
- pretrea.-tment o:~ the anim~ls by sodium butyrate
before innocula-tion o~ the EMC virus,
- trcatment by arginine or lysine butyrate simul-
-taneously with the injection o~ the virus~
Groups of control mice were treated by the Vi~lS
alone and -the so].vent (isotonic medium) alone.
By carryin~ out -two series of experiments with
groups o~ 15 mice9 it was observed tha-t, a~-ter 60 days~
- in the control groups, 94~0 (85 out of ~0) o~
the animals were dead,
- in -the groups trea-ted with sodium butyrate9 90~0
(~1 out of 90) of thc animals died; i-t was no-t
possible to speak o~ protec-tion (the significant
percentage ~enerally named p is higher than 95 %
it being recalled that the definition o:f "p" can
be found in the work o~ D. Schwar-t2, "S-ta-tistical
Me-thods for the Use o~ Doctors ~nd Biologists",
edited by Flammarion),
- in the group5 treated wi-th lysine butyra-te~ 75~0
(34 out of 45) o~ -the a~imals died (p < 0001),
- in the groups -treated with arginine bu-tyra-te, 77~0
(58 ou-t of 75) o~ the anima.ls died (p ~ 0.()1)~
3~
- in -the groups treated either with arginine buty-
ra-te, or with lysi~e butyrate, simultaneously
with the injeGtion of -the virus, no protec-tive
ac-tion was detected,
The experimen-ts showing the antitl~oral effect of
the basic amino acid butyrate when it is applied either alone,
or parallel with interferon and/or an immunostimulant agent~
have been described.
It is emphasized in fact tha-t the basic amino acid
butyrate~ particularly of arginine or of lysine, has a signi-
ficant antitumoral activi-ty when i-t is applied alone 9 this
activity becoming more marked on simultaneous administra-
tion with one at least of the two above-mentioned o-ther ac-
tive substances~
The tumor used in the experiments r~hich ~ollow is
a mlltant of the Crocker sarcoma 180 TG which re3ists treat-
ment by puric derivatives.
The experimental animals were again ~wiss mice o.E
the above-mentioned breed.
They were innoculated with the sarcoma intraperi-
toneally using a suspension 106 cells/0.5 mlO The amount
innoculated was 0.5 ml~
The experiments were carried out using;
- arginine or lysine butyrate,
- interferon,
- corynebac-terium parvum (or CP) of the Merieux
6 trainO
The arginine butyrate was applied in the :~orm and
amount similar to what was indieated above :for the antiviral
3~
o
activi-ty.
It was the same for the lysine butyrate~
The CP was administered in the experimen-ts which
follow in the form of a single dose applied at -the time of the
~a~t, or three days a.fterwards and were in -the :~orm o~ aln-
poules o:E 2 ml comprisinS 4 mg dry weight of germs k:illed
wi-th ~ormol and heat; 0.1 ml of the subs-tance was injected into
the mouse in-traperitoneally.
Nine independent experiments each bearing on several
groups of 15 animals were carried out~
In the first of these experimen-ts (control group),
the tumor alone was gr~fted on the anima.lsO
The anim.als of the other groups were also grafted
with -the above-said -tumor; they were in addi-tion -treated:
- either wi-th arginine butyrate (second experiment),
- or with lysine ~utyrate tthird experimen-t),
- or wi-th interferon (~ourth experiment)~
_ or with CP (f`ifth experiment),
- or wi-th arginine butyrate plus interferon (six-th
experiment?,
- or with lysine bu-tyrate plus interferon (seven-th
experiment),
- or wi.th arginine butyra-te plus CP (eighth experi-
ment),
c5 - or with ar~inine butyrate plus CP plus interferon
(ninth experiment)~
In the second and third experiments, the ba.sic
amino acid butyra-te was applied intraperitoneally in -the pro-
3'7
11
portion of 0.5 ml of a fifty times millimolar solution per
mouse (that is to say in the proportion of respectively 0.28 mg
and 0.24 mg per g of mouse), thrice weekly (at 48 hour inter-
vals) or three weeks.
In the ourth exper.iment, interferon was applied in
the proportion o:E 20,000 to 50,000 I.U. in a volume of 0.5 ml
administered to the animal three times weekly (at 48 hour
intervals) for three weeks.
In the fifth experiment, the CP was applied in a
single dose of 200 micrograms/0.1 ml per animal, administered
intraperitoneally at the same time or three days after the
cell grat.
In the sixth to ninth experiments, there was applied,
according to the case, the basic amino acid butyrate, the
interferon and/or the CP at the same doses, at the same
rhythm and at the same moments as when these products were
applied alone in the previous experiments.
The results o~ these experiments are apparent on
examining appended ~igures 1 to 9 which are histograms cor-
responding respectively to experiments 1 to 9.
These histograms show, as a unction of time ~number ofdays T), the number N of surviving animals and, among these
surviving animals, the number of animals bearing detectable tumors,
Thus the number of surviving animals is plotted, for
a given day, as ordinates in the form of a column showing the
number of animals and within which the number of animals
bearing tumors is shown by hatched portions.
From examination of the histograms of Figures 1 to 9,
it is apparent that:
37
12
- in the fir3t e}~periment, all the animals had a
tumor on the -tenth day; after 26 days, only 2
surviving animals out of 75 a-t -the s-tart remained,
both bea,rin~ tumors and which died on -thc 28th
day;
- in the second e~perimen~t, ~3 out of 75 .mimals
~ore tumors on the ten-th day; af-ter 50 days, there
remalned ~ animals of which 2 bore tumorsO A~-ter
a period of obse:rva-tion of 100 days, 3 mice sur-
vived finally from the cell ~aft;
in the -third e~periment~ 32 out of 45 animals bore'
tumors on the ten-th day; a.fter ~4 da,ys, therc re-
mai.ned 2 animals of.which 1 bore a tumor. ~-Etcr
a period of obscrvation of 100 day~, n ~in~le
animal sur,vived;
in -the ~our-th experimen-t, 3fi out of 60 animals
bore tumors on the ten-th d~,y and 5 ~urvived af-ter
100 days o~ observa.-tion;
- in -the :~ifth experiment, all ha.dtl~Ors on -the
tenth day and one only remained alive after the
period of observa-tion;
- in the sixth e~periment, only 13 animals ou-t of 75
bore -tumor~ on the ten-th day a.nd 10 mice survived
af-ter 100 days of observation;
- in -the seven-th experiment, 30 animals out of 45
bore tumors on the tenth day; after 49 aays, there
rem~ined 7 survivor3 of which none bore a tumor
and af-ter the period of observa-tion of 100 da.ys
9037
4 mice definitely survived ;
- in -the eighth experiment, 48 ou-t of 75 anima.ls
bore tumors on the -tenth day and 1~ mim<lls survived after
100 days of observa-tlorl;
- ln the ninth e7;pcrirnellt~ 25 out of 75 animals
bore tumors on -the tenth day and 33 anima.ls s~urvi~red a.f-te.r a
period of observa.-tion of 100 days.
0-ther experimen-ts show that -the amounts administered
can be considerably reduced.
With regard to an-tiviral a.c-tivi.-ty, these ex-periments
were carried out using solu-tions of arginine bu-tyra-te titrating
rcspcc~tively:
25 mmoles/litre
12.5 mmoles/li-tre
6,25 mmoles/li-trc
3.15 mmoles/li-tre
1.5 n~oles/litre
a~d for each experiment 0.5 ml o~ the solutlons per mouse of
25 ~ were administered.
Interferon was adminis-tered a-t the ra-te of 20,000
to 25,000 internationa.l units per mousc intraperitoneally in
a volume of 0.5 ml and the immunostimulan-t, namely corynebac-
teri~n parvum~ Merieux strain9 lNas injec-ted i.n to -the animal
intraperitoneally in the ra-tio of 200 ~g/0.1 ml mouse.
The antiviral action was studied u~der the same
condi-ti.ons as aboveO
~ In -the graphs (see Figures 10 to 13), there is shown
the development o~ survival (numbe.r of surviving mice M as a
14
func-tion oF -thc numbcr of days clapsed T) in group of 15
mice infected with the Er,lC virus (100 ~50 of virus, 0.5 ml
intraperi.toneally) to which had been administered:
within -the scope of the expe.rimcnt of Figure 10,
a.rgirlinc bu-tyratc a.t -the conccntrations ind-lcated r..bov~,
associa.-ted with inter.Eeron and wi-th cory~lebacterium pa.rvum:
the curves C1 to C5 correspond. respec-tively to conccntra-tions
of 25, 12.5, 6.25, 3~15 and 1.5 mmoles/litre; the curve C6
corresponds to the con-trol group -trea.-teci wi-tll i.nterferon alone
and the curvc C7 -to the untrea-ted control grollp;
- within the scope of the experimen-t of Fi~ure 11,
the arginine butyrate at the concentra.-tion indicated above,
assoclated wi-th the corynebac-terium parv-um alone: the c~ve-
C1to C5 correspond respectively to the a.bove-sai.cl conccn-tra-
tions; 7the curves C6 and C7 o~rrespond to the con-trol ~roups,
as indicated for Fi~lre 10;
- wi-thin -the scope of the experi]nent of Fi~ure 12
-the ar~inine butyrate at the concentra-tions indica-ted abov~3
associa-ted with interferon: the curves C1 to C5 correspond
r~5pec~tiVelJ t? the above-said concentr~tions; the curves
C6 and C7 correspond to thc control ~roups, as inclicated in
rc 10;
- withi.n the scope of the experiment of Fi~ure 13,
the arginine butyrate at the concentra.tions i.ndicated above,
used alone: the curves C1 to C7 a~re identified as above.
From ex~ina.tion of the graphs of Figrures 10 -to 13,
i-t re;ults that:
~2~33
- in the case of the association butyrate plus
immllnostiml1lant plus in-terferon a~d in -the case Or the a_so-
ciat;ion bu-tyra-te plus lmmullostimulcmt, -the most effec-tlve dose
of bul~r~-te is t;h,lt of 0,~7 m ~ ~ of mouse body wcig,ht;
' - in -the case of lhe assoclatioll Orc` b~ltyrate with
intcrferon L~d in tha-t of bu-tyrate alone~ -the mos-t ef~ective
dose of bu-tyrate was 0.0175 mg/g Or mouse body wei~h-t.
In respect o~ an-titumoral activi-ty, it h~s also
been shown that tthe amoun-ts adminis-t;ered could be loweredO
The correspondin experimen-ts were carried ou-t using
-the following concentra-tion~s and amounts;
- adminis-tration of 0.5 ml per mouse of 25 g of ~n
arL~:inine butyrate solution of concentration 12.5 mmoles per
lltre, tha-t; l~ to say 0.07 mg per ~ of mouse body weight;
- administration of 0~5 rnl per mouse of 25 g of ~m
arginlne butyrate solution of concentration 6~25 mmoles per
litre, ~hat ls to say 0.035 mg per g of mouse body wci,ght.
The animals received thc tumoral graft (106 Cxocker
1~0 TG cells/0.5 ml/mouse intrapcri-toneally) at day 0. Treat;-
ment ~Nlth the butyrate and the interferon ~20,000 to 25,000 I.IJ,
intraperl-toneally) was s-tarted on the third day. Thls -tre,at-
ment was contlnued,for three weel~s at -the ra-te of one bu-ty-
rate injection followed af-ter 24 hours by an interferon in-
jection? alterna-tely over -the whole period of -treatmen-tO
The results obtained are shown in -the histo~rams of
~igures 14 -to 19 which are established lil~e -those of Figures
1 to 9~
~ The histogram of FiO~ure 14 corresponds to the control
(graftea mice untreated) and shov~s -that all the mice
~Z~3
16
bore t~ors on -the tenth day and -that none survived beyond 31
days. The histogra.m of Fi~lre 15 (grafted mice treated with
interferon alone) shows -that inter~eron slightly slows down
the establi3hment of the tumor, only 7 out o~ 13 animals were
bea.rers o:E t~ors on the tenth day; 4 animals ou-t of 15 sur-
vived a.f-ter a. period of observation o~ 52 days).
The histograms of Figures 16 and 17 (mice grafted
and treated at the dose of o.O7 mg per g mouse body wei~ht
with butyrate respectively a.lone, Figure 16, and associa-ted
with interferon, Figure 17, show that the effec-t of the bu-ty-
rate alone is considerable since at this ~o~e no anima.l had
a tumor on the tenth day. At the end~of -the ob'servation
period, 9 ou-t of 15 mice survived of which 1 'had a tumor~
Similar results were obtained when the treatment was combined
with interferon (Figure 17). In this ca.se~ the slo~ing dol~n
of the establishment of the graft was considerable and 9 mice
survived after -the observa-tion periodt
~ he histograms of Figures 18 and 19 correspond to
the test of the butyra-te at the dose of 0.035 mg/g of mouse
body weight administered alone (Figure 18) and associated with
interferon (Figure 19).
The results obta.ined with this dooe of arginine
butyrate seem as good as for the double dose~ especially when
the amino acid is associated with'interferon (Figure l9). Very
few animals.were then carriers of tumors and in certain animals,
the la-t-ter regressed definitely. After an observation period of
52 days, 9 out of 15 mice survived~ whereas with butyrate alone
there remained ~ out of 15 of which 1 still bore a tumor.
.
37
17
. In general, it appears that in association with
in:terferon the lower doses of butyrate seem to possess
an effect equal to or superior -to that at ~reater doses.
The whole of the results shows that arginine or
lysine butyrate3 possess their own an-titumoral action which
is significant and comparable with that of interferon, at
least under the experimental conditions presen-ted here. It
should be emphasized -that the amounts of interferon administered
correspond to the optimum doseO The adminis-tration of the
basic amino acid butyrates with a single injection of CP
con~iderably increases -the effects. In addition, -the arginine
or lysine butyrates act in synergy wi-th the interferon. ~he
adminis-tration of arginine butyrate with an i~nunostimulant
agent and with interferon result in a considerable incre~se
in the total protection which approaches, ln -this ca.se9 50%
definitely surviving.
Similar e~periments to those which ha.ve just been
described7 which have been carried out with arginine propionate
(0.252 m~ per g of mouse) and arginine hexanoate (0.29 mg per
g of mouse) with respect to the antiviral properties of -these
products, have shoMn the survival read at the moment when,
in the series of control animals treated only with the vi.rus 9
mortality ceases, is
- 18 out of 30 animals in the case of arginine
propionate
- 11 out of 30 anima.ls in the case of arginine
hexanoate, a.nd
- 5 out of 30 çontrol animals which were only
trea.ted with the virv.s.
1~
~ in the case of the preceedin~ experiments, tJhis
survival is found to be considerably increased when interferon
is administered with the arginine propionate or arginine
hexanoa-te.
As is self-evident and 3S emerges already from the
foregoin~ the invention is in no way limited to those
types of a.pplication and embodiments which have been more
especially envisaged; it encompasses, on -the contrary, all
modifications.
