USE OF VOACAMINE AND RELATED COMPOUNDS TO ENHANCE THE ACTIVITY OF EXISTING MALARIA TREATMENTS

UTILISATION DE LA VOACAMINE ET DES COMPOSES APPARENTES POUR AMELIORER L'EFFICACITE DES TRAITEMENTS ANTIMALARIQUES EXISTANTS

English Abstract

Voacamine, voacamine isomers, metabolites and derivatives, and related
compounds can be used to, in effect, reverse multi-drug resistance in malaria
and
are non-toxic. The compounds can be used in combination with known drugs
such as chloroquine, arthemesin and qinghaosu to prevent or treat malaria.

Claims

CLAIMS
1. A method of preventing or treating malaria comprising the step of
exposing malaria cells to an effective concentration of a compound of the
formula
<IMG>
or

<IMG>
wherein R1 is a methyl group or hydrogen and R2, R3, R4 and R5, which are the
same or different, are CH2OH, CH3, OCH3, COOCH3, OH or hydrogen in
combination with at least one additional known principal drug used for
preventing
or treating malaria.
2. The method of claim 1 wherein said principal drug is selected from
the group consisting of chloroquine, arthemesin, qinghaosu and mixtures
thereof.
3. The method of claim 1 wherein said compound is used at a dosage
level of from about 100 to about 300 mg per day.
4. The method of claim 2, wherein said compound is used at a dosage
level of from about 100 to about 300 mg per day.
5. The method of claim 1, wherein R1 occupies location "S".
6. The method of claim 1, wherein said compound is selected from the
group consisting of voacamine, a voacamine isomer, a voacamine metabolite and
a voacamine derivative.
7. The method of claim 6, wherein said compound is used at a dosage
level of from about 100 to about 300 mg per day.
8. The method of claim 5, wherein said principal drug is selected from
the group consisting of chloroquine, arthemesin, qinghaosu and mixtures
thereof.
16

9. The method of claim 6, wherein said principal drug is selected from
the group consisting of chloroquine, arthemesin, qinghaosu and mixtures
thereof.
10. The method of claim 7, wherein said principal drug is selected from
the group consisting of chloroquine, arthemesin, qinghaosu and mixtures
thereof.
11. The method of claim 7, wherein said principal drug is selected from
the group consisting of chloroquine, arthemesin, qinghaosu, 8-aminoquinoline,
amodiaquine, arteether, artemether, artemsinin, artesunate, artesunic acid,
artelinic acid, atovoquone, azithromycine, biguanide, chloroquine, chloroquine
phosphate, chlorproguanil, cycloguanil, dapsone, desbutyl halofantrine,
desipramine, doxycycline, dihydrofolate reductase inhibitors, dipyridamole,
halofantrine, haloperidol, hydroxychloroquine sulfate, imipramine, mefloquine,
penfluridol, phospholipid inhibitors, primaquine, proguanil, pyrimethamine,
pyronaridine, quinine, quinidine, quinacrineartemisinin, sulfonamides,
sulfones,
sulfadoxine, sulfalene, tafenoquine, tetracycline, tetrandine, triazine or
derivatives
thereof.
12. A composition for preventing or treating malaria comprising:
a compound of the formula
17

<IMG>
18

wherein R1 is a methyl group or hydrogen and R2, R3, R4 and R5, which are the
same or different, are CH2OH, CH3, OCH3, COOCH3, OH or hydrogen in
combination with at least one additional known principal drug used for
preventing
or treating malaria.
13. The composition of claim 12, wherein said principal drug is selected
from the group consisting of chloroquine, arthemesin, qinghaosu and mixtures
thereof.
14. The composition of claim 12 wherein said compound is used at a
dosage level of from about 100 to about 300 mg per day.
15. The composition of claim 13, wherein said compound is used at a
dosage level of from about 100 to about 300 mg per day.
16. The composition of claim 12, wherein R, occupies location "S".
17. The composition of claim 12, wherein said compound is selected
from the group consisting of voacamine, a voacamine isomer, a voacamine
metabolite and a voacamine derivative.
18. The composition of claim 17, wherein said compound is used at a
dosage level of from about 100 to about 300 mg per day.
19. The composition of claim 15, wherein said principal drug is selected
from the group consisting of chloroquine, arthemesin, qinghaosu and mixtures
thereof.
20. The composition of claim 16, wherein said principal drug is selected
from the group consisting of chloroquine, arthemesin, qinghaosu and mixtures
thereof.
19

21. The composition of claim 17, wherein said principal drug is selected
from the group consisting of chloroquine, arthemesin, qinghaosu and mixtures
thereof.
22. The composition of claim 18, wherein said principal drug is selected
from the group consisting of chloroquine, arthemesin, qinghaosu,
8-aminoquinoline, amodiaquine, arteether, artemether, artemsinin, artesunate,
artesunic acid, artelinic acid, atovoquone, azithromycine, biguanide,
chloroquine,
chloroquine phosphate, chlorproguanil, cycloguanil, dapsone, desbutyl
halofantrine, desipramine, doxycycline, dihydrofolate reductase inhibitors,
dipyridamole, halofantrine, haloperidol, hydroxychloroquine sulfate,
imipramine,
mefloquine, penfluridol, phospholipid inhibitors, primaquine, proguanil,
pyrimethamine, pyronaridine, quinine, quinidine, quinacrineartemisinin,
sulfonamides, sulfones, sulfadoxine, sulfalene, tafenoquine, tetracycline,
tetrandine, triazine or derivatives thereof.

Description

CA 02655893 2009-02-20
USE OF VOACAMINE AND RELATED COMPOUNDS
to enhance the activity of existing malaria treatments
BACKGROUND OF THE INVENTION
This invention relates to compositions and methods for preventing and/or
treating malaria.
In particular, the invention relates to the use of voacamine, voacamine
isomers, metabolites, derivatives and related compounds for use in the
prevention and/or treatment of malaria. The compounds are used alone or in
combination with known anti--malarial drugs to potentiate the effectiveness of
such drugs against drug resistant malarial cells.
A number of different drugs have been found to be effective against
malaria. However in many cases, the initial success of such drugs in the
treatment and/or prevention of this disease is followed by total failure.
Drugs that
initially work become totally ineffective after a period of time. An initial
period of
remission is often followed by a period of frustration during which nothing
seems
to be effective against the disease. Death becomes inevitable. Such a
phenomenon is commonly referred to as multi-drug resistance. A malarial cell
that initially responds to treatment with one or more drugs becomes resistant
to
treatment by not only the drugs previously used, but also any other malarial
treatment drugs.
Martin Odula and Milhous (Martin et al, Science, Feb. 28, 1987) disclosed
the treatment of such multi-drug resistance in malaria by using verapamil. In
"Reversal of Chloroquine Resistance in Plasmodium falciparum by Verapamil",
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CA 02655893 2009-02-20
Martin et al., report that verapamil in combination with chloroquine was
effective
against malaria cells, but verapamil alone had no effect on malaria.
The problem with this approach is that verapamil is a calcium channel
blocker. While calcium channel blockers are therapeutic in the treatment of
hypertension at moderate levels, they are toxic at levels high enough for use
with
known anti-malarial drugs. Consequently, researchers throughout the world
continue to press for techniques for, in effect, reversing multi-drug
resistance. A
successful clinical technique for reversing multi-drug resistance in malaria
could
be one of the most important breakthroughs in the fight against malaria.
GENERAL DESCRIPTION OF THE INVENTION
The object of the present invention is to provide a treatment for use
against certain multi-drug resistant parasitic diseases. In addition to having
been
observed in malaria, multi-drug resistance is a phenomenon, which has been
observed in other parasitic diseases such as Entamoeba histolytica (amoebic
dysentery), Trypanosoma (African sleeping sickness), Leishmania and AIDS
pneumonia.
Accordingly, the invention relates to a method of preventing or treating
malaria comprising the step of exposing malaria cells to an effective
concentration of a compound of the formula
2

CA 02655893 2009-02-20
MeO0 6 H
9
7 1
Ix`12 ( H N, -~''1
13 N 2 21
H$ 20
R3 14 1s
19' 3U' 14 17' H 18 (1)
2r 1 1r
19= N 14 =
3' 2, 1 1 = `~.
i 13'
10'
5' 6
7. a s~ R,
9 R5
~= 8 7 5
10 H 3
11 12 1 2 16 21 R4
R3 a 13 N t2)
' 20 19 18
H 1
14
or
R4 3
a13 N /
3 21
R1 15
`' 19
3

CA 02655893 2009-02-20
wherein R, is a methyl group or hydrogen, and R2, R3, R4 and R5, which are the
same or different, are CH2OH, CH3, OCH3, COOCH3, OH or hydrogen in
combination with at least one additional known principal drug used for
preventing
or treating malaria.
According to another aspect, the invention relates to a composition for a
method of preventing or treating malaria comprising the compound of the
formula
McOO $ H
9 8 5
7 i
11 ~ ~ H R1
12 1 1~~yI
13 N 2 21
H i5 20
19
15' R3 14
14' H 18 18' 24' 17' 1
21` 12'
16, 11=
3' 2' 13'
10'
5' 7` R 2
R2 8__ R5
10 t H 3 N
11 12 ,g 1 2 16 21 R4 CIA/
R N R 20 19
H
14
or
4

CA 02655893 2009-02-20
R4 3
a13 N N a 21
Ri 15 '~.
19
wherein R, is a methyl group or hydrogen, and R2, R3, R4 and R5, which are the
same or different, are CH2OH, CH3, OCH3, COOCH3, OH or hydrogen in
combination with at least one additional known principal drug used for
preventing
or treating malaria.
The inventor has determined that voacamine, voacamine isomers,
metabolites and derivatives, and related compounds act to, in effect, reverse
multi-drug resistance in malaria, and do not show any of the toxicity problems
of
verapamil.
Moreover, voacamine and the dimeric related compounds found in
Peschiera laeta enhance vinblastine-mediated cytotoxicity in multi-drug
resistant
tumor cells (You M. et al., 1994, Journal of Natural Products). These dimeric
alkaloids may also be effective at modulating the sensitivity of chloroquine
resistant Plasmodium strains to this drug (Federici et al., 2000, Planta
Medica).
Voacamine, voacamine, voacamine isomers, metabolites and derivatives,
and related compounds are also specifically effective against malaria,
including
multi-drug resistant strains, even in the absence of primary treatment drugs.
Voacamine, voacamine isomers, metabolites and derivatives are as effective
against multi-drug resistant malarial strains as against drug sensitive
strains.
5

CA 02655893 2009-02-20
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is an isobologram showing the effectiveness of voacamine and
chloroquine at 50% inhibition concentrations against sensitive and resistant
malarial strains; and
Figure 2 is an isobologram showing the effectiveness of voacamine and
quinghaosu at 50% inhibition concentrations against sensitive and resistant
malarial strain.
DETAILED DESCRIPTION OF THE INVENTION
In the preferred embodiment, the compounds of the present invention
have the formula (1), (2) and (3) listed above. The compounds include
voacamine, voacamine isomers and derivatives, and related compounds. In all of
the examples, R, is a methyl group or hydrogen.
Variation within the group occurs in that R2, R3, R4 and R5 may be a
methyl, methoxy, hydroxyl, carboxymethyl or hydrogen and the isomeric
configuration of the compounds at the C-1 position may be either R (rectus) or
S
(sinister). In addition, hemandezine includes a carboxymethyl group at the C-5
position, a substitution that does not appear to be significant in the
operability of
the compound. The specific manner in which the family members vary is set
forth in Table V below, wherein the compounds are compared to two known
drugs for activity against drug sensitive and drug resistant strains of P.
falciparum
malaria.
A specific in vivo dosage for each member of the voacamine family for
reversing malarial multi-drug resistance and/or for specifically treating
and/or
preventing malaria has not been established. However, such dosage can be
established through routine clinical experimentation by referencing the
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CA 02655893 2009-02-20
concentrations at which the various compounds have exhibited 50% inhibition as
set forth in Tables I through V herein. These concentrations have been found
to
be from about 0.1 to about 3 micromolar. Such concentrations can be achieved
in vivo by administering dosages of from about 100 to about 300 mg/day. It is
known that at these concentrations, the voacamine family of compounds is
substantially non-toxic. The preferred method for administering the drug is
orally,
although other methods such as injection may be used.
One of the mechanisms whereby voacamine may sensitize chloroquine
resistant strains may involve changes in rates of accumulation of chloroquine
in
the vacuole of chloroquine resistant parasites. The calcium channel blocker
verapamil that has been known to reverse chloroquine resistance in strains
such
as K1 and W2 as a result of changes in membrane permeability exemplifies this
phenomenon.
Prior studies of voacamine, voacamine isomers, metabolites, derivatives
and related compounds for various other uses have indicated a minimal toxicity
at
doses of 2000 and 5000 mg/day. Voacamine and several voacamine derivatives
were screened for calcium channel blocker activity, and such activity was
found
to be minimal. Thus, the toxicity problems associated with higher doses of
calcium channel blockers such as verapamil have not so far been observed in
members of the voacamine family.
The effectiveness of voacamine, voacamine isomers, metabolites,
derivatives and related compounds in reversing malarial multi-drug resistance
was determined by comparing the antimalarial action of voacamine, voacamine
isomers, metabolites, derivatives and related compounds and chloroquine alone
and in combination against a P. falciparum malarial strain that is sensitive
to
7

CA 02655893 2009-02-20
chloroquine and another strain resistant to chloroquine. A similar study was
conducted using voacamine and qinghaosu. Chloroquine and qinghaosu are
commonly used anti-malaria drugs.
The dose (ICso) of each drug or each drug combination required to effect a
50% inhibition in the malarial activity of each strain was determined by
establishing a dose response curve for each drug.
The non-resistant (D6) strain and cloned Indochina (W2) strain of
P. falciparum were used. The former is sensitive to chloroquine and the latter
is
resistant to chloroquine. The two strains of the parasite were cultured
according
to the candle jar method of Trager and Jensen (Science, 1979, 193: 673-675).
In
a given experiment, 4-day-old Petri dish cultures (approx. 10% parasitemia)
were
diluted with medium containing an amount of non-infected type A human
erythrocytes to obtain a culture with a final hematocrit of 1.5% and
parasitemia of
0.5-10%. The resulting culture was ready for addition to microtitration plates
with
96 flat-bottom wells.
The testing procedure used was similar to that described elsewhere
(Desjardins et al., 1979, Antimicrobial Agents and Chemotherapy, 16: 710-718,
1979). Briefly, the final volume added to each of the 96-well microtitration
plates
was 250 pl and consisted of 25 pI of complete medium with or without the
primary
drug (chloroquine or qinghaosu), 175 pl of either the parasitized culture or a
non-
parasitized human erythrocyte control, and 25 pI of complete medium with or
without voacamine, 25 pl radioactive 2,8-3H-hypoxanthine (0.5 DCi). The
microtitration plates were incubated in a candle jar for an additional 18 hrs,
at
37 C.
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CA 02655893 2009-02-20
As the malaria parasite grows 3H-adenosine is metabolized and
incorporates into polymeric RNA and DNA. The labeled polymers are trapped on
glass fiber filters and unincorporated material is washed away. In the absence
of
drug there is 100% incorporation of the labeled material. When drugs interfere
directly or indirectly, an inhibitory dose of 50% (IC50) can be calculated
(Van Dyke
et al., 1987, Exp. Parasitol. vol. 64: 418-423).
Voacamine ad the voacamine family of compounds completely reversed
resistance to chloroquine in chloroquine-resistant malaria. When voacamine,
voacamine isomers, metabolites, derivatives and related compounds are added
to chloroquine, they supplement and potentiate the antimalarial activity. When
voacamine is added to qinghaosu, it provides long-acting and synergistic
activity
to qinghaosu. This can be seen in Tables I, II, 111, and IV while isobolograms
(Figures 1 and 2) of voacamine and chloroquine as well as voacamine and
qinghaosu reveal the synergistic and potentiating activity of voacamine when
added to chloroquine or qinghaosu. Remarkably when 3.0 pMolar voacamine is
added to 0.1 NMolar chloroquine, the IC50 of chloroquine can be lowered 43-
fold.
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TABLE I
IC50 (nM) OF VOACAMINE (VOA) AND CQ FOR EACH DRUG ALONE AND IN COMBINATION*
DRUG COMBINATION**
SINGLE DRUG VOA (1.0 M) VOA (2.0 M) VOA (3.0 M)
MALARIA*** VOA CQ CQ (0.3 M) CQ (0.2 M) CQ (0.1 M)
S. STRAIN 238.13.7 28.5.5 56.9 8.2 (VOA) 114.1325.1 (VOA) 223.3 35.8 (VOA)
15.9+x.7 (CQ) 13.5 2.9 (CQ) 8.2 1.5 (CQ)
R. STRAIN 290.5 24.7 185.8 4.9 79.5 13.7 (VOA) 125.5 16.1 (VOA) 254.6 39.6
(VOA)
25.6 3.2 (CQ) 9.1 2.1 (CQ) 3.9 0.5 (CQ)
*The data in the table above are the mean values S.D. (nM) from three
experiments except where noted
**Ratios of VOA/CQ in the drug combinations are 10:3, 10:1 and 30:1
respectively.
***S and R strains represent CQ-sensitive (D6) and resistant (W2) strains of
P.falciparum respectively.
TABLE II
IC50 (nM) OF VOA AND QHS FOR EACH DRUG ALONE AND IN COMBINATION*
DRUG COMBINATION**
SINGLE DRUG VOA (1.0 M) VOA (2.0 M) VOA (3.0 M)
MALARIA*** VOA QHS QHS (0.3 M) QHS (0.2 M) QHS (0.1 M)
S. STRAIN 298.2 59.8 38.3 4.7 87.2 9.5 (VOA) 113.8 5.6 (VOA) 239.8 45.3 (VOA)
25.9 2.5 (QHS) 14.6 0.8 (QHS) 9.7 2.3 (QHS)
R. STRAIN 305.1 29.2 57.6 4.5 78.9 14.5 (VOA) 98.1 17.3 (VOA) 296.9 54.1 (VOA)
22.8 3.1 (QHS) 9.3 1.5 (QHS) 5.7 1.2 (QHS)
*The data in the table above are the mean values S.D. (nM) from three
experiments except where noted
**Ratios of VOA/QHS in the drug combinations are 10:3, 10:1 and 30:1
respectively.
***S and R strains represent CQ-sensitive (D6) and resistant (W2) strains of
P. falciparum respectively

CA 02655893 2009-02-20
TABLE III
EFFECT OF COMBINATION OF VOACAMINE AND CHLORQUINE ON P. FALCIPARUM
SFIC *
1.OpM (VOA) 2.0 M (VOA) 3.OpM (VOA)
MALARIA** TRIAL 0.31LM (CQ) 0.2 M (CQ) 0-1 M (CQ)
S. STRAIN 1 0.76 0.69 0.75
2 0.65 0.76 0.69
3 0.79 0.53 0.78
MEAN 0.73 0.04 0.66 0.08 0.74+0.03
S.D.
R. STRAIN 1 0.62 0.54 0.70
2 0.64 0.60 0.78
3 0.43 0.29 0.54
MEAN 0.56 0.10 0.47 0.12 0.71 0.15
S.D.
*SFIC represents sum of fractional inhibitory concentration as described by
Berenbaum (II), SFIC is equal
to one in cases of additive effects of the drugs, higher than one in cases of
antagonism and lower than one in
synergistic action.
**S and R strains: chioroquine sensitive (06) and resistant (W2) strains of P.
falciparum.
TABLE IV
EFFECT OF COMBINATION OF VOACAMINE AND QINGHAOSU ON P. FALCIPARUM
SFIC*
I.OiM (VOA) 2.0 M (VOA) 3.OpM (VOA)
MALARIA** TRIAL 0.3 M (QHS) 0.2 M (QHS) 01 M (QHS)
S. STRAIN 1 0.79 0.70 0.75
2 0.71 0.44 0.78
3 0.80 0.69 0.83
MEAN 0.77 0.08 0.61 0.12 0.78+0.05
S.D.
R STRAIN 1 0.65 0.56 0.85
2 0.79 0.68 0.67
3 0.61 0.50 0.71
MEAN 0.68 0.07 0.58 0.08 0.74+0.14
S.D.
*SFIC represents sum of fractional inhibitory concentration as described by
Berenbaum (11), SFIC is equal
to one in cases of additive effects of the drugs, higher than one in cases of
antagonism and lower than one in
synergistic action.
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When the inhibiting activity of two drugs e.g. A and B are compared, the
middle point of the dose response curve is usually chosen as the basis for
comparison. This point is known as the inhibitory dose that occurs at the
point of
50% inhibition of the response to be measured (inhibitory concentration at 50%
inhibitory response = IC5o). An isobologram is developed by comparing the IC50
of one drug against the other (i.e. drug A against drug B). We start by
putting the
IC50 of Drug B at the top of the Y-axis marked 1Ø The IC5o of drug A is
placed at
the position 1.0 on the X-axis. Combinations of drug A and drug B are mixed
and
tested that are below IC5o of either drug and the points are located on a
graph. If
the two drugs are additive there is a straight line between the Y, Xo (drug B)
and
Yo X, (drug A). If the line or curve bends below the straight line the drugs
are
synergistic or potentiating. If the line bends above the straight line the two
drugs
are antagonistic (Figures 1 and 2).
Voacamine was also compared to several of its derivatives for their
effectiveness against a chloroquine sensitive and a chloroquine resistant
strain of
P. falciparum malaria. The test procedure was basically the same as outlined
above. The structural formulas of the derivatives are formulas (1), (2) and
(3).
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TABLE V
CHEMICAL STRUCTURE-ANTIMALARIAL ACTIVITY OF DIMERIC BISINDOLE AND BASIC
ALKALOIDS AGAINST PLASMODIUM FALCIPARUM IN VITRO
aDrug Substituents Linkage IC50 10( 7 M) Ratio
C3 C4 C10 CIO' C16 C16' C19 S" R" (S/R)*
VOA H Me - OMe CO7Me CO2Me - C3-CI1' 2.4 2.9 0.8
DCV H Me - Me CO2Me - - C3-C11' 4.9 8.2 0.6
NDV H H - Me CO2Me CO2Me - C3-Cll' 4.7 9.6 0.5
VDN H Me - OMe CO2Me CO2Me - C3-C9' 5.1 9.8 0.5
TAB H Me - OMe CO2Me H - C3-Cll' 6.5 9.5 0.7
ERV H Me - OMe CO2Me CO2Me - C3-C11' 6.8 8.8 0.8
VOBb =0 Me - - CO2Me - - - 4.7 8.2 0.6
CORb H H H H CO2Me - H - 5.0 2.8 1.9
aDimeric Bisindole Alkaloids (Voa = Voacamine; DCV = Decarbomethoxyvoacamine;
NDV = N6Demethylvoacamine; VDN = Voacamidine; TAB = Tabemamine; ERV =
Ervahanine)
bBasic tertiary Alkaloids (VOB = Vobasine; COR = Coronaridine)
`IC50 of a drug against sensitive strain of P. faleiparum is divided by IC5o
for resistant strain.
"S and R represent chioroquine-sensitive and resistant strain of P falciparum.
The results of Table V show that voacamine and its dimeric derivatives are
far more effective against either the chloroquine sensitive malarial strain or
the
chloroquine resistant strain than the basic tertiary alkaloids. Coronaridine
and
vobasine were the best of the non-dimeric compounds, 2.8 x 10-7 and 8.2x10'7
moles were required respectively to effect a 50% inhibition in activity of the
resistant strain, as compared to the ICso values from 13.3x 10-7 up to 50.0x10-
7
moles of the other non-dimeric voacamine family of compounds.
The results of Table V also illustrate the members of the voacamine family
having at least one of the R2 and R3 substituents consisting of CH3, OCH3 or
COOCH3 that are the most effective against the chloroquine resistant malarial
strains. When R2 is a CH3, OCH3 or COOCH3 substituent, the voacamine family
13

CA 02655893 2009-02-20
members are actually as effective against the chloroquine resistant malarial
strain
as they are against the chloroquine sensitive malarial strain. This result
suggests
that the family members would also be the most effective members in affecting
multi-drug resistance reversal. Thus, in the preferred voacamine family
members
at least one of R2, R3, R4 and R5 is CH3, OCH3 or COOCH3, preferably at least
R2
being CH3.
The results suggest that the compounds either inhibit the expression of the
glycoprotein pump responsible for removal of the therapeutic drug from the
cell or
actually reverse or inhibit the pumping action of the glycoproteins or calcium
channel associated with such multi-drug resistant cells. Instead of pumping
all
the toxic drug out of the cell, it appears that a lesser concentration of the
toxic
drug is being pumped out of the cell. At present, these are the only
reasonable
explanations for these surprising results, since the only known significant
difference between the multi-drug resistant cells and the corresponding drug
sensitive cells is the substantially greater percentage of P-glycoprotein
associated with the multi-drug resistant cell.
14