Note: Descriptions are shown in the official language in which they were submitted.
13~3~iZ
AVARONE AND AVAROL PHARMACEUTICAL
COMPOSITIONS, AND PROCESS
BACKGROUND OF INVENTION
1. Field of Invention
_ _ _
Antiviral and antitumor compositions comprising
avarone or avarol and method of employing avarone and avarol
for their antiviral and antitumor activity; method of
killing virus ànd ameliorating, alleviating, or palliating
tumorous and/or cancerous conditions therewith; method of
production of the active principles avarone and avarol;
employment of the same as such or in pharmaceutical composi-
tions containing the same for their antibacterial and/or
antimycotic activities. In brief, pharmaceutical composi-
tions embodying avarone or avarol, method of treating with
avarone or avarol, and employment of the said active princi-
ples for their antiviral and anticarcinogenic activities as
well as their antibacterial and antimycotic activities.
2. Prior Art
.
;20 Avarone and its hydroquinone derivative avarol are
natural substances found in the marine sponge Dysidea avara
(L. Minale, R. Riccio and G. Sodano, Tetrahearon Letters
1974, 3401-3404; S. de Rosa, L. Minale, R. Riccio and G.
Sodano, J. Chem. Soc. PerXin I, 1408-1414 (1976)). These
compounds were isolated rom a diethyl ether extract using
column chromatography on silica gel (L. Cariello, M. de
Nicola Giudici and L. Zanetti, Comp. Biochem. Physiol. 65c,
37-41 (1980)). With regard to biological effects it has so
far only been known that avarol in very high concentrations
~130 ~m) affects the cells of the sea urchin cmbryo, causing
~developtnental abberations~ (L. Cariello et al., ibid.).
rhe derivatives described in the literature are, among
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~3~33C~
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others, the 3'-methylamino and the 4'-methylaminoavarone (G.
Cimino, S. de Rosa, S. de Stefano, L. Cariello and L.
Zanetti, Experientia 38, 896 (1982) as well as the avarol
dimethyl ether and the avarol diacetate (S. de Rosa et al.,
ibid.). The rnethylaminoavarones affect the cells of the sea
urchin embryo in the same way as avarol, but only in concen-
trations which are ten times higher. Additional biological
effects of avarone derivatives have to date not been known.
THE PRESENT INVENrION
It has now been found that avarone and avarol have
pronounced and unpredictable antitumoral, antibacterial,
antiviral and antimycotic properties. Owing to the
aforementioned properties, these substances are suited
for the treatment of carcinoses and viral and other infec-
tious diseases, either as such or in the form of a prodrug
or precursor or any of the foregoing in the form of a
pharmaceutical composition where present together with a
pharmaceutically-acceptable diluent or carrier.
OBJECTS OF THE INVENTION
It is an object of the present invention to provide
novel pharmaceutical compositions embodying avarone or
avarol or a pro-drug or precursor thereof or therefor. It
is a further object of the invention to provide a novel
method of treating, eliminating, alleviating, palliating,
or ameliorating undesirable turnorous or cancerous, viral,
or other infectious conditions by the employment of avarone
or avarol or a pharmaceutical composition containing the
same. An additional object of the invention is the provi-
sion of a process for producing the active principles
avarone and avarol. Still additional objects will bccome
- 2 - MERZ 12
< l3~3a~z
apparent hereinafter, and still further objects will be
apparent to one skilled in the art.
SUMMARY OF THE INVENTION
What we therefore believe to be comprised by our
invention may be summarized inter alia in the following
words:
A pharmaceutical composition suitable for use as an
antiviral composition or antitumor composition comprising an
effective antiviral or antitumor amount of avarone or avarol
together with a pharmaceutically-acceptable pharmaceutical
carrier; such a pharmaceutical composition useful for
adversely influencing growth of tumor cells susceptible
thereto which comprises an effective amount of avarone or
avarol in admixture with a carrier which does not interfere
with the effect of the active compound; such a pharmaceu-
tical composition suitable for use as an antitumor composi-
tion comprising an effective antitumor amount of avarone or
avarol together with a pharmaceutically-acceptable pharma-
ceutical carrier; such a pharmaceutical composition suit-
able for use as an antitumor composition comprising an
effective antitumor amount of avarone together with a
pharmaceutically-acceptable pharmaceutical carrier; such a
pharmaceutical composition comprising an effective antitumor
amount of avarol together with a pharmaceutically-acceptable
pharmaceutical carrier; such a pharmaceutical composition
useful for adversely influencing growth of tumor cells
susceptible thereto which comprises an effective amount of
avarone or avarol in admixture with a pharmaceutically-
acceptable carrier; such a pharmaceutical cornposition
suitable for use as an antiviral composition comprising an
effective antiviral amount of avarone or avarol together
_ 3 _ MERZ 12
1303!~5Z
with a pharmaceutically-acceptable pharmaceutical carrier;
such a pharmaceutical composition suitable for use as
an antiviral composition comprising an effective anti-
viral amount of avarone together with a pharmaceutically-
acceptable pharmaceutical carrier; and such a pharmaceutical
composition suitable for use as an antiviral composition
comprising an effective antiviral amount of avarol together
with a pharmaceutically-acceptable pharmaceutical carrier;
also a method of combating a virus or tumor comprising
administering to the host or situs an effective antiviral or
antitumor amount of avarone or avarol; such a method o~
combating a tumor comprising adrninistering to the host or
situs an effective antitumor amount of avarone or avarol;
such a method of combating a tumor comprising administering
to the host or situs an effective antitumor amount of
avarone; such a method of combating a tumor comprising
administering to the host or situs an effective antitumor
amount of avarol; such a method of adversely influencing
growth of tumor cells susceptible thereto comprising the
step of administering thereto or to the host an effective
amount of a compound selected from avarone and avarol, such
a method of combating a virus comprising administering to
the host or situs an effective antiviral amount of avarone
or avarol; such a method of combating a virus comprising
administering to the host or situs an effective antiviral
amount of avarone; such a method of combating a virus
comprising administering to the host or situs an efective
antiviral amount of avarol; and such a method wherein the
compound is administered in ~he form of a pharmaceutical
cornpos.ition thereof, in which it is present together
with a pharmaceutically-acceptable carrier or diluent.
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i` ~3V~Z
.~
Also, a process for the procurement of avarone and avarol
which comprises the steps of cutting the fresh marine sponge
Dysidea avara into small pieces, extracting the same with
ethyl acetate, removing solvent, and separating the avarone
and avarol from a solvent mixture by column chromatography
over silica gel; such a process wherein the solvent m;xture
is one suitable for the separation of a hydroquinone from an
aromatic diol; and such a process wherein the solvent
mixture is a benzene-ethyl acetate mixture. Finally a
pharmaceutical composition suitable for use as an antiviral
composition, an antitumor composition, an. antibacterial
composition, or an antimycotic composition comprising an
effective antiviral or antitumor or antibacterial or
antimycotic amount of avarone or avarol together with a
pharmaceutically-acceptable pharmaceutical carrier and a
method of combating a virus or tumor or bacteria or fungus
comprising administering to the host or situs an effective
antiviral or antitumor or antibacterial or antimycotic
amount of avarone or avarol.
- S ~ MERZ ~2
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IDENTITY
The active antiviral and antitumor ingredients or
agents of the present invention have the formulas:
2-[(lR)-1,2,3,4,4a,7,8,8a~-octahydro-lB,2B,4aB,S-tetramethyl-l-
naphthylmethyl]-quinone
0~0
H CH3
~CH3 ,
C H3
Formula of Avarone
C2 H28O2; Mol wt: 312.20
C 80.73 %; H g.03 %; O 10.24 %
0 H 0 ~3 -~
H . CH3
~CH3
CIH CH3
Its derivative Avarol
C2~H30O2; Mol wt: 314.22
C 80.32 %; H 9.81 %; O 9.87 %
-6 - MERZ 12
~ 13~3~2
H_stor~
Sponges live in symbiosis with algae, fungi, bryozoae
and bacteria (Muller et al, F. Bacteriol. 1981, 145, pp.
548-558). Experimental data support the hypothesis that
the symbiotic relationship between sponge and non-sponge
cells is based on a growth-promoting (e.q., lectin) and a
growth-inhibiting principle (cytostatic agent). One
derivative of ~he cytostatic agent 1-~-D-arabinofuranosyl-
thymine, isolated from the sponge Cryptothethya crypta, has
already been established as an anti-cancer agent in the clinic
~ D-arabinofuranosylcytosine) (Muller et al., Jap. J.
Antibiotics 1977, 30 Supp. pp. S104-S120). In the course
of our screening program for antimitotic agents, it was
found that the sponge secondary metabolites Avarone and
Avarol exhibit antimitotic activity, which is differ-
ent than that of Vincristine [OncovinR], Colchicine, or
4'-Demethylepipodophyllotoxin-9-(4,6-0-ethylidene-~ -D-
glucopyranoside) [EtoposideR].
Isolation and Synthesis
Avarol has been isolated from the marine sponge Dysidea
avara, which is ubiquitous near the Atlantic coast of
Europe, in the Mediterranean and around the Maldive Islands
(Minale et al., Tetrahedron Lett. 1974, No. 36, pp. 3401-
3404). According to our present process, ground, chopped,
or otherwise comminuted material is extracted with ethyl
acetate. The organic phase is dried over MgSO4 and then
evaporated to dryness yielding a tar-liXe residue. This
material is taken up in benzene and purified by silica gel
column chromatography using benzene/10% ethyl acetate as
solvent. The Avarol fractions are concentrated; purified
Avarol is obtained after several crystallizations from
MERZ 12
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.
acetone-methylene chloride. Yield: 2.7 g of ~va~ol from l
kg of fresh material. Avarone may be obtained from its
corresponding hydroquinone Avarol by Ag20 oxidation, but
it is preferably obtained by column separation according to
our new process, represented by Example 1 hereinafter.
Description
Avarone: Yellowish-brownish crystals; m.p. 62-64C.
(hexane); slightly soluble in water; solubility in dimethyl-
sulfoxide > 100 mg/ml at 23C.; stable in solution when
stored at 4C. for up to 1 year.
Avarol: Whitish crystals; m.p. 146-148C. (chloro-
form); other characteristics same as for Avarone.
M E ~ Z 1 2
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PRODRUGS OR PRECURSORS AND THEIR PREPARATION
.
The compounds avarone and avarol may also be employed
or embodied in pha~maceutical compositions according to the
invention in the form of compounds which convert to avarone
or avarol after administration to the living animal body.
Such compounds are commonly referred to today as prodrugs or
precursors, and representative examples include esters of
avarol and alkylamino derivatives of avarone. As already
indicated, some of these compounds are known in the prior
art, whereas others are made in a known manner corresponding
thereto. Representative of such prodrugs and precursors,
and their preparation, are set forth in the following.
The active ingredients of the present invention, and
their precursors or prodrugs, are accordingly avarone,
avarol, and thsir derivatives of the general formula I
R'
X~ R2
( I )
wherein
1. X1 and X is oxo with double bonds between C-1' and C-6',
and between C-3' and C-4';
R1 is hydrogen
and
R2 is alkyl amino having one, two, three or four C atoms;
or wherein
2. X1 and x2 is oxo with double bonds between C-1' and C-6',
and between C-3' and C-4';
R1 is alkyl amino having one, two, three or four C atoms,
and
R is hydrogen;
- 9 - ~ER~ l2
~ 31~J3~!5;2
or wherein
3. xl and x2 is hydroxy or acyloxy with 2-6 C atoms or
xl with x2 is diacyloxy with 4-6 C atoms with aromatic
r;ng;
and
R1 and R2 is hydrogen,
4. Avarone- As in 1 or 2, but both Rl and R2 being
hydrogen, and
5. Avarol - As in 3, both X1 and x2 being hydroxy, as
weli as salts of the alkylaminoavarones as conventionally
obtained by reaction with physiologically-tolerable acids.
The compounds of formula I thus comprise avarone
~R1=R2=H in Formula Ia) and alkylaminoavarone derivatives
thereof, as well as avarol (R1=R2=H in Formula Ib) and
esters thereof.
. . . _
.... ,. I . . .......... -- .
~6 R 0,~ ~
~ 6~?'
(lb)
- (laJ
The process for the manufacture of alkylaminoavarones
of the general formula Ia is characterized in that avarone
is reacted with alkylamine hydrochloride of the general
formula RNH2xHcl~ the resulting compounds being conver-
ted, if desired, into salts using physiologically-tolerable
acids. Compounds of the general formula RNH2xHCl can
be ethyl, propyl, isopropyl, n-butyl, isobutyl and tert.
butylamine hydrochloride (Reaction 1 hereinafter).
- 10 - MERZ t2 -
13~!3~
Preparation of avarone derivatives of the general
formula Ia by placing the substituent -NHR in 3' or 4'
position is characterized in that ~he reaction components
are reacted in 50% ethanol in the presence of pyridine
(G. Cimino, S. de Rosa, S. de Stefano, L. Cariello and
L. Zanetti, Experientia 38, 896 (1982)). The resulting
isomer mixtures of 3' and 4' alkylamino avarones can be
separated by column chromatography using silica gel. If
desired, these reaction products can be converted into
their salts by reaction with physiologically tolerable
acids. For this purpose suitable acids are hydrochloric
acid, hydrobromic acid, sulphuric acid, phosphoric acid,
tartaric acid, maleic acid, etc.
The process for the manufacture of avarol derivatives
` of the general formula Ib, wherein R1 and R2 are acyl,
is characterized in that avarol is acylated with an acyl
chloride of the general formula RCOCl or with a carboxylic
acid anhydride of the general formula RCOOCOR, with the
exception of the acetanhydride at the hydroxyl group of
the C-2' and/or C-5' position. For this purpose acid
chlorides or acid anhydrides of dibasic acids such as,
e.g., succinic acid, can be used, whereby one molecule
of avarol accounts for only one molecule of the derivative
of the dibasic acid (diacyloxy).
Compounds of the general formula RCoCi can be, for
example, straight-chain acylchlorides such as acetyl,
propionyl, n-butyryl, n-valeroyl and capronoyl chlorides, as
well as branched acyl chlorides like isobutyryl, isovale-
royl, or ethylmethylacetyl, and trimethyl acetyl chloride.
Compounds of the general formula RCOOCOR can, for example,
be straight-chain acid anhydrides such as propionic acid,
.~ERZ 12
-- 11 --
13~3~Z
`V
butyric acid, valeric acid and capronic acid anhydrides as
well as branched acid anhydrides like isobutyric acid,
valeric acid, or ethylmethyl acetic acid and trimethyl
acetic acid anhydrides (Reaction 2 hereinafter).
The preparation of the avarol derivatives of the
general formula Ib by introducing substituent -COR is
characterized in that the reaction components are reacted in
the presence of pyridine (S. de Rosa, L. Minale, R. Riccio
and G. Sodano, J. Chem. Soc. Perkin I, 1976, 1408-1414;
Organikum, VEB Deutscher Verla9 ~er Wissenschaften, 13th
Edition, Berlin 1974, pp. 441-446).
H R
REACTIC~ 1 ~N~
Oq~
~ ~0
0~ RNH2' HCI ~ ~ H
~ (Pyridine) \ O~--R
~_ ~0
AC~ION 2 R
R-C-CI C = O
or
R- 1l O~
~OH (Pyridine) ~`1
=O
R
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13~3~
.
PREPARATION
Preparation of the active principles avarone and
avarol of the present invention from known starting mater-
ials is effected according to the following specific
Examples and pursuant to the following general procedure.
Accordingly, additional subject matter of the invention
comprises a process for the manufacture of avarone and
avarol characterized in that the fresh marine sponge Dysidea
avara is ground, cut into small pieces, or otherwise com-
minuted, and then extracted with ethyl acetate, solvent
being removed from the extract, which extract is preferably
reduced to dryness, and the residue being chromatographed
over a silica gel column using suitable solvent systems for
separating the diketone (hydroquinone) compound from the
aromatic diol, such as benzene/ethyl acetate or the like,
including, for example, an aromatic solvent such as benzene
or an aliphatic solvent such as hexane, together with up to
about twenty percent (V/V) of an aliphatic solvent such as
diethylether or ethylacetate. By the employment of this
method avarone and avarol can be conveniently separated and
isolated.
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~3g33~
DETAILED DESCRIPTION OF THE INVENTION
The following Examples are given for purposes of
illustration only, but are not to be construed as limiting.
Examples 2a to 2d of the following examples were obtained
according to ~eaction 1, whilst examples 3 to 4 were prepared
according to Reaction 2. Example 1 relates to the preparation
of avarone and avarol from the natural product.
Example 1
Avarone and avarol
Extract 3 kg of the sponge with 250 ml of ethyl acetate. Dry
the resultant extract over magnesium sulfate and filter.
~vaporate the filtrate to dryness. Take up the residue in
benzene and subject to chromatography using a silica gel column
and benzene as extraction agent. ~varone can be found
in the extract whilst avarol is retained on the column.
Extract avarol with a mixture of benzene and ethyl acetate
(90:10, V:V). Evaporate the extract to dryness. Subsequently,
pure avarol is obtained by crystallization from dichloromethane-
acetone. Purify avarone by recrystallization from benzene.
Avarone Avarol
Melting point:62-64C ~Aelting point: 146-14~C
- 14 - MERZ 12
13e~3a?SZ
E mple 2
3'-ethylamino avarone and 4'-ethylamino avarone
a) Add 2.5 g of ethylamino hydrochloride and 5 ml of pyridine
to a s~lution of 500 mg avarone in 1ooo ml of 50~ ethanol,
and distill off the ethanol under water-jet vacuum after
20 hours. Extract the aqueous residue with dichloromethane
and chromatograph the reduced dichloromethane extract using
a silica gel column and dichloromethane as extraction agent.
In the course of this process 3'-ethylamino and 4'-ethylamino
avarone is obtained.
In the same way the following products have been obtained:
b) 3'-propylamino and 4'-propylamino avarone
c) 3'-isopropylamino and 4'-isopropylamino avarone
d) 3'-n-butylamino and 4'-n-butylamino avarone
Example 3
Avarol diacetate
a) Dissolve 500 mg of avarol in 20 ml of absolute pyridine,
and add 1 g of acetyl chloride in portions to the solution
under shaking. Treat the mixture as usual, evaporate to
dryness and extract the residue with boiling heptane.
Cn cooling the ester crystallizes. It is then recrystallized
from hexane.
Meltir.g point: 62-64C
In the same way the following products have been obtained:
b) Avarol dipropionate
c) Avarol divalerianate
d) Avarol ditrimethyl acetate
- 15 - MERZ 12
~3~3~Z
Example 4
Avarol dicapronate
a) Dissolve 300 mg of avarol in 25 ml of absolute pyridine,
and add o.6 g of caproic acid anhydride in portions to the
solution whilst shaking. Treat the mixture as usual, evaporate
to dryness, and extract the residue with boiling heptane.
Recrystallize from.acetone and subsequently frcm hexane.
In the same way the following products have been obtained:
b) Avarol diisovalerianate
c) Avarol diethyl methyl acetate
d) Avarol succinate
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13~3~iZ
.
P13ARMACEUTICAL COMPOSI'rIONS
___ ~__ _
The active ingredients of the invention, together with
a, conventional adjuvant, carrier, or diluent, may be placed
into the form of pharmaceutical compositions and unit
dosages thereof, and in such form may be employed as solids,
such as tablets or filled capsules, or liquids, s~ch
as solutions, suspensions, emulsions, elixirs, or capsules
filled with the same, all for oral use; in the form of
suppositories for rectal administration; or in the form of
sterile injectable solutions for parenteral (including
intravenous or subcutaneous) use. Such pharmaceutical
compositions and unit dosage forms thereof may comprise
conventional ingredients in conventional proportions, with
or ~ithout additional active compounds or principles, and
such unit dosage forms may contain any suitable effective
amount of the active ingredient commensurate with the
intended daily dosage range to be employed. Tablets con-
taining ten (10) milligrams of active ingredient or, more
broadly, ten (10) to one hundred (100) milligrams, per
tablet, are accordingly suitable representative unit dosage
forms.
METHOD OF TREATING
Due to their high degree of activity and their low
toxicity, together presenting a most favorable therapeutic
index, the active principles of the invention may be admin-
istered to a subject, e.g., a living animal body, in need
thereof, for the treatment, alleviation, or amelioration,
palliation, or elimination of an indication which is suscep-
tible thereto, or representatively of an indication set
forth elsewhere in this application, preferably concurrently,
simultaneously, or together with a pharmaceutically-acceptable
MERZ 12
- 17 -
13~3(:~2
carrier or diluent, especially and preferably in the form of
al pharmaceutical composition thereof, whether by oral,
rectal, or parenteral (including intravenous and subcutane-
ous) or in some cases even topical route, in an effective
antibacterial, antimycoticl antiviral, or antimitotic,
cytostatic, and/or antimutagenic amount. Suitable dosage
ranges are 1-1000 milligrams daily, preferably 10-500
milligrams daily, and especially 50-500 milligrams daily,
depending as usual upon the exact mode of administration,
form in which administered, the indication toward which the
administration is directed, the subject involved and the
body weight of the subject involved, and the preference
and experience of the physician or veterinarian in charge.
M E R Z 1 2
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~ 3~Z
EXAMPLES OF REPRESE~lTATIVE PI~ARMACEU'rICAL COI~POSITIONS
With the aid of commonly used sol~ents, auxiliaryagents and carriers, the reaction products can be processed
into tablets, coated tablets, capsules, drip solutions,
suppositories, injection and infusion preparations, and the
like, and can be therapeutically applied by the oral,
rectal, parenteral and additional routes. Representàtive
pharmaceutical compositions follows.
a) Tablets suitable for oral administration which
contain the active ingredient may be prepared by conven-
tional tabletting techniques.
b) For suppositories, any usual suppository base may
be employed for incorporation thereinto by usual procedure
of the active ingredient, such as a polyethyleneglycol
which is a solid at normal room temperature but which melts
at or about body temperature.
c) For parenteral (including intravenous and subcu-
taneous) sterile solutions, the active ingredient together
with conventional ingredients in usual amounts are employed,
such as sodium chloride, sodium dihydrogen phosphate,
disodium edetate (ethylenediaminetetraacetic acid disodium
salt), benzyl alcohol, sodium hydroxide to adjust pH, and
double-distilled water q.s., according to conventional
procedure, such as filtration, aseptic filling into ampoules
or IV-drip bottles, and autoclaving for sterility.
Other suitable pharmaceutical compositions will be
immediate}y apparent to one s~illed in the art.
The following examples are again given by way of
illustration only and are not to be construed as limiting.
- 19 - MERZ 12
l3~jl3a~;~
Exa~ ablet formulation
__ __
A suitable formulation for a tablet containing 10
milligrams of active ingredient is as follows:
Mg.
Active Ingredient---------------10
Lactose-------------------------18
Potato starch-------------------38
Gelatin------------------------- 2
Talcum-------------------------- 2
Magnesium stearate------------- 0.1
. - Tablet formulation
Another suitable formulation for a tablet is as
follows:
. Mg
Active Ingredient---------------10
Potato starch-------------------40
Polyvinylpyrrolidone------------ 5
Sugar coated and colored.
. Example 3. - Capsule formulation
A suitable formulation for a capsule containing 10
milligrams of active ingredient is as follows:
Mg.
Active Ingredient---------------10
Corn starch---------------------90 '
Lactose-------------------------50
Talcum-------------------------- 2
filled in a gelatin capsule.
Example 4. - Solution for injection
A suitable formulation for an injectable solution
- 30 containing one percent of active ingredient is as follows:
Active Ingredient---------------mg-- 12
Sorbitol------------------------mg-- 40
Sterile water to make-----------ml--
Example 5. - Liquid oral formulation
A suitable formulation for 1 liter of a liquid mixture
containing 2 milligrams of active ingredient in one milli-
liter of the mixture is as follows:
- 20 - ~E~2 12
13~3~
G.
Active Ingredicnt~ 2
Saccharose--------------------- 250
- Glucose------------------------ 300
d-Sorbitol--------------------- 150
Agar-agar----------------------0.15
Methylparaben------------------ 0.5
Propylparaben------------------0.05
Orange flavor------------------ 10
Tartrazine yellow.
Purified water to make a total of 1000 ml.
Exam~le 6. - Liquid oral formulation
Another suitable formulation for 1 liter of a liquid
mixture containing 2 milligrams of active ingredient
in one milliliter of the mixture is as follows:
G.
Active Ingredient------------- 2
Tragacanth-------------------- 7
Glycerol---------------------- 50
Saccharose----------- -------- 400
Methylparaben----------------- 0.5
Propylparaben----------------- 0.05
Black currant-flavor---------- 10
Red No. 2 C.I. 184------------- 0.02
Purified water to make a total of 1000 ml.
Example 7. - Liquid oral formulation
Another suitable formulation for 1 liter of liquid,
mixture containing 2 milligrams of active ingredient
in one milliliter of the mixture is as follows:
G.
Active Ingredient-------------- 2.4
Saccharose--------------------- 400
Bitter orange peel tincture---- 20
Sweet orange peel tincture----- 15
Purified water to make a total of 1000 ml.
- 2l - MER2 12
13~
PHARMACOLOGY - SUMMARY
The active principles of the present invention, avarone
and avarol, and pharmaceutical compositions thereof and
method of treating therewith, are characterized by unique
advantageous and unpredictable properties, rendering the
~subject matter as a whole", as claimed herein, unobvious.
The compounds and pharmaceutical compositions thereof have
exhibited, in standard accepted reliable test procedures,
the following valuable properties and characteristics:
Antiviral
~ Antitumor
I Antimitotic
. ¦ Cytostatic
Antimutagenic
Antilymphoma
. Antileukemic
Anti human malignant.brain tumor
Antibacterial
Antimycotic
and are accordingly of utility in the treatment, elimina-
k,.~, tion, palliation, alleviation, and amelioration of respon-
sive conditions9 infections, and infestations by application
:. . or administration to the host or to.the situs, or even to
the microorganism itself, as when bacterial, fungal, or
viral infestations are involved.
,
- 22 -- MER8 12
13~3~
PHARMACOLOG~
. .
Pharmacological Actions (A) - Antitumor
The reaction products according to the general formul~
I and particularly avarone and avarol, show valuable pharma-
cological properties. The antitumor activity has been shown
in vitro using the L5178y and L1210 lymphoma cell system in
mice. These cells were suspension cultures, as described
previously (W.E.G. Muller and R. Zahn, Cancer Res. 39, 1102
(1979)).
The EDso concentrations of the compounds (inoculation:
4000 cells/ml; incubation time: 72 hours) ranged from o.7
to 6.7 ~m for both tumor cell strains. In this test avarone,
avarol and the avarol esters were the most effective substances.
The action mechanism could be demonstrated in incorporation
studies. In the presence of the reaction products in ED50
concentrations the incorporation of L3H]thymidine into the
DNA, of L3H]uridine into the RNA, of ~ H~ phenylalanine i~to
proteins, and of L3H~mannose into glycoproteins is promoted.
This effect could be shown by autoradiographic methods (K. Habel
and N.P. Salzman, Fundamental Techniques in Virology, Academic
Press, New York 1969, p. 242) to be a partial synchronization
of the cells during the G2-M phase.
In cytological studies by using Giemsa staining and acridine orange
staining of the mitotic chromosomes (R. Rigler, Acta Physiol. Scand.
67 (suppl. 26?), 1 (1966), it ~las found that the reaction products
affect the mitotic index of L5178y cells. Therefore, the pharma-
cological activity of avarone and its derivatives is that of a
mitotic toxin. This action mechanism has also been demonstrated in
biochemical studies: Avarone and its derivatives inhibit the poly-
merization of microtubuli in the substoichiometric range as was
shown in viscosimetric studies by S.D. Mac.Lean-Fletcher and T.D.
Pollard (J. Cell Biol. 85, 414-428 (1980l).
The polymerization of the microtubuli is inhibited on the
level of protofilament elongation. By employing higher
concentrations of the reaction products the polymerization of
microtubuli during other cell phases is inhibited in addition to
the inhibition of the polymerization of microtubuli during the
mitotic phase. There are no chromosome aberrations after the ad-
ministration of therapeutic doses of avarone and its derivatives.
F - 23 - MERZ 12
13~3~2
of special importance for the application of the reaction products
in the chemotherapy of carcinoses is the fact that there is no
cytostatic effect on non-tumor cells in culture when being used
in therapeutically~applicable concentrations.
This result was obtained from tests on lymphocyte cultures:
Spleen lymphocytes were obtained from 6- week --old NMRI mice.
The erythrocytes were eliminated from the suspension by
treatment with ammonium chloride. The spleen lymphocytes were
kept in RPMI 1640 medium/20% fetal bovine serum in a density
l~ of 1.5 x 107 cells/ml for 72 hours in the presence of 2 ~g/ml
concanavalin A. 18 hours prior to the termination of the test
~3HJthymidine was added. On incubation with 10 pm of the
reaction products no impairment of the DNA rate of synthesis
was determined. In the presence of 15 ~m there was only a
15% inhibition of the incorporation rate fL3~ thymidine
into the DNA.
The antitumor action of avarone and its derivatives has also
been shown in vivo in experimental animals. The tests were
carried out as described previously (W.E.G. Muller, R.K. Zahn,
A. Maidhof, H.C. Schroder, M. Bachmann and H. Umezawa,
J. Antibiotics 37 , 239 (1984)). NMRI mice that had been
inoculated with L5178y lymphoma, and - after having developed
the tumor - were treated for.5 days with,e.g., o.3 ~g/kg avarone
i.p. per day, lived considerably longer than the untreated
mice. 50% of the untreated animals had died after 18 days,
whereas 50~ of the treated animals were still alive on the
26th day.
The subacute toxicity values ranging from 35 to 75 mg/kg at 5-day
i.p. treatment of mice are so favourable that the application
of the reaction products in the chemotherapy of carcinoses
is to be expected.
-ANTIBACTE~lAJ.
The antibacterial action of avarone and its derivatives was
established in in-vitro tests by using the ag~r dilution method
(~.E. Scully, K. Jules and H.C. Neu, Antimicr. Ag. Chemother.
23, 907 (1983). The bacteria were used in volumes of 105
colony forming units.
- 24 - MERZ 12
13(~3C1~2
Ihe minimum inhibitory concentration (~-IIC) ofe.g. avarol
was determined to be o.4 ,um in cocci like Micricoccus
pyrogenes, o.9 ~m in gram-negative bacteria like, for
example, Escherichia coli, and 1.3 ym in gram~positive
bacteria such as Bacillus cereus. This high in-vitro
activity is within the range of the in-vitro antitumor
activities. Owing to the low subacute in-vivo toxicity
in mice the chemotherapeutic index (TI=LD50 in the mouse
divided by the sensitivity of the germ) for,e.g., avarol
is between 100 and 140, dependent on the microorganism
used.
-ANTIVIRAL
Avaron and its derivatives show an antiviral effect in
BHK cell cultures that have been infected with herpes
simplex virus of types I and II. The viral concentration
(multiplicity of infectiosity) was adjusted to o.5 to
1.5 plaque for~.ing units per cell. 2 hours after infection
the cultures were washed, and the reaction products added.
24 hours after infection the virus titre (p.f.u./o.2 ml)
was determined. With concentrations ofle.g.~avarol of 1 pm
type I replication of 9 x 104 (control) was reduced to
1 x 103 (treated cultures), and type II replication of
6 x 105 (control) was diminished to 7 x 103 (treated cultures).
-ANTIMYCOTIC
The antimycotic activity was determined in vitro with the aid
of the Broth Dilution Method on Saccharomyces cerevisiae (E.
Lennette (ed.), Manual of Clinical ~icrobiology, ed. by
American Society of Microbiology, Washington 1980, p. 648-
649). The minimum inhibitory concentration was determined for~
e.g.)avarol to be o.7 ~m, the 50% inhibitory concentration being
about 1.6 ~ .
Owing to the pronounced antibacterial, antiviral and antimycotic
activities of the products avarone and avarol, it is clear that
they will be useful in the treatment of infectious diseases.
- 25 - MERZ 12
F
13V3C~
.
Pharmacolo ical Actions (B) - Antitumor
,~ 9 _ __ __ _
Avarone and also Avarol are antimitotic agents which
cause at ED50 concentrations an increase of mitotic
indices from 6.5 to 11.9 [10.4~, using L5178y mouse lymphoma
cell system (Muller et al., Bas. Appl. Histochem. 1985,
29, in press). In in vitro studies, applying the ~nethods
of viscosimetry and electron microscopy, it was demonstrated
that the compounds inhibit assembly of brain microtubule
protein at an at least stoichiometric concentration ratio
(luller et al., Bas. Appl. Histochem. 1985, 29, in press).
Moreover, evidence is available that the new antimitotic
agents inhibit protofilament elongation rather than lateral
association of tubulin during protofilament formation. It
is suggested t~at the compounds interfere with polymeriza-
tion of tubulin both in interphase and during mitosis.
Binding studies in vitro demonstrated Avarol (radiolabelled
as 13HI-Dihydroavarol) to interact with tubulin at sites
different from those known for Vincristine, Colchicine and
EtoposideR (Table).
T A B L E
Binding affinity of 13l~-Avarol (labelled [3HI-Dihydroavarol)
(1 ~M) to tubulin, as measured by binding assay technique
none plus 30 ~M plus 30 ~M plus 30 ~M
¦ Vincristine Colchicine EtoposideR
. I
no tubulin <4 ¦ <4 <4 <4
1 yM tubulin 61 ¦ 57 59
10 ~Mtubulin 96 ¦ 93 1 97 91
- 26 - MERZ 12
~3~3C~
In cell culture studies it was established that Avarone
and its analogue Avarol exhibit potent cytostatic activities
both on L5118y mouse lymphoma cells (T-cell lymphoma)
and on L1210 mouse leukemia cells (B-cell derivative)
(Muller et al., Comp. Biochem. Physiol. 1985, 80C, pp.
47-52` and Cancer Research 1985, 45(10), in press). The
following ED50 concentrations (concentration which causes
a 50% inhibition of cell growth) were determined for
Avarone (Avarol); L5178y cells: 0.62 M (0.93 M) and for
L1210: 0.95 M (1.1 M). The corresponding ED50 concen-
trations in experiments with non-lymphoid cells~ (e.q.,
melanoma cells, HeLa cells, human fibroblasts, and human
gingival cells) were determined to be 15 to 120-fold higher.
In L5178y cells, Avarone was the most cytotoxic of the
compounds tested; Avarol and Dihydroavarol were moderately
cytotoxic, and the methylamino derivatives of Avarone were
effective at higher concentrations.
In order to define the activity spectrurn of Avarone and
Avarol further, their influence on DNA synthesis in
and B lymphocytes was investigated. As mitotic inductors we
used concanavalin A (ConA) for T lymphocytes from both mouse
spleen and human peripheral blood, lipopolysaccharide (LPS)
for murine B lymphocytes, and pokeweed mitogen (PWM) for
human q~ and 8 lymphocytes ~Muller et al., European J.
Cancer and Clinical Oncology - in press).
Studying the effect on mouse spleen lymphocytes, we
found the following EDso concentrations (inhibition of
t3H] dThd incorporation rate~ by 5096), for Avarone:
non-activated cultures, 2.9 M; ConA-stimulated lymphocytes,
1.9 ~ and LPS-stimulated cells, 4.3 M; and for Avarol:
2 7 - ~5~F~Z 12
i3~3~
non-activated cultures, 3.8 M; and ConA- or I,PS- stimulated
lymphocytes, 2.4 M and 5.9 M, resp. Interesting was the
finding that at low concentrations both Avarone and Avarol
enhanced DNA synthesis in non-activated lymphocytes and
especially in LPS-stimulated lymphocytes. ~he highest
stimulatory effect was measured in the drup ranges 1-2 M.
This means that the two secondary metabolites from Dysidea
avara can break - dose dependently - the restrictive
control mechanism(s) of DNA synthesis in particular eukaryo-
l0tic cells.
-- TOXICITY
The in vivo toxicity Img compound/kg) Avarone (Avarol)
on male NMRI mice is as follows; acute toxicity: LDso
181.2 (269.2), LD10 111.1 (156.4) and subacute toxicity:
LDso 172.1 (218.4), LD10 10g.7 (138.6) (Muller et al.,
Cancer Research 1985, 45(10), in press).
- ANTILEUKEMIC ACTIVITY
The antileukemic activity of Avarone and Avarol was
studied in vivo using the L5178y cell system in NMRI mice
20(Muller et al., ibid.). The median life span of the L5178y
lymphoma-bearing control mice was 14.3 days. Both Avarone
and Avarol were determined to increase the life span of the
tumor-bearing mice considerably. The animals were i.p.
injected with the compounds for 5 consecutive days starting
at day 1 or aay 8. In general, the treatment starting at
day 1 after tumor inoculation was superior to that beginning
at day 8, as can be deduced fom the values for the % ILS
(increase in median life span): Schedule day 1-5 (day 8-12)
for 10mg Avarone/~cg/injection: 146% (69%) and for Avarol:
3087% (34%). The schedule of days 1-5 resulted even in a
-- 28 -- MERZ 12
13~)3~`~i2
dose-dependent cure of 20-70% of the tumor-bearing mice
both with Avarone and Avarol. Using this regimen, the
dosage of 10 mg Avarone/kg/day received the hiyhly active
activity rating, which bases on the determined log10 kill
values. Avarol was somewhat less efficient. The 90~
effective doses of both Avarone and Avarol on tumor growth
were estimated according to Skipper and Schmidt tCancer
Chemotherapy Rep. 1962, 17, pp. 1-173~ and found to be
for Avarone 9.4 mg/kg/day and for Avarol 31 mg/kg/day.
From these data and the LD10 values, the therapeutic
ratios were calculated for Avarone to be 11.7 and for
Avarol, 4.5. These values are in the range of those deter-
mined for cyclophosphamide, daunomycin and methotrexate
(Rubidomycin. Berlin, Springer-Verlag 1969).
-ANTIBACTERIAL-ANTIFUNGAL
In a screening we found that Avarone and to a smaller
extent also Avarol were active against a variety of grampo-
sitive bacterial species. The highest activity was deter-
mined for Pneumococcus mucosus and Erysipelothrix insidiosa
~MIC, 0.781 mg/l). The antibacterial activity can be
augmented 2 to 4-fold by lowering the pH in the culture
medium from 7.0 to 6Ø In addition, Avarol and to a
smaller extent also Avarone displayed an antifungal activity
on Trichophyton species and Microsporum canis (MIC, 15.6-
62.5 mg/1), while Avarone but not Avarol was active on
Aspergillus niger; no activity was found against Candida
species ~uller et al., Zbl. Bakt. 198S, in press).
-ANTIMUTAGENIC
Avarone and its analogue were determined in the Ames
test to be neither direct nor indirect ~S-9 activatin,g
MERZ 12
- 29 -
. 13~3C~Z
system) mutagens (Muller et al., Cancer Res. 198S, 45(10),
in press, and Mut. Res. Letters 1985, 144, pp. 63-66).
I?urthermore ~varone and its analogues were no -inducers of
benzo(a) pyrene monooxygenase (BaPMO) activity in experi-
mental fish. At doses of 50 or 100 mg/kg of Avarone and its
analogues, a BaPMO activity of 30.2 pg of 3-OH-BaP formed
per mg protein x min was measured; the enzyme in the control
animals showed an activity of 35 pg/mg x min. For the
assessment of the potential antimutagenic property, Avarone
and Avarol were incubated in combination with benzo(a)pyrene
in the Ames-microsomal assay. Benzo(a)pyrene alone was
found to induce in the presence of S-9 fraction 1325 his+
revertants at a concentration of 10 ~M. Applied in combina-
tion with Avarone or Avarol, the mutagenic effect of benzo-
(a)pyrene was drastically reduced. Addition of 5 ~ M of
either of the two compounds reduced the number of the
revertants to 22 26%, while at 30 ~ M and 150 ~M a reduction
to 14% and 11%, respectively, was det~rmined. Hence, both
Avarone and Avarol are powerful antimutagenic agents exhi-
biting an activity as strong as the known cytochrome P-450
dependent monooxygenase inhibitor benzoflavone. The assump-
tion, drawn from the data of mutagenicity testing, that both
Avarone and Avarol are potent inhibitors of BaPMO, was
confirmed by enzymic studies. Concentrations of 50 ~ M
reduced the activity to 18% (Avarone) and to 20% (Avarol),
respectively.
The hitherto elucidated characteristics provide Avarone
and Avarol with promising properties indicative of potential
utility for an application thereof in human cancer treat-
rnent. They are; (a) the compounds are present in a high
yield in thP sponge Dysidea avara, which is very abundant;
MERZ 12
- 30 -
~3~33(~S'2
(b) Avarone and Avarol display potent antileukemic activity
in vitro; 8c) they cause biphasic and differential effects
on DNA metabolism of human and murine T and B lymphocytes;
(d) the cytostatic agents are highly active also as anti-
leukemic agents in vivo; (e) they act as antimitotic com-
pounds on the level of microtubule formation; (f) the two
agents were determined to be neither direct mutagens nor
premutagens; (g) moreover, they display antimutagenic
activity and (h) the two compounds display also antibac-
terial and antifungal activity against a limited range of
microorganisms.
PHARMACOLOGICAL ACTIONS (C) - ANTIVIRAL
The antiviral activity of Avarone and Avarol is not
restricted to DNA containing viruses only, but is also
pronounced towards RNA containing viruses. The compounds
inhibit sensitively the growth of oncogenic RNA viruses.
The studies were performed with Schmidt-Ruppin D strain
of Rous Sarcoma Virus (RSV) in cell culture assays. The
detailed methods for testing the compounds were described
earlier in (A. Totsuka, W.E.B. Muller and R.R. Zahn:
~leomycin, action on growth of oncogenic RNA viruses and
on cell transformation. Archives of Virology 43, 169-179;
t975). For infection, secondary cultures of chic~ embryo
fibroblasts were used. lrhe cells were infected with RSV at
a concentration of 10 3 focus forming units (FYU). One
hour later the cultures were supplemented with different
concentrations of the two compounds. 48 hours later the
virus yield per cell was determined.
Result: At a concentration of:
1 micromolar of Avarone the virus yield was reduced by
72% and
- 3l - M~RZ 12
` ~3~3~52
1 micromolar of Avarol by 68~.
The virus yield in the inected cultures, which were not
treated by the compounds, was 251,188 FFU per 1,000,000
cells.
The proliferation of non-infected cells was not influenced
by Avarone and Avarol at the concentrations (1 micromolar)
used.
The described inhibition of oncogenic RNA viruses
in intact cell system is also confirmed by subcellular
studies. As a te~sting parameter, the key enzyme for virus
multiplication - the reverse transcriptase (RNA-depenaent
DNA polymerase) - was chosen. This enzyme was isolated from
Rauscher murine leukemia virus (RMI,~) and from human T-
lymphotropic retrovirus (HTLV-III) as described earlier
(W.E.G. Muller, R.K. Zahn, H.J. Seidel: Inhibitors acting
on nucleic acid synthesis in an oncogenic R~A virus.
Nature, New Biology 232 143-145; 1971). The detailed
description of the test procedure of the reverse transcrip-
tase is given in the same publication.
Results: At a concentration of 1 microgram per ml the
reverse transcriptase from RMLV was inhibited
by 59% and the same enzyme from HTLV-III by
67%.
PHARMACOLOGICAL ACTIONS (D) - ANTITUMOR (Human Brain)
The antitumor activity of Avarone ana Avarol is also
pronounced towards malignant human brain tumors.
A glioblastoma line was used for the tissue culture
studies. The cells derived from a patient with primary
and metastatic malignant glioblastoma turnor. Biopsy mater-
ial was disaggregated and cultivated in MEM-medium plus 20%
fetal calf serum. The cells were seeded at a concentration
- 32 - MERZ 12
13~3~2
.
of 500,000 cells per 3.5 cm petri dish and incubated for
48 hours in the absence or presence of Avarol or Avarone.
Then the cell number was determined. The detailed descrip-
tion of the test procedure was given earlier (A. Totsuka,
W.E.G. Muller, R.K. Zahn: Bleomycin, action on growth of
oncogenic RNA viruses and on cell proliferation. Archives
of Virology 48~ 169-179; 1975).
Results: At a concentration of 3.5 microgram per ml the
inhibition of the cell growth was:
for Avarone: 86%and
for Avarol: ?'%-
The future application of Avarone and/or Avarol inhuman chemotherapy of brain tumors is very promising,
becausé the compounds can penetrate the blood-brain-barrier.
The experiments were performed in vivo.
The experiments were performed with dihydro-avarol
(tritium labelled; specific radioacti~ity: 3.2 Ci/mmol).
As test animals Balb/c mice (male; 25 g) were chosen.
The radioactive Avarol was injected intra-peritoneally
(i. p.) at a concentration of 10 mg/kg. 1 hour thereafter
the concentration in the organs was determined by the
radioactivity.
Results:
Organ Concentration of tritium-labelled
dihydro-avarol (microgram per g
tissue)
Liver 12.3
Lung 7.4
Skin 2.8
Brain 4.2
These data indicate that the compound is transported
into the brain after i.p. injection. Together with the
tissue culture data, a curative effect of Avarol and/or
Avarone towards brain tumors in vivo can be expected.
- 33 - MERZ 12
13~3~SZ Merz 12
EURTHER
DETAILED PHARMACOLOGICAL E~MPLES
EXAMPLE I
ABSTRACT
The two novel antlmltotic and potent antileukemlc agents avarone
and avarol were determined to lnhibit the L3H]-dThd lncorporation
rates o~ both murllle spleen and human peripheral blood lympho-
cytes wlthin the concentratlon range 2 - 6 ~M. The mitogens con-
canavalln A (ConA; for T lymphocytes), llpopolysaccharlde (LPS;
ror murine B lymphocytes) and pokeweed mitogen (P~1M; ror human T
and B lymphocytes) were used to stimulate DNA synthesis in the
lymphocyte fractlons. The EDso concentratlons, causing a 50~ re-
duction o~ [3H]-dThd lncorporation, were signiflcantly lower ln
the experiments with avarone than in those with avarol. Moreoyer
i~ was established that the DNA synthesis Or ConA-activated lyr-
phocytes was more sensitively innlbited by the compounds than
that Or I.PS- or PMI'-activated cells, or non-activated cells. In
additlon lt ~as elucidated, that at low concentrations (1 - 2 ~
avarone and avarol caused a stimulation Or dThd incorporation or.-
ly in LPS or PWM-activated lymphocytes. Based on these results it
is assumed that both antileukemic agents difrerentially afrect
also the dirrerent hematologic neoplasms.
a ~ - 34 - MERZ 12
Merz 12
13~)3~SZ
l~;TRODUCTION
Basic studles to elucldate the symblotlc relatlonship betueen
sponges and thelr species-speclflc bacterlal rlora led to the
dlscovery Or the sesqulterpenold qulnone avarone and lts hydro-
qulnone derlvative avarol 11, 2]. These two secondary metabolites
from the marine sponge Dysldea avara were determlned to be lnhl-
bltors Or mltosls Or eukaryotlc cells 12] possibly due to an in-
terference Or these compounds wlth the process Or tubulln poly-
merlzatlon 13]. Recently we notlced that both avarone and avarol
exhlbit potent antlleukemlc actlvity not only in vitro 12] but
also ln vlvo L4 ]. An lnterestln~ reature Or these cytostatlc
a6ents ls thelr hlghly selectlve inhlbitory actlvity on lympho-
cytes ln vltro 14]. Dose-response experiments wlth the T cell
lymphoma cell llne L5178y 15] revealed that avarone inhibits cell
growth at 0.62 ~M, and avarol causes the same efrect at 0.93 ~M
14]- The corresponding EDso concentr2tions in in vitro experi-
ments wlth non-lymphold cells (e.g., melanoma cells, HeLa cells,
human rlbroblasts and human glnglval cells) were determined to be
15 to 120-rold higher.
In the present study we determined the lnrluence Or avarone
and avarol on normal lymphocytes in vltro. In the rlrst sectlon
Or thls paper, the influence on the mltotlc index was studled. In
the second part, the errect on DNA synthesis ln T and B lympho-
~ytes was lnvestl~ated. As mitotlc lnductors we used concanavalln
A [ConA] ror T lymphocytes rrom both mouse spleen and human perl-
pheral blood 16], llpopolysaccharlde 1LPS ] ror murlne B lympho-
cytes 17] and pokeweed mitogen ~P~'M] for human T and B lympho-
cytes 16]. The results revealed that 2varone and avarol prereren-
tl211y lnhlblt DNA synthesls ln T cells. Moreover lt was round
that at non-cytoctatlc concentratlons the two compounds enhance
DtJA synt~.esls ln B lymphocyte~.
F - 35
13~3~`S2 Merz 12
AT~RI~LS AI~D METHODS
~aterlals
Concanavalln A [ConA] (no. C 5275), lipopolysaccharlde 1LPS] (no.
L 4130) and pokeweed mito~en (no. L 9379) were obtalned rrom Si6-
ma, St. Louls, MO (USA); lmethyl-3H]thymldlne (spec. act. 87 Cl/
mmole) from the Radlochemlcal Centre, Amersham ~En~land); colcl-
mide rrom Ciba-Geigy, Wehr (Germany) and phytohema~glutlnln (PHA
15) rrom Deutsche Wellcome, Burgwedel (Germany).
Avarol was lsolated rrom Dysidea avara (2), whlch was col-
lected ln the Bay Or Kotor (Yugoslavla). Avarone was obtainedrrom lts correspondlng hydroquinone avarol by Ag20 oxidatlon (2).
Cultlvation o~ lymPhocytes rrom mouse spleen
Spleen lymphocytes were prepared rrom 5 - 6 weeks old male out-
bred NMRI mlce as descrlbed [8]. The erythrocyte-rree and macro-
phage-contalning lymphocytes (2.5 x 106 cells) were placed in a
inal volume Or 200 al on mlcrotiter plates and incubated ror 7~
hr ln Dulbeccosbmminlmum essential medium (DMEM), supplemented
wlth 2 mM glutamine and lO S retal calr serum. 18 hr prlor to the
end Or the incubation 0.1 ~Ci of [3H~-dThd was added to each cup.
~ere lndicated 2 ~g/ml o~ ConA~or 20 ~g/ml Or LPS were added to
the cultures. The cytostatic agents avarone and avarol were dls-
solved ln dimethyl sulroxide and added at tlme zero to the cul-
tures. The flnal concentration Or dlmethyl sulroxlde was 0.1 ~;
at this concentratlon dlmethyl sulroxide has round not to in~lu-
ence the ~3H]-dThd lncorporation rate. Incorporation Or 13H]-dThd
has determined as descrlbed [9].
Each value came rrom 6 parallel experiments. ~ne EDso con-
centratlons causln~ a 50 Z reduction Or [3H]-dThd lncorporatlon
were estlmated by logit regression [lO].
PreDaration Or human lymphocytes rrom clrculatin~ blood
From freshly drawn heparlnized blood (lO IU Or heparln sodlu~
salt/ml blood) the lymphocytes uere isolated accordln~ to ~he
descrlbec procedure by Flcoll-fionpccon lll]. The resultlr.~ l~m-
c - 36 -
Merz 12
~3~3C~
phocytes were suspended at 2 density of 4 x 106 cells/ml DMEJ~l,
~upplemented wlth 10 Z retal calr serum. 1 x 106 cells ln 200 ~1
were placed on mlcrotiter plates and processed as descrlbed
above. The cultures were lncubated either ln the absence or pre-
sence Or mlto~en (2 ~g/ml Or ConA or 3 ~g/ml Or PWM).
Determlnatlon Or mltotlc lndex
Purirled human perlpheral blood lymphocytes were incubated ror 70
hr ln DME~ supplemented wlth 10 ~ fetal calf serum at a density
Or 1 x 106 cells/ml ln the presence Or colcimide ~0.1 ~/ml) and
phytohemagglutinin (2 ~g/ml). Durlng the last 3 hr Or lncubatlon,
dlfrerent concentrations Or avarol were added to the 5-ml assays.
The cells were then processed as rollows: 20 mln exposure to hy-
potonlcity achleved by adding 3 volumes Or distilled water to tne
cultures; fixation was ln 60 Z acetlc acld-0.1 N hydrochlorlc
acid ror 15 min; stalning with 2 ~ acetlc orcein. Squash prepa-
ratlons were made on slllconlzed slldes.
For the determlnation Or the mitotlc lndex, 1000 to 1500
cells were analyzed per assay.
Statistical evaluation
T-tests to determlne the signlricance Or the growth lnhlbltlon
errects ln the presence or absence Or mitogens were perrormed ac-
cordlng to Student [10].
d F - 37 -
13~3~S2
In the accompanying drawings:
Figs. l and 2 are graphs with Fig. l illustrating
the effect of avarone (A) and avarol (B) on
[3H]-dThd and Fig. 2 illustrating the effect
of avarone (A) and avarol (B) on 13H]-dThd;
Fig. 3 is a graph illustrating the infrared absorption
spectrum of avarol (KBr);
Fig. 4 is a graph illustrating the reversiblilty of
the inhibition of cell growth caused by avarol; and
Fig. 5 is a copy of autoradiographs of L5178Y cells
treated with O (A) or 0.85~M of avarol (B).
Fiq. l. Effect of avarone (A) and avarol (B) on 13H]-dThd
incorporation into murine spleen lymphocytes in the absence
(- ), or the presence of 2 ~g concanavalin A/ml (o--o)
or 20 ~g lipopolysaccharide/ml (x..... x). Addition of
13H]-dThd: 18 hr prior to the end of the incubation.
Avarol or avarone was added at time zero. Means of quadriplicate
experiments are presented; the S.D. varied between 6 and 8 %.
The values for a 50 % inhibition of 13H]-dThd incorporation
of the corresponding dose-response experiments are given
as vertical lines.
Fig. 2. Effect of avarone (A) and avarol (B) on 13H]-dThd
incorporation into human peripheral blood lymphocites in
the absence (- ), or the presence of 2 ~y concanavalin
A/ml (o--o) or 3 ~g pokeweed mitogen/ml (x..... x). Further
details are given in the legend to Fig. l.
3 .a
Merz 12
13~3C~5;~
~ESULTS
Increase Or mltotlc lndex by avarol treatment
Arter a 3 hr lncubatlon Or perlpheral blood lymphocytes ln the
presence Or avarol, the mltotlc lndex substantlally lncreased. At
dru6 concentratlons Or 1.5 ~M, 3.0 ~ or 6.o ~M the mitotic lndex
lncreased to 6.27, 8.91 or 9.45. The mitotlc lndex Or the con-
trols (absence Or avarol) ~as 3.51.
Errect Or avarone and avarol on mouse spleen lymDhocytes
In the absence Or mltogen the ~3H~-dThd incorporation rate ln the
lymphocyte cultures was determlned to be 1.3 + 0.1 x 10 3 dpm/2.5
x 106 cells per 18 hr. Addltlon Or 2 ~g ConA/ml or 20 ~g LPS/ml
to the cultures augmented the lncorporatlon rate to 21.7 + 1.~ x
103 dpm or 45.8 + 3.6 x 103 dpm/2.5 x 106 cells per 18 hr resp.
As summarlzed ln Fig. 1, avarone was the more potent lnhibl-
tor Or DNA synthesls (measured by the [3H]-dThd lncorporatlon
rate) than avarol lrrespectlvely Or the actlvatlon state Or the
lymphocytes. The rollowing EDsO concentratlons were estimated
from the dose-response experiments; ror avarone: non-actlvated
cultures, 2.9 + 0.2 ~M; ConA-stlmulated lymphocytes, 1.9 + 0.2
~M; and LPS-stlmulated cells, 4.3 + 0.3 ~ ; and ror avarol: non-
activated cultures; 3.8 + 0.3 ~M; and ConA- or LPS-stlmulated
lymphocytes, 2.4 + O.2 ~; and 5.9 + 0.4 ~M, resp. In all three
cases the dirrerences ln-the lnhibitory potencles between avarcne
and avarol were stat~stlcally slgnlficant (P-value: c 0.001).
Interesting was the rlndlng (~lg. 1) that both avarone and
avarol at low concer.tratlons enhanced DNA synthesls ln non-actl-
vated lymphocytes and especially ln LPS-stimulated lymphocyte
The highes~ s~lrnul2tory errect was measured ln the dru6 r2n6es
1 - 2 ~M; mhxlmal stlmulatlon ror avarone was determlned to be
203 ~ (control: 100 ~; P value versus control c 0.001) at a con-
centratlon Or 1.8 ~M, and ror avarol, ~68 ~ (P: c 0.001) at the
same concentration.
e - 38 -
Merz 12
13~3C?~Z
Frfect on human perlpheral blood lymphocytes
The basis lncorporation rates ln the human lymphocyte cultures
were as follows; wlthout mltogen, 1.4 + 0.2 x 103 dpm/l x 106
cells per 18 hr and ln the presence Or 2 ~g ConA/ml or 3 ~g PW~,/
ml, 17.4 + 1.4 x 103 dpm or 35.9 + 2.6 x 103 dpm/l x 106 cells
per 18 hr, resp.
As alreaày determined ln the experlments with mouse spleen
cells, avarone exhlblted a slgnlrlcantly hlgher lnhlbltory poten-
cy (P: ~ 0.001) also on human lymphocytes than avarol. The ~ol-
lowlng EDso values were determined ror avarone (avarol): non-ac-
tlvated lymphocytes, 3.2 + 0.3 (4.9 ~ 0.4) ~M; ConA-stimulated
cells, 2.3 + 0.2 (4.0 + 0.3) ~M and PWM-stlmulated cells, 4.2 +
0.3 (5.8 + 0.4) ~M (Fig. 2).
Just as ln the experlments with murine lymphocytes, the
human n~n-actlvated and PWM-actlvated lymphocytes were also deter-
m~ to have an au~E~ted dThd-lncorporation rate at low avarone/ava-
rol concentratlons (Flg. 2). Agaln, thls enhanclng errect on DNA
synthesls was measured between 1 and 2 ~ . Compared to murine
lymphocytesl the degree Or stimulation Or human lymphocytes wlth
avarone/avarol was less pronounced and amounted ror PWM-actlvated
cells at 1.8 ~M Or avarone (avarol) 136 % (126 %) [control: 100 %;
P value versus control ~ 0.001~.
f - 39 -
F
Merz 12
13~3~S2
DISC'JSSIOI~
The data reported ln thls contrlbutlon conrlrm earller rinding
[3] showing that avarone and avarol cause mitotlc arrest Or lym-
phocytes in vltro due to an interrerence with the mlcrotubule sy-
stem. The maln concluslons dra~ln rrom thls paper are that (a) D~A
synthesls Or T lymphocytes and Or B lymphocytes ls dlrrerentlally
lnhlblted by both antlmltotlc drugs and (b) a dose-dependent stl-
mulatlon Or DNA synthesls by these drugs occurs only ln B lym~ho-
cytes.
Avarone and avarol were round preferentlally to lnhlblt D~
synthesis Or T lymphocytes rrom both mouse spleen and human pe,ri
pheral blood. The T lymphocytes rrom spleen were determlned to be
2.3 to 2.5-rold more sensitlvely lnhlblted (statistically si6ni-
flcant; P-value: ~ 0.001) than the B lymphocytes. In the~ruture
we have to elucldate whether the observed antlleukemlc actlvlty
ln vlvo ~4~ ls restricted to T-cell derlved leukemlas only.
In the last few years lt has been become clecr that cytoske'-
letal protelns (actln and tubulin) are also regulator elements Or
gene expression ln multlcellular or6anlsms [for revlew see: ~2].
Dlsruptlon Or microtubule archltecture or detorlatlon Or mlcro-
tubule runctlons by mlcrotubule polsons may result ln moàulatlon
Or gene expresslon, elther ln the catabollc ~13] or anabollc di-
rectlon [14]. Uslng Swlss 3T3 cells lt was shown that colchlclne
enhances the rete Or hormone-dependent DNA synthesls [14, 15].
The presented data ascrlbe avarone and avarol such ~n actlvltJ
also. An lnterestin6 feature Or both antlmltotlc drugs, thct
might âttaln therapeutlcal lmportance, ls their dirrerentlal ef-
fcct on cells. Only DNk syn~hesls ln B lymphocytes rrom bo~h
mouse spleen (stlmulateà with the B cell mito~en LPS) anà hu~ar.
perlpheral blood (stimulated by the T and B cell mlto~en PW~.) waC
poslt'vely afrected (P-value: c 0.001) a~ low concentrations Or
avarone or avarol. me DNA synthesls ln T lymphocytes has not
stimulated by the drugs. It is not known whlch blochemlcal
e~ents, th2t 2re sensltive to the runctlon21 state Or the mlcrc-
t~ules, are involved ln the .estrictlve control Or DN~ synthe-
g F -40 -
Mer2 12
13~)3~
sls. ~lthou6h mlcrotubulec are appzren~l'y not p.esent ln the nuc-
leus~ tubulin has been detected amon6 the,non-histone protelns
116]. Further experimental evldence sus6ests an lnteractlon Or
mlcrotubules with the nuclear pore-comple~. [17]. Thus, mlcrotu-
bule rormatlon under experimental condltions has been shown to
occur rrom the pores towards the nuclear Sâp [18 ]. Results rrom
studles wlth microtubule-dlsruptlng dru~s, e.g., colchiclne ~14],
vlnblastlne [19] and avarone/avarol ~3; and this paper] suggest
that an lncrease Or the concentratlon Or rree tubulin stimulates
mltotic growth. Based on our recent data we assume that changes
ln the array Or mlcrotubules and/or alteratlons Or the concentra-
tlon Or free tubulln dimers may impalr posttranscrlptlonal con-
trol mechanlsms at the level Or nuclear-envelope nucleoslde tri-
phosphatase L20], essentlal ror nuclear-cytoplasmic transport Or
poly(A)+mRNA 121].
The hitherto elucldated characteristics (a) prereren-
tially antlleukemic 14], (b) T-lymphocyte-speclric and (c) antl-
mutagenic [22], provide the cytostatic a~ent(s) avarone/avarol
wlth promising properties utillzable ror an application also ln
human cancer treatment.
h - 41 -
F
Merz 12
13~)3QSZ
~EFEfiENCES
1. MINALE L, RICCIO RJ SODANO G. Avarol, a novel sesaulterpenoid
hydroqulnone wlth a rearran~ed drlmane skeleton rrom the
spon~e Dlsidea avara. Tetrahedron Lett 1974, no. 38, 3401-
3404.
2. ~JULLER WEG, ZAHN R~, GASIC MJ, DOGOVIC N, MAIDHOF A, BEC~ER
C, DIEHL-SEIFERT B, EICH E. Avarol, à cytostatically active
compound rrom the m.arlne spon~e D~slaea avara. Comp Biochem
Physlol 1985, 80C, 47-52.
3. ~ LLER WEG, ZAHN R~, MAIDHOF A, DIEHL-SEIFERT B, SACHSSE W,
GASIC MJ, SCHR~DER HC. Inhlbltion Or mltosls by avarol, a n2-
tural product isolated rrom the sponge Dysidea avara. Bas Ap-
pl Hlstochem 1985, ln pres~.
4. MULLER WEG, MAIDHOF A, ZAHN RK, SCHR~DER HC, GASIC MJ, HEIDE-
MANN D, BERND A, KURELEC B, EICH E, SEIBERT G. Potent antl-
leukemic actlvlty Or the novel cytostatic agent avarone and
lts analosues ln vltro and ln vlvo. Cancer Res 1985, in
press.
5. FRADE R, KOVRILSKY FM. Prellminary characterizatlon Or a gly-
coproteln havin~ Fc receptor propertles extracted rrom a T
cell lymphoma (L5178y). Eur J Immunol 1977, 7, 663-666.
6. LIS H, SHARON N. The biochemistry Or plant lectlns (phyto-
hemagglut1nlns). Ann Rev Biochem 1973, 42, 541-574.
7. Y.OLB JPB, QU~N PC, POUPON ~F, DES~.Y~r~.RD C. Carra~eenan stimu-
lates populatlons Or mouse "B" cell-~ mostly nonoverlappln~
with those s~lmul2ted wlth LPS or ~ex.tr2n sulrate. Cellul 1~-
~unol 1981, 57, 348-360.
i - 42 -
Merz 12
~303~
8. Lr~'HhUSEN G, SCHR~DEP~ HC~ SCHUSTEr ~ ihlDHOF P" UlqEZAW~ H,
MULLER ~EG. Potentlatlon Or the bleomycin, ara~lnoruranosyl-
cytoslne and adrlamycln-caused lnhlbltion of DN~ synthesls ln
lymphocytes by bestatln ln vltro. Eur J Cancer Clln Oncol
1985, ln press.
9. LEYHAUSEN G, SEIBERT G~ MAIDHOF A~ M~)LLER h~EG. Dlfrerentlal
stlmulation Or lyrnphocyte cell ~srowth ln vitro by cephalo-
sporlns. Antlmlcrob Agents Chemother i984, 26, 752-756.
10. KOLLER S. Statlstlsche Auswertmethoden. In: Biochemisches
Taschenbuch (ed. by H.M. Rauen), pp. 959-1045. Sprln~er-Per-
lag, Berlln (1964).
11. ~OYUM P.. Separatlon Or leukocytes rrom blood and bone marrow.
Scand J Clln Lab Invest 1968, 21 SUDP1., 1-114.
12. MULLER WEG, BERND A, SCHR~DER HC. Modulatlon Or poly(A)
(+)mRNA-metabolizlng and transportlng systems under special
conslderatlon Or microtubule proteln and actln. Molec Cell
~lochem 1983, 53/54, 197-220.
13. WALKER PR, WHITFIELD JF. Cytoplasmlc mlcrotubules are essen-
tial for the rormation of` membrane-bound polyrlbosomes.
J Blol Chem 1985, 260, 765-770.
14. OTTO AM, ULRICI'. MO, ZUI~.BE A, ASUA LJ. I~lcrotubule-dlsruptins
a~ents afrect two dlrrerent events reEulatln6 the lnltlatlon
Or Dh'A synthesls ln Shlss 3T3 cells. Proc Natl hcad Scl US~.
1581, 78, 3063-3067.
'5. FR~EDKIl~,' M, LEGG A, ROZEI~GURT E. Enhancement Or DN~ synthecis
by colchicine ln 3T3 mouse fibrobl2sts stimulated wlth ~rowth
~actors. Er.ptl Cell Res 1980, 129, 23-30.
f~`' - 43 -
Merz 12
13~3~S;~
16. ME~Y~O AS, Th;~ ~;B. ~iucle2r tubulin Or tlssue culture cell3.
Blochlm Blophys Acta 1980, 629, 359-370.
17. CHEMNITZ J, SAHNBERG K. Interrelatlonshlp between annulate
lamellae and the cytoplasmlc mlcrotubule complex in tumor
cells ln vlvo and ln vltro. Z Xrebsforsch 1977, 90, 175-186.
18. GRAY EG, WESTRUM LE. ~;lcrotubules associated wlth nuclear
pore complexes and coated plts in the CNS. Cell Tissue;Res
1976, l60, 445-454.
19. TENG MK, BARTHOLOMEhr JC, BISSELL MJ. Synergism between ant~-
microtubule agents and growth stlmulants ln enhancement o~
cell cycle traverse. Nature 1977, 268, 739-741.
20. BERND A, SCHR~DER HC, ZAHN RK, M~LLER WEG. Modulatlon Or the
nuclear-envelope mucleoslde trlphosphatase by poly(A)-ricr.
mRNA and by mlcrotubule proteln. Eur J Blochem 1982, 129, 43-
49.
21. BACHMANN M, BERND A, SCHR~DER HC, ZAHN RK, M~LLER WEG. The
role o~ proteln phosphoklnase and proteln phosphatase durins
the nuclear envelope nucleoslde trlphosphatase reactlon. ~lo-
chim Biophys Acta 1984, 773, 308-316.
22. ~RELEC B, ZAHN R}~, GASIC MJ, BRITVIC S, LUCIC D, ~ULLE~ WEG.
Antimuta~enlc actlvlty Or the novel antlleukemlc agents av~-
rone and avarol. Mutation Res 1985, in press.
k - 44 -
F
13~?3~
The following briefly describes Fig~res 1 and 2, but
somewhat more detailed than the reference thereto on page
37~l:
F~_l. Effect of avarone (A~ and avarol (B) on ~3H]-dThd
incorporation into murine spleen lymphocytes in the absence
(- ), or the presence of 2~g concanavalin A/ml (o--o) or
20~g lipopolysaccharide/ml (x..... x). Addition of [3H]-dThd:
18 hr prior to the end of the incubation. Avarol or avarone
was added at time zero. Means of quadruplicate experiments
are presented; the S.D. varied between 6 and 8%. The values
for a 50% inhibition of [3H]-dThd incorporation of the
corresponding dose-response experiments are given as vertical
lines.
Fi~. 2. Effect of avarone (A~ and avarol (B) on [3H]-dThd
incorporation into human peripheral blood lymphocytes in the
absence (-- ), or the presence of 2~g concanavalin A/ml
(o--o) or 3~g pokeweed mitogen/ml (x..... x). Further details
are given in the legend to Fig. 1.
- 45 -
.
i3(~305i;~ Merz 12
~3H~drhd incorpora~ion ~/0)
o . o
o o o
o
~3
o~
o
.
FIG. 1 ~
. .
-- 46 --
13C)3C~;Z Merz 12
[3H~dThd incorporation (%J
O o
~~ r '~
: D
: ~ ' , ' ' ' .
FIG. 2
' . . . .
: ~ 47 -- .;
'' ' ' ', . . '''' ''~,".
. ~ -' . . . .
13~3~SZ `
.
In conclusion, from the foregoing, it is apparent
that the present invention provides novel, valuable, and
unpredictable applications and uses of the compounds
avarone and avarol, which compounds comprise the active
principles according to the present invention, as well as
novel pharmaceutical compositions thereof and methods of
preparation thereof and of treating therewith, all possessed
of the foregoing more specifically-enumerated characteris-
tics and advantages.
The high order of activity of the active agents of
the present invention and compositions thereof, as evidenced
by the tests reported, is indicative of utility ba`sed on
their valuable activity in human beings as well as in lower
animals. Clinical evaluation in human beings has not been
completed, however. It will be clearly understood that the
distribution and marketing of any compound or composition
falling within the scope of the present invention for use in
human beings will of course have to be predicated upon prior
approval by governmental agencies, such as the U.S. Federal
Food and Drug Administration, which are responsible for and
authorized to pass judgment on such questions.
It is to be understood that the invention is not to be
limited to the exact details of operation, or to the exact
compositions, methods, procedures, or embodiments shown and
described, as obvious modifications and equivalents will be
apparent to one skilled in the art, and the invention is
therefore to be limited only by the full scope o the
appended claims.
,, ~
4 g MERZ t2
_ ,~ _
