Note: Descriptions are shown in the official language in which they were submitted.
11249~9
The invention relates to novel heteropolyanion compounds
containing tungsten combined with antimony
The invention also relates to processes for the manu-
facture of such compounds. The novel compounds are useful as
active agents in the treatment of viral infections in man and
animals as well as of the pathological processes due to viruses.
The invention also relates to pharmaceutical compositions con-
taining a therapeutically effective amount of at least one of
said compounds.
Co-pending patent application no. 205.780 relates to
heteropolyanions possessing anti-viral properties, in particular
a compound of tungsten and antimony denoted by the reference
HPA 23.
In general to prepare the compound HPA 23 in the form
of its ammonium salt, an aqueous solution containing the SbII
ion is reacted hot with a lM solution of sodium tungstate and the
reaction medium is kept substantially at neutrality by the ad-
dition of concentrated ammonium hydroxide in a sufficient amount
to make the medium colourless, which results in the precipitation
of the desired ammonium salt which is recovered by filtration and
subsequently treated in the usual manner.
The reaction temperature is less than the boiling temp-
erature of the reaction medium, for example in the vicinity of
80C. The aqueous solution containing the SbI I ion is advan-
tageously prepared by dissolving SbC13 in a saturated solution of
NH4Cl.
As later studies have shown, the product HPA 23 is a
heteropolyanion corresponding to the formula:
~ 9 21 8~
In the form of its ammonium salt, the product has the formula:
[Na Sb9 W21 8~ (NH4) 18~
and it can be in various forms, notably a hydrate with 8 mole-
cules of water. ~
112~139
The present invention relates to novel heteropolyanion
compounds in which the position of the sodium atom of the com-
pound HPA 23 is vacant or occupied by other metallic ion. The
invention also relates to such heteropolyanion compounds in their
metallic form, that is to say the form of their salts with metals
or with ammonium.
According to a first aspect, the invention relates there-
fore to a novel general class of heteropolyanions of the formula
(I)
~ 9 21 86~ (I)
in which M can be absent or represent an alkali or alkaline-
earth metal, other than sodium, and n is equal to 19 if M is
absent, or to l9-p, if p is the charge of the M ion. The overall
negative charge of the ion is hence equal to 19.
The polyion (I) can notably be isolated in the solid
state in the form of its ammonium salt of formula (II)
[ 9 21 8~ ( 4)19 (II)
The IR spectrum of the compound (II) is shown in Figure 1
which has been divided into Figures la and lb for convenience of
presentation (concentration 6/600 mg, phase KBr, apparatus
Perkin-Elmer 225).
To prepare compound (II) an aqueous solution of ammonium
tungstate is taken to which is added hot (60-80 C) a solution of
Sb203 in HCl (or SbC13 in saturated NH4Cl), the addition of
concentrated ammonia then causing, after removal of an insoluble
part, the precipitation of the desired ammonium salt.
The aqueous solution of ammonium tungstate is prepared by
reacting tungstic anhydride W03 at boiling temperature with
ammonia.
The molar proportions to be respected between W03 and
Sb203 are 4.5 to 5 moles of W03 per mole of Sb203.
llZ4989
Other compounds containing heteropolyanion of formula (I)
correspond to formula (III)
~ 9 21 86~ 18
in which formula M represents an alkali or alkaline-earth metal
other than sodium, such as potassium, calcium or magnesium and M'
is an alkali metal such as cesium, rubidium or potassium or
alkaline-earth metal, such as calcium or barium, or again ammon-
ium or quartena~y ammonium.
In particular, the invention relates to a novel heter-
opolyanion compound corresponding to the formula (IV)
~K Sb9 W21 8~ 18 (IV)
This novel product may be considered as a 9-antimonio-III-21-
tungsto-VI-potassium compound. It can occur as such or in the
form of ammonium or metal salts, in particular of the alkali or
alkaline-earth metals. A typical example is the ammonium salt
of formula:
~ Sbg W21 86~ (NH4)18
Another particularly advantageous example is the potassium salt
of the heteropolyanion (IV), this salt corresponding to the
formula (VI)
rK Sb9 W21 86~ K18
The compound (VI) decomposes above 340C. Its IR
spectrum is shown in Figure 2 of the accompanying drawings which
have been divided into Figure 2a and 2b for convenience of
presentation.
The IR spectrum obtained has been produced on a Beckmann
IR-12 spectrophotometer with a KBr pastille.
The Raman spectrum of the compound (VI) is shown in
Figure 3. The following data were used:
compound (VI) in powder form
5145 A 400 mW
interference filter
llZ~9~9
PM 720 V resolution 4 cm
speed 50 cm l/min
Cod-rg PHO Spectrometer
The polyanion (II) can be applied as such in the form of
the distilled water solution. The polyanionic structure is
however instable in the presence of metallic ions in aqueous
solution and this observation enables the preparation according
to a first process of other compounds according to the invention.
In this regard it will be noted that the compound HPA 23 de-
scribed in copending application no. 205.780 can be obtained by
the treatment of the polyanion (II) in aqueous solution by means
of an 0.5 M solution of NaCl.
The compound (V) can be obtained from the polyanion (II)by treatment of the latter in aqueous solution by means of a
solution of KCl, for example 0.5 M.
Other compounds according to the invention can be pre-
pared in similar manner by the treatment of the polyanion (II)
by means of a solution of a salt of the metal that it is desired
to introduce into the ionic structure. By way of example, there
may be mentioned the compound of magnesium obtained by treatment
of the polyanion (II) with a solution of 0.5 M MgC12.
Of course, the compounds according to the invention can
also be prepared by direct synthesis, as will be illustrated by
the following examples.
The invented products can exist in the form of numerous hydrates.
All the hydrated forms of said products fall within the scope of
the present invention.
The polyanion (IV) is composed of a central bicyclic
cavity formed of Sb-O-W-O-Sb chains having at its centre a K+
ion. Around this cavity exist other atoms of W and Sb, the over-
all ratio r=W/Sb being in the vicinity of 2.4.
The invention relates also to all the isomeric forms of
the heteropolyanion concerned. It relates also to mixtures
4 --
llZ~ 9
obtainable by conversion of heteropolyanion compounds under the
influence of variations in the pH and in the nature of the solu-
tions in which they are placed.
As has been previously noted, the heteropolyanion (II) is
not stable in aqueous solution. To stabilise it, it should be
placed in aqueous solution in the presence of metallic ions which
are introducible inside the complex, these ions being preferably
sodium, potassium, magnesium or calcium ions.
In aqueous medium containing potassium ions, in partic-
ular a dilute aqueous solution of potassium chloride, the hetero-
polyanion (II) provides compound V (ammonium salt).
The HPA 23 compound can also be subjected to an ionic
exchange reaction in the course of which the potassium takes the
place of the sodium and ammonium ions. This can be carried out
simply by dissolving the compound HPA 23 in a concentrated
aqueous solution of potassium chloride. In this way a mixture
of the compound HPA 23, (V) and (VI) is obtained.
It is advantageous, however, to prepare the compound (VI)
by a direct process starting from SbC13, KCl and K2W04.
According to this process, a first solution is prepared,
containing the Sb ion, by dissolving SbC13 hot in a saturated
aqueous solution of KCl, said first solution is added to a second
solution consisting of an aqueous solution of K2WO4, the reaction
temperature being from 60-90 C, in particular about 80C, and at
the end of the reaction a saturated solution of potassium bi-
carbonate is introduced until the reaction medium is colourless
and its pH is about 7.
The compound (VI) precipitates in the course of the
reaction and continues to deposit during cooling. For purifi-
cation, the precipitate is filtered, then washed with a solution
of KCl, for example 0.5 molar, and finally recrystallised in
distilled water.
--5--
1~2~39
The proportions of the constituents of the reaction are
selected so that the heteropolyanion corresponding to the com-
pound (VI) is obtained. To this end, 1 part by weight of SbC13
is used per 3.4 parts of K2W04. For example, an aqueous molar
solution of K2W04 is used and the corresponding amount of SbC13
must be dissolved in the saturated KCl solution, is calculated.
Thus the present invention provides a process for pro-
ducing the heteropolyanion compound of the formula
Mm Sb9 W21 86 M n (III')
wherein M is selected from a group consisting of alkali metal
ions other than sodium and alkaline earth metal ions, M' is
selected from a group consisting of alkali metal ions, alkaline
earth metal ions, ammonium and quaternary ammonium ion, m is 0
or 1 and n is l9-p, wherein p is the charge of said M ion, said
process comprising adding to an aqueous solution of the tungstate
of M' a solution containing SbIII ions, then adding concentrated
ammonia or a saturated solution of a salt of M' to precipitate the
compound of the formula III' wherein m is 0 and M' is ammonium
or the compound of the formula III' wherein m is 1 and M and m
are as hereinabove defined, and optionally reacting the compound
of formula III' wherein m is 0 and M' is ammonium with an aqueous
solution containing an alkali metal ion other than sodium or an
alkaline earth metal ion to form the compound of the formula III'.
In a specific embodiment the invention provides a process
for producing the compound of the formula
Sb9 W21 8~ K ~ (VI)
III
comprising preparing a first solution containing the Sb ions
by dissolving SbC13 in a saturated aqueous solution of KCl, add-
ing said first solution to a second solution consisting of an
aqueous solution of K2W04, and introducing a saturated potassium
bicarbonate solution until the reaction medium is colourless and
its pH is in the vicinity of 7 to obtain the desired compound of
formula VI.
~4,r
1~ - 6 -
llZ~9~39
In a further embodiment the present invention provides a
process for producing the compound of the formula
~ Sbg W21 o ~ (NH4)18 (V)
comprising adding to an aqueous solution of ammonium tungstate a
solution of antimony trioxide Sb203 in HCl, then adding concen-
trated ammonia to precipitate the desired ammonium salt, and
treating the obtained ammonium salt in an aqueous solution by
means of a solution of KCl in order to obtain compound of formula
V.
The following examples illustrate the invention without
being in any way limiting.
EXAMPLE 1
Preparation of ¦Sb9 21 ~ ( 4)19 : compound of formula (II)
An aqueous solution of ammonium tungstate is prepared by
reacting at boiling temperature 81 g of tungstic anhydride (W03)
with NH3 (1 liter of 1.2 molar ammonia).
To this solution, is added gradually at 80C, a solution obtained
by dissolving 21;8 g of antimony trioxide (Sb203) in 100 ml of
6 M HCl.
At the end of the operation, about 20 ml of 12 M NH3 are
added. An insoluble precipitate forms and it is filtered hot
rapidly. The ammonium salt ~b9 W21 0 ~ ~NH4)19 crystallises in
the course of cooling from the supernatent solution.
The crystals obtained are filtered and then washed with a
saturated solution of NH4Cl.
EXAMPLE 2
Preparation of the compound K Sb9 W21 86 K18 : compound of
formula (VI)
A solution containing 12.2 g of SbC13 dissolved hot in
50 ml of a saturated solution of KCl is added gradually to 125 ml
of an aqueous molar solution of K2W04 previously brought to 80 C.
At the end of the operation, about 20 ml of a saturated solution
llZ49~9
of potassium kicarbonate îs added, so that the mixture is colour-
less and its pH close to 7.
The potassium salt of the heteropolyanion precipitates
in the course of the preparation and continues to deposit during
cooling. The precipitate is filtered and then washed with a
solution of 0.5 molar KCl and finally recrystallised in distilled
water. In this way about 25 g of the compound (VI) of the
formula indicated above are obtained. Its IR and Raman spectra
are shown in Figures 2 and 3 respectively.
The present invention relates also to the application of
the heteropolyanion compounds of formulas (II) and (III), in
particular the compound (VI), denoted below by the abbreviated
reference HPA 39, as medicament for the preventive and curative
treatment of viral infections in man and in animals and of the
pathological processes due to viruses. Thus, the invention also
relates to pharmaceutical compositions containing, as active
agents, the heteropolyanion (III) compounds, in particular the
compound HPA 39, in association with pharmaceutically acceptable
vehicle. In a particular embodiment, the pharmaceutical compo-
sitions according to the invention are conditioned in injectable
form, for example in aqueous solution (physiological solution) at
a pH in the neighbourhood of neutrality, for injection into man
or into animal.
In the first pharmacological tests reported below, the
heteropolyanion applied is the product of formula (VI) denoted by
the abbreviated reference HPA 39. The activity of HPA 39 was
compared with the sodium compound of the formula
~ a Sbg W21 8~ (NH4) 18'
the latter compound being denoted by the abbreviation HPA 23.
The tests were applied to the treatment of viruses of ~riend's
leukemia.
-8-
11249t39
Pharmacological Tests
In the experiments, DBA2 mice, male or female, aged from
2 to 3 months, of a weight close to 20 g were used.
A polycythemia strain of Friend's virus (VFP) was used
maintained for some years in the laboratory. The viral stock was
constituted by an acellular supernatent liquid obtained by cen-
trifugation of ground leukemic spleens, formed in a 0.5 M PBS-
saccharose solution. Titration of the stock was done in vivo,
according to the method of Reed and Meunch (American Journal of
Hygiene 1938, 27, 493-497). The measurement was expressed as
SD50 ( Spleen enlarging dose 50%").
Experimental Procedure:
The Friend's virus was injected in the mouse by the intra-
peritoneal route. The animal then developed a characteristic
splenomegaly. Death appearing three weeks after the viral innoc-
ulation, at the doses normally employed, the animals were sacri-
ficed on the 21st day after infection, for study of the
splenomegaly. The spleens were taken out and then weighed indi-
vidually. Any spleen whose weight exceeded 250 mg was considered
as pathological.
The HPA 23 and HPA 39 heteropolyanions were diluted
usually in physiological serum or distilled water.
They were administered like the virus by the intraper-
itoneal route in a single dose or in repeated doses at times
varied with respect to the viral innoculation period.
Results: 1. Protective Effect of HPA 39 against Friend's
Leukemia.
Table 1 below summarises the results of two consecutive
experiments wherein the mice, innoculated with 20 SD50 of VFP,
received a single dose of HPA 39 ranging from 0.5 mg to 2 mg, the
same day as the virus.
The survival percentage was established on the 62nd day
after the viral injection.
_g_
~1249~B9
2. Study of the Response to HPA 39 as a function of
the Innoculated Vira'L Dose.
-
From the point of view of the splenomegaly, a study wasmade of the effect of a single injection of 2 mg of HPA 39 in the
case where the animals were innoculated with increasing doses of
VFP ranging from 5 to 50 SD50 per animal.
The results are summarised in the following Table II.
3. Test of Treatment of Friend's Leukemia by Repeated
Injections of HPA 39.
In this experiment, animals previously innoculated with
20 SD50 of VFP were used. The effect of 5-day treatments com-
prising one daily injection of 0.5 mg of HPA 39, were studied.
Day 0 (Do) being the day of infection, three periods were
selected:
from D + 3 to D + 7, from D + 10 to D + 14, from D + 17 to D + 21.
In order to know the stage of the disease at the beginning of
each treatment, 10 control animals were sacrificed on days +lO
and +17.
In parallel, the toxicity of this treatment on non-
leukemic animals was studied. Although the short-term toxicity
seems very low, it should be noted that, 60 days after the end of
the treatment, 50% of these animals died.
Table III below gives the average value of the spleen
weight 21 days after infection, as well as the average weight of
the spleens at the beginning of each treatment.
4. Comparative Test of HPA 23 and HPA 39 on
Splenomegaly induced by the VFP.
Table IV below summarises the results obtained with a
single injection of 1 mg of each of these products on the splen-
omegaly induced by VFP 21 days after the injection of 20 SD50.
TABLE I
Effect of HPA 39 on Friend's Leukemia-
--10--
/
~12~E~
Dose HPA 39 Average weight Limiting Survival Toxicity
injected of spleens (g) values % t*)
D + 21 (g) D + 21
0 exp 1 = 1.349 0.402-~2.63 NF
exp 2 = 0.897 0.390-~1.598 40
0.5 mg per exp 1 = N.F. N.F. NF
mouse
IP exp 2 = 0.711 0.211-~2.036 70
1 mg per exp 1 = 0.3220.252-~0.443 NF
mouse
IP exp 2 = 0.258 0.190-~0.348 100
2 mg per exp 1 = 0.3080.225-~0.405 NF
mouse
IP exp 2 = 0.262 0.208-~0.291 100
* The to~icity represents the percentage of mortality in lots of
10 non-leukemic animals treated with a dose as indicated.
TABLE II
Dose-effect Relationship of HPA 39 with respect to VFP
Viral HPA 39 Average Spleen Limiting ~alues
Innoculum injected Weights (g) (g)
(IP) (mg IP)
5 SD50 0 0.6190.228-_1.552
2 0.2530.158-~0.310
10 SD50 0.7260.323-~0.979
2 0.2690.160-~0.331
20 SD50 0 1.0520.239-~1.989
2 0.4090.233-~0.412
50 SD50 1.6511.328-~2.180
2 0.3500.113-~0.721
~lZ4~8"
TABLE III
Effect of HPA 39 in the Treatment of Friend's Leukemia
Period of Average Spleen Average Spleen Limiting
Treatment Weight at the Weight (g) Values
(5x0.5 mg) beginning of D + 21(g)
the Treatment
0 1.2020.408-~2.594
D + 3-~D + 7 not done 0.610* 0.204-~1.577
D + 10-~D + 14 0.204 0.656* 0.250-~1.250
D + 17-~D + 21 0.500 0.532** 0.263-~0.988
*The increase in weight of the spleen manifests the continuing
development of the leukemia.
**Arrest of the leukemia.
TABLE IV
Comparative Anti-viral Effect of HPA 23 and HPA 39: structure-
activity relationship
TreatmentAverage Spleen Limiting
(1 mg - IP) Weight Values (g)
01.426 0.740-~2.063
HPA 230.320 0.222-~0.391
HPA 390.294 0.259-~0.335
The results reported in Table IV show that the products
HPA 23 and HPA 39 result in a very marked reduction in splen-
omegaly, which demonstrates their effectiveness in inhibiting the
development of Friend's leukemia. In addition, HPA 39 manifests
an activity higher than that of HPA 23.
As a whole, the above results make it evident that HPA 39,
administered to mice infected with Friend's leukemia virus, show
no effect of toxicity. HPA 39 arrests the development of the
leukemia: thus, a dose of 2 mg of HPA 39 per mouse permits, for
-12-
1~2~39
100% of the infected mice, survival up to the 62nd day, the day
at which they were sacrificed.
An advantageous property of the product HPA 39 has also
been established, namely that this product possesses a synergic
action when it is administered at the same time as interferon.
To prove this synergy of HPA-interferon, four groups of mice were
used and the products tested were administered under the following
conditions:
Interferon: pre-treatment 6 hours before infection - intraperi-
10toneal route 5000 U/mouse.
HPA 39: 3 mg/mouse - intraperitoneal route immediately after the
innoculation of the virus.
Encephalomyocarditis virus of the mouse - 50 lethal dose/mouse;
innoculation subcutaneous route (SC)
Group I: 10 mice received the virus and served as
controls
Group II: 10 mice received interferon and, 6 hours afterwards,
the virus
Group III: 10 mice received the virus by the SC route and HPA 39
20by the intraperitoneal route
Group IV: 10 mice received interferon and then, 6 hours after-
wards, the virus and HPA 39.
Results on the 15th Day
Mortality
Group I: Control 8/10
Group II: Interferon alone 8/10
Group III: HPA 39 alone 8/10
Group IV: HPA 39 + interferon 3/10
The results recorded above show clearly the superiority of
the HPA 39-interferon association with respect to the results ob-
tained with one of the substances alone.
Other "in vivo" tests were carried out to test various
products according to the invention and various comparison
--13--
llZ~9~9
products of the prior art for their preventive action against
Friend's virus-induced leukemia in the DBA-2 mouse (according to
the technique described above).
By way of comparison HPA 23 and two organic cryptates were
tested:
Cryptofix 222*(Merck): formula C18H36N206
(MW: 376)
Cryptofix 211*(Merck): formula C14H29N206
(MW: 288.35)
To gr~ups of ten DBA 2 mice previously innoculated with
Friend's virus, were administered each of the products described
in the dose of 1 mg per mouse. The control group only received
the virus.
The mice were sacrificed 21 days after the innoculation of
the virus. The spleens were taken out and then weighed individ-
ually. Each spleen weighing more than 250 mg was considered as
leukemic. The results of these experiments are assembled in
Table V. As this Table shows, all the products according to the
invention are capable of reducing in the mouse, splenomegaly
induced by Friend's virus, although the known cryptates are in-
active, and even toxic.
In addition, the compound (VI) or HPA 39 is more active
than HPA 23.
Further pharmacological tests were performed for testing
the in vivo activity of the HPA 39 compound.
1. FRIEND'S LEUKEMIA
Friend's leukemia is induced in a DBA2 mouse by the poly-
cythemiant strain of virus.
The criteria selected to follow the development of the
disease were splenomegaly and survival.
The animals were sacrificed 21 days after injection of the
virus. The spleens were taken out and weighed individually.
* Trademarks
~12~39
EEfect of HPA 39 on Splenomegaly
As shown in Table VI, HPA 39, injected in a single dose
by the intraperitoneal route the same day as the virus (20 SD50),
considerably reduced splenomegaly. In this experiment, the doses
ranged from 1 to 5 mg. These three doses had substantially the
same activity. It is noted, however, that there is a certain
toxicity at 5 mg.
Table VII summarises an experiment where HPA was tested,
under the same conditions as previously, against increasing doses
of virus. It is clear that, whatever the viral innoculum, HPA 39
preserves all its activity.
Effect of HPA 39-on Survival
The survival was followed of groups of animals (20) in-
noculated by 20 SD50 of virus and treated the same day, by a
single dose of HPA 39 of 0.5, 1, and 2 mg.
The median survival of the control group was 56 days, that
of the group treated by 0.5 mg, 128 days, that of the group
treated by 1 mg, 151 days, and finally, that of the group treated
by 2 mg, 108 days.
200 days after the beginning of the experiment, 25~ of the
animals treated by 1 or 2 mg were still living.
Time of Action of HPA 39
Table VIII shows the results of treatment tests by HPA 39
on splenomegaly.
The animals all received Do of the experiment, 20 SD50 of
Friend's virus. In the first part of the Table, tests of sequen-
tial treatments of 5 injections of 0.5 mg per day of HPA 39 are
reported.
Under these conditions, HPA 39 preserves notable activity
on splenomegaly, but less important than after a single injection
of the substance the same day as the virus. In the second part of
Table VIII, tests of the effect of a single dose of 2 mg of sub-
stance injected on different days with respect to the viral
'., ~S--
11249~9
innoculation are reported.
Only one injection 2 days after the virus preserved a
certain effectiveness, but however the protection level obtained
is less than that obtained on injections effected the same day as
the virus.
Effect of HPA 39 on the Intrasplenic Virus Ratio
Compared were the virus ratio in the spleen of the control
animals with that found in the spleen of the animals treated at
Do by a dose of HPA 39. Although the treated animals had a spleen
weight very much less than the controls, the virus level is com-
parable in the two groups. The technique used had been the S+L-
titration of ground spleen.
II. ENCEPHALOMYOCARDITIS
The effect of HPA 39 on the lethal effect of encephal-
omyocarditis virus in the mouse was also tested.
Strains V77 L3 and S3 (passage in the culture for the
strain L3 and in vivo for the strain S3),VR129 L3 and S3 were
used.
The substance was injected in a single dose (3 mg) by the
intraperitoneal route 30 minutes before the innoculation of
50 LD50 of virus which, itself, is done by the subcutaneous route.
A distinct increase in the number of animals surviving was
observed relative to the control animals which had not received
an injection of HPA 39.
-16-
l~Z4989
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/: .
1124~9
(1) Polyanion (II) signifie~ the polyanion [Sbg W21 86~
(NH4)19 in solution in distilled water, the metal M being
absent
(2) Synthesised: signifies a compound prepared by direct
synthesis as opposed to the similar compound prepared
by treatment of the Polyanion (II) by means of a
solution containing a metallic ion such as K or Mg.
-18-
11249~9
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112~39
TAB~E VI~
HPA 39: EFFECT ON SPLENOMEGALY INDUCED BY DIFFERENT DOSES
OF FRIEND'S VIRUS.
Friend's HPA 39 Average WeightLimits
Virus of Spleen (g) (g)
5 SD 0.619+0.427 1 551
2 mg 0.253+0.043 0 3510
10 SD50 0.697+0.268 1 120
2 mg 0.269+0.050 o:336
.
O 1.102+0.644 10 2909
20 S~50
2 mg 0.409+0.298 1 2413
l . .
O 1.651+0 r 535 2 0814
50 SD50
2 mg 0.350+0.155 0 278
. 20 .
., . ,, . .. . .. , . ~ .. . ..
l~Z4989
TABLE VIII
HPA 39: EFFECT OF VARIOUS TREATMENTS ON SPLENOMEGALY
INDUCED BY FRIENDS VIRUS.
Treatment Dose Average Weight of Limiting
Spleen ~ type Values
Deviation
0 1.202+0.6430.488-2.5943
D~ ~ D17 5 x 0.5 mg 0.610+0.4400.204 - 1.577
~lO ~14 5 x 0.5 ~g 0.658+0.2990.250 - 1.250
~17 21 5 x 0.5 mg 0.532+0.289o, Z 63_D,95~
0 1.438+0.5500.727-2.493
~0 ~ 2 mg 0.244+0.0400.185-0.308
D 7 2 mg 1.488+0.4a30.762-2.033
....
D 4 2 mg 1.827+0.3041.221-2.170
D_2 2 mg 1.668+0.5690.764-2.233
.
D+2 2 mg 0.560+0.3780.202-1.270
. .. .. .
D~4 2 mg 1.179+0.5480.257-1.748
.
D+7 2 mg 1.17G+0.7400.390-2.402
. 21 .
