Note: Descriptions are shown in the official language in which they were submitted.
ORIGIN OF INVENTION
The invention described herein was in part made in
the course of work under a grant or award from the
National Institute of Health, Department of Health,
Education and Welfare.
BACKGROUND OF INVENTION
Leukemia is an acute or chronic disease of unkown
cause in man and other warm-blooded animals. It is
characterized by an abnormal increase in the number
of immature leukocytes in the tissues of the body and
in the circulating blood. The disease apparently
affects the blood-forming organs, and is classified
according to the type of leukocyte that is being
proliferated abnormally. The disease is one of a
number of forms of neoplastic disease, and the
development of drugs for amelioration or curing the
disease has occupied the attention of research organ-
izations for many years, and until most recently
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without appreciable success.~ Today, many forms of
leukemia can be effectively treated with drugs. In
the case of combination chemotherapy with acute
lymphocytic leukemia in children a large percentage
(50 - 60%) of five year survivals are obtained, and
the disease is now classified as curable.
DF,SCRIPTION OF THE INVENTION
In accordance with the invention, it has been deter-
mined that leu~emia, as well as other malignancies,
including ascitic tumors, can be ameliorated in
warm-blooded lower animals by the administration of
10-deazaminopterin, a nontrivial analogue of metho-
trexate, the current drug of choice for the treatment ~~
of these malignancies in the clinic, and it is ex-
pected that 10-deazaminopterin will have a similar
effect in humans.
10-deazaminopterin has the structure:
'
J ~ ~3COIIIICU
CO~H
A~ 3
. ~ .
~ . . .
'.. . . . ' -
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The relationshlp between this ~ompound and the N-10 methyl
derivati~e o~ ?,minopterin, methotrexa~e, ls apparent ~rom the following:
N~N~CN,I ~3 CH,
H2 N CH5~
/~OOH
4-Amino-4-deoxy-10-deazapteroic acid, a key intermediate for
6ynthesis of 10-deazaminopterln, was fir6t prepared byDeGraw, Brown,
Kisliuk and Gaumont, Journal of Medicinal Chemistry 14 866 (1971)/.
DeGraw, Tsakotellis, Kisliul~, and Gaumont, Journal of Heterocyclic
Chemistry 8 105 (1971) had reported the potent growth-inhibitory activity
of 10-deazapteroic acid and its tetrahydro derivative against Streptococcus
faecium, a folate-dependent organism. Activity was greatly enhanced by
reduction to the tetrahydro compound . Accordingly, it was thought that the
2, 4-diamino-pteridines ~hould be investigated, because they would be
expected to be more capable of cell penetration, and among the 2, 4-diamino-
pteridines prepared was 4-amino-4-deoxy-10-deazapteroic acid, the com-
pound shown under the Scheme I, Series d, at page 867 of the article.
In the Journal of Miedicinal Chemistr~ 17 552 (1974) DeGraw,
-- . :
Kisliuk, Gaumont, Baugh and Nair reported on the synthesis and anti~olate ~:
activity of 10-deazaminopterin. The antimlcrobial and antitumor activities
of the powerful dihydro~olic reductase tnhibitor6 aminopterln and it~
N-10 methyl derivative, methotrexate, are well known, and numerous
analogues have been made to further tmprove the potency, cell paletration
~nd to~lcity properties of these compounds. As part ~ a continuing program,
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to investigate structure-activity relationships in
folic acid analogues, DeGraw et al. were interested
in the effects of replacement of the nitrogen atom
in the side chain of aminopterin, and reported on the
synthesis and biological activity of 10-deazaminopterin
in this paper. Continuing work with 10-deazaminopterin
has now led to the discovery of its antileukemic
activity and to its efficacy in treating various
~scites tumor systems.
The process of treating leukemia and ascitic tumors
according to this invention comprises administering
to a warm-blooded animal having an abnormal proportion
of leukocytes or other evidences of the malignancy,
a therapeutic nontoxic amount of 10-deazaminopterin,
said material being employed as such or in the form of
a pharmaceutically acceptable salt thereof. These
salts are formed with one or more free NH2 groups of
10-deazaminopterin.
The acid addition salts are preferably the pharmaceutically
acceptable, nontoxic addition salts with suitable acids,
such as those with inorganic acids, for example,
hydrochloric, hydrobromic, nitric, sulphuric and
phosphoric acids, and with organic acids, such as
organic carboxylic acids, for example, glycolic,
maleic, hydroxymaleic, malic, tartaric, citric,
salicylic, o-acetyloxybenzoic, nicotinic and iso-
nicotinic acid, and organic sulphbnic acids, for
108572g
example, methanesulphonic, ethanesulphonic,
2-hydroxyethanesulphonic, toluene-p- 8 ulphonic,
and naphthalene-2-sulphonic acid.
An acid addition salt can be converted into the
free compound according to known methods, for
example, by treating it with a base, such as
with a metal hydroxide or alkoxide, for example,
an alkali metal or alkaline earth metal hydroxide,
for example, lithium hydroxide, sodium hydroxide,
potassium hydroxide or calcium hydroxide; with a
metal carbonate, such as an alkali metal or an
alkaline earth metal carbonate or hydrogen carbonate,
for example sodium, potassium or calcium carbonate
or hydrogen carbonate; with ammonia; or with a hydroxyl
ion exchange resin, or with any other suitable reagent.
An acid addition salt may also be converted into
another acid addition salt according to known methods;
for example, a salt with an inorganic acid may be
treated with a metal salt, for example a sodium,
barium or silver salt, of an acid in a suitable
diluent, in which a resulting inorganic salt is
insoluble and is thus removed from the reaction
medium. An acid addition salt may also be con-
verted into another acid addition salt by treatment
with an anion exchange preparation.
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The 10-deazaminopterin can be administered ~ se,
or in association with a pharmaceutically acceptable
diluent or carrier. The invention accordingly also
provides a pharmaceutical composition in dosage
unit form comprising from 0.1 to about 500 mg of
10-deazaminopterin, per dosage unit, together with
a pharmaceutically acceptable nontoxic inert carrier
or diluent therefor.
lO-Deazaminopterin or salt thereof can be administered
to the animal by any available route, including oral
and parenteral (intravenous, intraperitoneal, sub-
cutaneous, and intramuscular) administration. The
amount administered is sufficient to ameliorate the
leukemia or the ascitic tumor and will depend upon the
type of malignancy, the species of animal, and the
weight of the animal. For example, in human adminis-
tration, a dosage of 10-deazaminopterin within the range
from about 0.1 mg/kg to about 500 mg/kg per day should
be sufficient. Dosages in the higher part of the range,
approaching 500 mg/kg, are normally administered in
con~unction with leucovoran (dl-5-formyl tetrahydrofolate)
to avoid toxicity. In the treatment of lower test
animals, a similar dosage range is therapeutic. The
upper limit af dosage is that imposed by toxic side
effects, and can be determined by trial and error for
the animal to be treated, including humans.
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To facilitate administration, lO-deazaminopterin
or salt thereof can be provided in composition
form, and preferably in dosage unit form. While
the compound can be administered Per se, it is
normally administered in conjunction with a pharma-
ceutically acceptable carrier therefor, which
dilutes the compound and facilitates handling.
The term "pharmaceutically acceptable" means
that the carrier (as well as the resulting
composition) is sterile and nontoxic.
The carrier or diluent can be solid, semisolid,
or liquid, and can serve as a vehicle, excipient,
or medium for 10-deazaminopterin. Exemplary
diluents and carriers include lactose, dextrose,
sucrose, sorbitol, mannitol, starches, gum acacia,
calcium phosphate, mineral oil, cocoa butter, oil of
theobroma, alginates, tragacanth, gelatin, syrup,
methyl cellulose, polyoxyethylene sorbitan
monolaurate, methyl- and proply-hydroxybenzoate,
talc or magnesium stearate.
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For con- enience ln handllng, the 10-deazamlnopterln and carrier
or diluent can be'enclosed or encapsulated in a capsule, fiachet, cachet,
5 gelatin, paper or ot1-,er contalner, especlally when Intended for ufie In
dosage unlts. The dosage unlts can for example take the form of tablets,
capsules, suppos~t~ries or cachets.
The ~ollowing Examp1es lllustrate various forms of dosage unlts
in whlch the 10-deazaminopterin or salts thereo~ can be prepared:
EXAMPLE 1
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Tablet formulation _Mg/tablet
10-deazamlnopterin 15
Lactose 86
Corn starch (dried) 45 5
Gelatln 2. 5
agnesium stearate 1. 0
I'he 10-deazaminopterin is powdered and passed through a
mesh sleve and well mlxed wlth the lactose and 30 mg of the corn starch,
both passed throu~h a sieve.
The mixed powders are massed with a warm gelatin fiolution,
prepared by ~tlrrlng the gelatln ln water and heatlng to form a 10~'C w/w
601ution. The mass is granulated by passing through a sieve, and the
moist granules drled at 40C.
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The dried granules are regranulated by pa~sing through a sie~e
and the balance of the starch and the magnesium stearate ~ added and
thoroughly mixed.
The granules are compressed to produce tablets each we~ghing
150 Mg.
EXAMPLE 2
Tablet formulatlon M~/tsblet
10-deazamlnopterin, 100
Lactose ~9
Corn 6tarch (dried) 80
Gelatin 4. o
Magnes~um stearate 2. 0
The method of preparation ls identical wlth that of Example 1
except that 60 mg of starch ls used ln the granulation process and 20 mg
durlng tabletting.
XAMPLE 3
Capsule formulatlon M~/ca~sule
10-deazamlnopterln 250
Lactose 160
The 10-dea&amlnopterln ~nd lactose are passed throu~h a
aleve and the powders well ml~ed tog~ther beîore filllng Into hard gelatin
capsules of sultable sl&e, BO tbat each capsule contain~ 400 mg of mked
powder~.
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EXAMPLE 4
Suppositorles ~/supposltory
. lO-deazaminopterin 50
Oil o~ Theobroma
~50
The 10-deazaminopterln Is powdered and pQssed through a
sleve and trlturated wlth m~lten oil of theobroma at 45C to form a smooth
suspenslon.
The mixture is well stirred and poured ~nto molds, each of
nomlnal 1 g capacity, . to produce suppos itor ies .
~5
Cachets ~/cachet
lO-deazamlnopterin 100
Lactose
400
The 10-deazaminopterln ls p~ssed through a mesh sieve,
lS mlxed wlth lactose prevlously sleved and fitted lnto cachets of sultable ~lze80 that each contains 500 mg.
Intramuscular In~ectlon
~sterll suspen6lon In aqueous vehlcle)
10-deazamlnopterln 10
ao 80dlum cUrate 5. 7
~odlum carboxymethylcellulose (low v16coslty grade) 2. 0
Methyl para-hydroxybenzoate 1. 5
Propyl para-hydroxybenzoate 0. 2
Water for Iniection to 1. 0 ml
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Intraperltoneal lntraveneous or subcutaneous lnjectlon
~ .
10-deazaminopterln, hydrochloric acld addition salt 15
Sodium cltrate 5-7
Sodium carbaxymethylcellulo~e (low vlsco~lty grade) 2. 0
Methy1 para-hydroxybenzoate 1. 5
Propyl para-hydroxybenzoate 0.2
Water for lnjection to 1. 0 ml
The followlng Example Illustrates admlnlstration of 10-
deazamlnopterln uslng a standardized test procedure:
EXAMPLE 8
~odium hydroxlde (0. 2 ml of 0.1 N) was added to 5 mg of
10-deazamlnopterln. Dlstl11ed water was then added, the pH adjusted to 7. 0,
and the solutlon then diluted wlth distilled water to 10 ml. The resultlng
so1ution and dllutlons thereo~ ~vere admlnLstered In allquots of 0.1 ml by
Intraperltoneal lnjectlon lnto L1210 leukemlc BD (2) Fl female mlce
(A.R. Schmld, Madison, Wi~). Injectlons ~ivere glven ~nce per day, three
tlmes per week ~Ionday, Wednesday, ~rlday) ~tartlng one day after tumor
20 transplantatlon (106 cells/mouse). I~erapy was contlnued untll death of
the anlmals.
~ or comparl~on purposes, and as a conkol, a parallel ~erles of
tests was carrled out slmultaneously uslng L1210 leukemlc BD (2) F~ fernale
mlce, under e~cactly the same test corldltlons, admlnlsterlng methotre~cate
25 lnstP~d of 10-deazamlnopterln.
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The procedure for te~tlng, and the maintenance and tran~plantation
of the L1210 leukemia, is ln accordance wlth the method of Hutchlson, D.J.,
Rnbinson, D.C., Martin, D., Ittensohn, O.L. andDillenberg, Journal
Cancer Res. 22 57-72 (1~62). The antileukemic actlvlty of 10-deazamlnopterln
5 waæ evaluated against methotrexate In terms oI the Increase ln median life span
obtained at various dosa~es, up to the maximum tolerated level, when compared
to untreated cont~ols. l~oxiclty of various dosages was evaluated by the extent
of welght loss and eventual death, wlth no evidence of tumor.
Representatlve results obtained against the L1210 leukemla are
10 as Iollows:
TABLE I
~qice Dosage Median llfe span Increased llfe span Weight change
(Exp. 'B X No. ) (m~/kg) (days)
10-deaza~erin:
None
2 x 10 (control) 6.8 - 0-5 - + 15
2x5 0.1875 7.2 + 0.6 3.0 +10
2x5 0.375 8.~ + 1.0 2~.7 ~7
3x5 O.i5 10.7 ~+ 1.458.3 ~15
3 x 5 1.5 14.7 + 1.8116.5 +11
2x5 3.0 18.0 ~ 1.9164.8 ~5
2 x 5 6.01 21.8 - 1.5 210.0 -
Methotrexate (control):
2x5 ~.75 ~ 1.0 46.0 +11
2x5 1.5 12.7+~1.8 86.5 ~.13
3 x 5 3.0 17.5 ~ 2.~152.8 ~8
x 5 6.0- 18.3 +- 3.51~9.0
Slight toxlcity
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Examples 9 - 12, which follow, deal with the treatment
of ascites tumors of one species of another. The
nature of the tumor, the condition of its treatment
in the mice host animals, and the results obtained
from the treatment are indicated by the data of the
several tables and the notes appended to each. The
results of these treatments are summarized at the
conclusion of Example 12.
EXAMPLE 9
S180-J ascites
Dosa~.e' ~Icthotrexate _ _ 10-Dea~aminopterin
mg/~g x 5 MST ILS Toxie MST ILS Toxic
s.c. (d~ys? (%)Deaths t (days) , (X) Deaths t
-- 12.1 + 1.1 -- 0/15 12.1 + 1.1 -- 0/15
3 11.7 + 1.7 0 0/10 13.0 + 1.1 8.7 0/10
6 15.2 , 0.9 24.9 0!15 21.2 + 1.7 74-~ 0/15
9 18.3 + 2.0 51.3 0/15 26.3 i 0.7 117.3 0115
12 19.8 + 1.8 64.0 0/15~31.4 + 2.5 ~59.6x 0~15
18 . 19.4 + 2.7 60.6 6/1018.6 + 2.4 54.0 7/10
* One dose (8 . C. ) every 2 days for a total of 5 doses.
t Toxic animals weigh 13-15 g at death (orig. wt. 20 ~).
x Two 60-day survivor6.
MST ~ Medium 6urvival time + standard deviation.
ILS - Increased life ~pan.
Each dose tested 2-3 times.
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EX~iMPLE 10
P815 ascites
Dosa~e* Methotrcxa~e 10-Deazam$nopterin
,mg/k~. x 5 MST ILS Toxlc ~IST ILS Toxic
s.c. _ (days) (X)Deathst (days) (X) Deathst
-- 8.8 ~ 0.9 -- 0/10 8.8 + 0.9 -- 0/10
3 -- __
6 15.4 + 1.1 75.0 0/10 18.2 + 1.3 107.5 0/10
9 17.6 + 1.6 100.30/10 19.0 + 0.9 116.3 0/10
12 18.4 + 1.3 109.10/10 19.2 + 1.2 118.4 0/10
18 . -~
, * One dose (s.c.) every 2 days for a total of. 5 doses.
,Toxic animals weigh 13-15 g at death (orig. wt. 20 g).
,MST ~ Mediu~ survival time + standard,deviatlon.
,ILS ~ Increased life span.
~ach dos~ge testcd twice.
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EX~PLE 11
L1210 ~scites
Dosage* ~Scthotrexate ~ b~za~i~opterin
mg/kg x 5 MST lLSToxic MST ILS Toxic
s.c. Routc (davs) (Z) Deathst(days) (%) Deathst
-- 6.6 + 0.7 -- 0/20 6.6 + 0.7 --0/20
3 S.C. 11.5 + 1.174 o/io13.8 ~ 0.7 108 0~10
6 14.4 + 0.8118 0/1015.2 ~ 1.5 . 131 0/10
9 15.8 + 0.9 139 0/15 17.8 + 0.8 168 0/15
12 16.7 + 1.1 148 0/15 18.2 + 1.3 176 1/15
3 Oral9.4 + 1.1 42 0/15 11.7 + 1.0 77 0/15
4.5 9.6 + 0.8 45 0/20 13.3 + 0.9 101 0/20
6.0 10.4 + 0.7 58 0/15 14.7 r 1.3 122 0/15
. .
* One dose every 2 days for a total of 5 doses.
t Animals weigh 13-lS g at death (orig. wt. 20 g).
MST ~ Medium survival time + standard deviation.
ILS ~ Increased life span.
Each dose testcd 2-4 timQs
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EXAMPLE 12
Ehrlich ascites
Dosage* Methotre.xate ~O-Deaz~minoPterin
mg~kg x 5 MST ILSToxic ; ~IST ILS Toxic
s.c. (days) (X)Deathst __5~ L__ (X) Deathst
-- 17.2 + 2.1 -- 0/1017.2 + 2.1 -- 0/10
3 -- __
6 19.8 + 2.3 15.1 0/1023.2 + 1.8 34.8 0/10
9 20.8 + 3.0 20.9 0/1027.2 ~ 2.2 58.1 0/10
12 20.2 + 1.9 17.0 0/1028.2 ~ 2.9 64.0 0/10
18 -- --
* One tose (s.c.) every 2 days for a total of 5 doses.
t Toxic animals weigh 13-15 g at death (orig. wt. 20 g).
MST ~ ~edium 6urvival time + standard deviation.
ILS - Increased life span.
Each dose tested twice.
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In summarizîng the data of Examples 9 - 12, it will
be seen that 10-deazaminopterin (dAM) was more
active than methotrexate (MTX) in all of the
ascites tumor systems examined. Optimum sc
dosages for each agent were 6-12 mg/kg when given
once q2d x 5. Maximum ILS (dAM/MTX) was +172%/+144%
against L1210, +117~/o/+103% against P815, +61%/+19%
against Ehrlich and >+160%/+64% against S180 with
long-term survivors after dAM. Maximum ILS (dAM/MTX)
at the same dosage and schedule against L1210 was
+203%/+182% by ip administration and +122%/+58~o
orally. The acute LD50 ip or sc was 65 mg/kg (dAM)
and 96 mg/kg (MTX). Plasma and tissue (tumor, small
intestine and marrow) pharmacokinetics were similar
for dAM and MTX. The increase in efficacy of dAM over
MTX at optimum dosages was associated with a greater
persistence of exchangeable dAM in tumor, but similar
persistence in drug-limiting normal tissue. Greater
persistence of dAM versus MTX in tumor was accounted
for by differences in membrane transport, specifically
in regard to the saturability (Km) for influx, favor-
ing greater accumulation of dAM. Both agents compete
for the same carrier mechanism, but carrier in tumor
exhibits a greater affinity for dAM.
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