DNA polymerase inhibitors, mikanolide and dihydromikanolide

Details DNA polymerase inhibitors, mikanolide and dihydromikanolide DNA polymerase inhibitors, mikanolide and dihydromikanolide

2002-09-27

2004-07-27

Tate, Christopher R. (Department: 1654)

Drug, bio-affecting and body treating compositions

Plant material or plant extract of undetermined constitution...

C424S774000

Reexamination Certificate

active

06767561

ABSTRACT:

The present invention relates to, as medicaments and in particular as anti-proliferative, anti-viral and anti-parasitic agents, mikanolide, dihydromikanolide and dihydromikanolide enriched extracts. A subject of the invention is also obtaining them by extraction from the
Mikania micrantha
plant by a process described hereafter.
The plants have been a source of pharmaceutical products and ingredients in traditional medicine for a long time (Phillipson, J. D.,
Trans. R. Soc. Trop. Med. Hyg.
(1994), 88 Suppl. 1, S17-S19). The medicaments formed by natural products or their synthetic analogues play a very important role.
For example, in oncology, products such as taxol (Paclitaxel®), vincristine (Oncovin®), vinorelbine (Navelbine®), teniposide (Vumon®), and several analogues of camptothecin (CPT) soluble in water (Pezzuto, J. M.,
Biochem. Pharmacol
. (1997), 53, 121-133) are found. Other products originating from plants are moreover being studied for the prevention of the recurrence of stenosis.
In the field of cancer treatment, for example, camptothecin is a pentacyclic alkaloid isolated from
Camptotheca acuminata
wood (Wall, M. E.,
Med. Res. Rev
. (1998), 18, 299-314). Initially, CPT was identified as an inhibitor of tumor proliferation in human cell models. Subsequently, the action mechanism of CPT has been identified as a topoisomerase I enzyme poison involved in the replication of DNA. An increasing number of CPT analogues (topotecan, CPT-11, BN-80915) are used in anti-cancer treatment in man (Lavergne, O. et al.,
J. Med. Chem.
(1998), 41, 5410-5419).
Another example is taxol, a terpenic alkaloid isolated from
Taxus brevifolia
. Taxol is a powerful cytotoxin which inhibits tubulin, a protein involved in mitosis. This is a particularly effective product in ovarian cancer.
Moreover, the vinca alkaloids were first extracted from
Catharanthus roseus
. They were first used ethnobotanically for indications other than that of cancer treatment.
As far as the prevention of the recurrence of stenosis is concerned, studies indicate that it could be obtained thanks to a protein originating from
Saponaria officinalis
, saponin, recombinated with fibroblast growth factor (FGF). The latter allows targeting of the vascular cells due to its affinity for the FGF receptor (Lin P H et al.,
Atherosclerosis
(April 1998), 137(2), 277-289).
The method for discovering such products is fractionation of plant extract molecules followed by the evaluation of their biological activities. But compared with the number of plant types, species and varieties, and the enormous quantity of molecules produced by these plants, the identification of a molecule and its biological activity remains a very difficult task to carry out.
Mikania micrantha
is a plant which is pandemic in tropical regions. It proliferates in an extremely rapid fashion, to such an extent that it is nicknamed “a-mile-a-minute”.
Mikania scandens
and
cordata
are often confused with it. These three species contain in particular mikanolide (I), dihydromikanolide (II), scandenolide (III) and dihydroscandenolide (IV).
The extraction of mikanolide, dihydromikanolide, scandenolide and dihydroscandenolide from
Mikania scandens, Mikania cordata
and
Mikania micrantha
was described in the following references respectively: Herz,
Tetrahedron Lett
. (1967), 32, 3111-15; Kiang,
Phytochemistry
(1968), 7(6), 1035-7; and Cuenca,
J. Nat. Prod.
(1988), 51(3), 625-6. The solvents used include chloroform and diethylether which cannot be used industrially because of their toxicity or inflammability.
Ethnobotanically,
Mikania scandens
and
cordata
have been and are still used by traditional medicine as an anti-asthmatic, analgesic, anti-rhumatic, anti-inflammatory, anti-cancer, febrifuge and vermifuge agent in Central America where its common name is Guaco. It is only in the last decade that some of these properties have been studied scientifically, in particular
Mikania cordata
extracts in the field of inflammation.
Recently, the Applicant has tested the anti-proliferative activity and found that, in a surprisingly, a dihydromikanolide and mikanolide enriched extract or pure forms of the latter have a powerful activity. In addition, dihydromikanolide and mikanolide inhibit the replication of DNA by inhibiting the DNA polymerase enzymes necessary for the multiplication of the eukaryotic and prokaryotic cells as well as viruses.
According to the invention, the said extract, obtained from
Mikania micrantha, Mikania scandens
and
Mikania cordata
leaves, comprises more than 50%, and preferably more than 60% mikanolide and dihydromikanolide. Even more preferably, said extract comprises more than 70% mikanolide and dihydromikanolide. Preferably, said extract comprises moreover less than 10%, and more preferably less than 5%, scandenolide and dihydroscandenolide. Furthermore, extracts comprising more than 20% dihydromikanolide, and more preferably, more than 30% dihydromikanolide are also preferred.
The said extract can be prepared according to the extraction method described hereafter starting from
Mikania micrantha
, without however excluding the species
Mikania scandens
and
Mikania cordata.
A subject of the invention is therefore firstly a process for the preparation of an extract according to the invention, characterized in that it comprises the following successive stages:
grinding up and drying of
Mikania micrantha, Mikania scandens
and
Mikania cordata
leaves;
addition of a solvent chosen from toluene, ethyl acetate, toluene and ethyl acetate mixtures, toluene and acetone mixtures, ethyl acetate and heptane mixtures, and heptane and acetone mixtures in proportions of 7:3 to 3:7 to the ground leaves of
Mikania micrantha, Mikania scandens
and
Mikania cordata;
filtration and recovery of the filtrate;
concentration of the extract of the primary extract solution in order to obtain a concentration of 2.5 to 10% in dry extract;
dividing the concentrated extract obtained in the preceding stage between the extraction solvent and a mixture comprising 10% to 50% methanol or ethanol in water;
washing the alcohol-water phase with n-hexane or heptane;
elimination of the alcohol of the alcohol-water phase;
extraction of the aqueous phase obtained with ethyl acetate;
drying of the ethyl acetate phase with a drying agent;
evaporation of the solvents and recovery of the dry extract;
purification of the dry extract obtained by a physico-chemical separation method; and
obtaining, after drying, a dry extract according to the invention.
According to the invention, the physico-chemical separation method can be, for example, filtration on silica gel, or also, according to a preferred variant, crystallization from methanol, ethanol or acetone.
If filtration on silica gel is chosen, the eluents used are preferably heptane-ethyl acetate mixtures in proportions comprised between 9:1 and 1:1.
In the case where crystallization is chosen, it is preferably carried out at a temperature comprised between −10 and 5° C. for a duration preferably comprised between 2 and 16 hours.
Moreover, according to a preferred variant of the above process, dividing the concentrated extract is carried out using a mixture comprising 25 to 30% methanol in water. Preferably, the lower aqueous phase will be heated to facilitate phase separation, for example at a temperature of 30 to 50° C.
The dry extract as obtained by the above process can be dihydromikanolide-enriched by conversion of the mikanolide to dihydromikanolide by catalytic hydrogenation. Subsequently, re-crystallization allows the pure compound to be obtained.
A subject of the invention is also therefore a process for the preparation of a dihydromikanolide-enriched extract characterized in that it consists of hydrogenation of the dry extract described previously, dissolved in ethyl acetate, at a temperature comprised between 10 and 35° C., in the presence of a hydrogenation catalyst under a pressure of 1 to 2 hydrogen atmospheres for a duration preferably comprised between 4 and 16 hours. Preferably,

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