U.S. patent application number 17/307722 was filed with the patent office on 2021-08-26 for use of avermectin derivative for increasing bioavailability and efficacy of macrocylic lactones.
The applicant listed for this patent is INSTITUT NATIONAL DE RECHERCHE POUR L'AGRICULTURE, L'ALIMENTATION ET L'ENVIRONNEMENT, THE ROYAL INSTITUTION FOR THE ADVANCEMENT OF LEARNING/MCGILL UNIVERSITY. Invention is credited to Anne LESPINE, Cecile MENEZ, Roger PRICHARD.
Application Number | 20210261594 17/307722 |
Document ID | / |
Family ID | 1000005556856 |
Filed Date | 2021-08-26 |
United States Patent
Application |
20210261594 |
Kind Code |
A1 |
LESPINE; Anne ; et
al. |
August 26, 2021 |
USE OF AVERMECTIN DERIVATIVE FOR INCREASING BIOAVAILABILITY AND
EFFICACY OF MACROCYLIC LACTONES
Abstract
The present invention relates to the use of avermectin
derivative as a drug for the treatment of parasitic infections. The
avermectin derivative is represented by the formula (I) where: (i)
R.sup.1 is chosen from the group constituted of
--CH(CH.sub.3).sub.2, --CH(CH.sub.3)CH.sub.2CH.sub.3, or
cyclohexyl, (ii) X represents --CH.sub.2--CH.sub.2--, or
--CH.dbd.CH--, (iii) R.sup.2 is chosen from ##STR00001## or an OH
group, (iv) R.sup.3 is OH or NOH, (v) represents a single bond when
R.sup.3 is OH, or a double bond when R.sup.3 is NOH, as an
inhibitor of a membrane-bound protein which transports exogenous
compounds out of target cells.
Inventors: |
LESPINE; Anne; (Toulouse,
FR) ; PRICHARD; Roger; (Sainte Anne-de-Bellevue,
CA) ; MENEZ; Cecile; (Toulouse, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INSTITUT NATIONAL DE RECHERCHE POUR L'AGRICULTURE, L'ALIMENTATION
ET L'ENVIRONNEMENT
THE ROYAL INSTITUTION FOR THE ADVANCEMENT OF LEARNING/MCGILL
UNIVERSITY |
Paris
Montreal |
|
FR
CA |
|
|
Family ID: |
1000005556856 |
Appl. No.: |
17/307722 |
Filed: |
May 4, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15988073 |
May 24, 2018 |
11021508 |
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17307722 |
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14352669 |
Apr 17, 2014 |
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PCT/EP2012/070704 |
Oct 18, 2012 |
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15988073 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/365 20130101;
A61K 45/06 20130101; C07D 493/20 20130101; A61K 31/475 20130101;
A61K 31/7048 20130101; A61K 31/277 20130101; C07H 17/08 20130101;
C07D 493/06 20130101; A61K 31/366 20130101 |
International
Class: |
C07H 17/08 20060101
C07H017/08; A61K 31/7048 20060101 A61K031/7048; A61K 45/06 20060101
A61K045/06; A61K 31/277 20060101 A61K031/277; A61K 31/365 20060101
A61K031/365; A61K 31/475 20060101 A61K031/475; A61K 31/366 20060101
A61K031/366; C07D 493/06 20060101 C07D493/06; C07D 493/20 20060101
C07D493/20 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 18, 2011 |
EP |
11306348.1 |
Claims
1. A pharmaceutical composition comprising a compound of formula I
##STR00015## wherein (i) R.sub.1 is selected from the group
consisting of --CH(CH.sub.3).sub.2, CH(CH.sub.3)CH.sub.2CH.sub.3,
or cyclohexyl (ii) X represents --CH.dbd.CH-- or
--CH.sub.2--CH.sub.2--, wherein X is --CH.sub.2--CH.sub.2-- only
when R.sub.1 is cyclohexyl; (iii) R.sub.2 is --OH, and (iv) R.sub.3
is OH or NOH; a second active ingredient selected from the group
consisting of a macrocyclic lactone antiparasitic agent, an
antitumoral agent, an antiviral agent, an anti-epileptic agent, an
antibacterial agent, and an antifungal agent, wherein the second
active ingredient is a compound other than a compound of formula I;
and, a pharmaceutically acceptable carrier,
2. The pharmaceutical composition according to claim 1, wherein (i)
R.sub.1 is selected from the group consisting of
--CH(CH.sub.3).sub.2, and --CH(CH.sub.3)CH.sub.2CH.sub.3, (ii) X
represents --CH.dbd.CH--, (iii) R.sub.2 is --OH, and (iv) R.sub.3
is OH, said compound corresponding to the compound of formula I(c):
##STR00016##
3. The pharmaceutical composition according to claim 1, wherein (i)
R.sub.1 is cyclohexyl, (ii) X represents --CH.dbd.CH--, (iii)
R.sub.2 is --OH, and (iv) R.sub.3 is OH, said compound
corresponding to a compound of formula I(d): ##STR00017##
4. The pharmaceutical composition according to claim 1, wherein (i)
R.sub.1 is cyclohexyl, (ii) X represents --CH.sub.2--CH.sub.2--,
(iii) R.sub.2 is --OH, and (iv) R.sub.3 is .dbd.NOH, said compound
corresponding a compound of formula I(e): ##STR00018##
5. The pharmaceutical composition of claim 1, wherein the second
active ingredient is chosen from an antiplasmodium, or an
antileshmania agent.
6. The pharmaceutical composition of claim 1, wherein the second
active ingredient is an antiviral agent.
7. A method of treatment of infections comprising administering to
a subject in need thereof a pharmaceutical composition of claim
1.
8. The method of claim 7, wherein the infection is a viral
infection, a bacterial infection or a fungal infection wherein the
second active ingredient is an antiviral agent, antibacterial agent
or an antifungal agent, respectively.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. patent
application Ser. No. 15/988,073, filed May 24, 2018, which is a
continuation of U.S. patent application Ser. No. 14/352,669, filed
Apr. 17, 2014, which is a National Stage entry of International
Application No. PCT/EP2012/070704, filed Oct. 18, 2012, which
claims priority to European Patent Application No. 11306348.1,
filed Oct. 18, 2011. The disclosure of the priority applications
are incorporated in their entirety herein by reference.
[0002] The present invention relates to the use of avermectin
derivative in association with macrocyclic lactones as a
formulation for the treatment of parasitic infections.
[0003] Parasitic infections are the most frequent diseases for
livestock or domestic animal. In spite of recent advance in
veterinary pharmaceutical research, it is always necessary to find
out more efficient and safe drug or formulation to fight against
parasitic infections. For humans, the parasitic infections such as
onchocerciasis caused by infection by Onchocerca volvulus,
lymphatic filariasis caused by Wuchereria bancrofti, Brugia malayi,
and Brugia timori, or tropical parasitic diseases are still
frequent diseases in developing countries. By the way, though
endemic in some developing countries, intestinal strongyloidiasis
and cutaneous parasitic diseases also pose a threat to the
developed world.
[0004] The macrocyclic lactones (ML) are a family of broad spectrum
anti-parasitic drugs that was developed in early 80s and have been
widely used for the treatment of both internal and external
parasites in pets, in livestock and in humans. MLs are also
efficient for treating parasitic diseases caused by benzimidazole-,
levamisole-, and pyrantel-resistant strains of nematodes.
[0005] MLs are a family of compounds isolated from soil
microorganisms belonging to the genus Streptomyces. Macrocyclic
lactones comprise avermectins and milbemycins. Avermectins comprise
ivermectin, abamectin, doramectin, eprinomectin or selamectin,
while milbemycins comprise moxidectin, nemadectin, and milbemycin
oxime.
[0006] The principal action of MLs in parasitic nematodes is to
increase membrane permeability to chloride ions by interacting with
the glutamate-gated chloride channel subunit. The glutamate and
.gamma.-aminobutyric acid (GABA)-agonist activities of the MLs are
the mechanisms that lead to the paralysis and death of the treated
parasites at nanomolar concentration. In fact, MLs maintain open
the glutamate-gated channels that blocks pharyngeal pumping and
inhibits of feeding, which is one of the effects that cause the
death of parasite. By the way, ivermectin leads also to activation
and paralysis of body muscle in Haemonchus contortus (Sheriff et
al., Vet. Parasitol., 2005, 128(3-4), 341-346), and inhibits worm
reproduction in Onchocerca volvulus (Schulz-Key, Acta Leiden, 1990,
59(1-2), 27-44).
[0007] However, in the last years, widespread MLs resistance has
been observed in some nematode parasites of sheep, goats and
cattle. The cause and mechanism of MLs resistance are yet not
completely understood, but recent research has showed that MDR
(multidrug resistance) transporters are a group of protein implied
in the MLs resistance. MDR transporters are membrane proteins
belonging to the ABC (ATP binding cassette) family, and whose main
function is the ATP-dependent transport of a number of structurally
unrelated exogenous compounds. Due to their expression in the
plasma membrane, they function as a permeability barrier for the
passage of xenobiotic across the cell membrane by actively
expelling them out of the cells. MDR transporters have been
considered as one of the causes of chemotherapy effectiveness
restriction, when the tumor cells overexpress these transporters.
MDR transporters also limit the entry of MLs into human target
organism and affect the efficiency of MLs as antiparasitic. In
addition, the expression of MDR transporters in intestine, liver
and kidney allows them to detoxify these tissues, and ultimately
eliminate the substrate drugs out of the systemic circulation,
exerting a protecting action against their toxicity but also
restricting their therapeutic efficacy.
[0008] P-glycoprotein (Pgp), localized in the apical membrane, is
one of MDR transporters. The main function of Pgp is the active
efflux of various structurally unrelated exogenous compounds to
protect both vertebrate and invertebrate organisms against
potentially toxic molecules. Pgp can transport its substrate from
the baso-lateral side to the apical side of epithelia and
endothelia. Pgp plays also an important role in blood-brain
barrier, since it can limit the concentration of xenobiotics in the
brain. The overexpression of Pgp is one of cause of drug resistance
observed during avermectin treatment for parasitic infections or
some tumor chemotherapy.
[0009] Later, other multidrug resistance proteins MRP1, 2 and 3
(ABCC1, 2 and 3) are also discovered. They are also involved in
multidrug resistance and provide complementary and overlapping
activities as multispecific drug efflux pumps
[0010] The more recently discovered Brest Cancer Resistance protein
is ABCG2, which assists Pgp to prevent unwanted material in the
circulation from passing into the brain. Homologues of MDR
transporters exist also in parasite, and the selection and/or
modulation of expression of their gene could be one of the reasons
of the resistance of parasite to MLs.
[0011] Different methods have been developed in the past years to
overcome the effectiveness restriction due to the efflux pumps of
administrated drug. One of them is the use of MDR transporters
inhibitors which can block efflux pumps of administrated drugs to
improve intracellular concentration of active ingredient. Some Pgp
ligands have been reported in the past, such as cyclosporine A, its
derivative PSC833 (Valspodar.RTM.), the antidiarrheal opioid drug
loperamid, or verapamil. However, unfortunately, till today,
because of important toxicity of these MDR transporters inhibitors,
neither of them can be applied in pharmaceutical use.
[0012] MLs have been firstly observed as substrates of MDR
transporters. Later, it was found that MLs, in particular
ivermectin, are also inhibitors of MDR transporters.
[0013] In spite of the fact that ivermectin can efficiently inhibit
MDR transporters, it can not become a candidate drug, because of
its important neurotoxicity if it penetrates in the brain at high
concentration. In fact, ivermectin interacts with GABA receptors, a
complex situated in nervous system. The abnormal function of GABA
receptors can lead to neurologic, mental, vegetotropic, somatic,
hormonal and other disorders. Since inhibition of MDR transporters
is still the most promising method to restrain chemotherapy
resistance; and macrocyclic lactone are still key molecules for
treating parasitic infections, it is necessary and urgent to find
new safe and efficient inhibitors of MDR transporters.
[0014] The objective of the present invention is to provide an
inhibitor of multidrug resistance proteins.
[0015] Particularly, the present invention concerns the use of an
avermectin derivative compound of formula I
##STR00002##
[0016] wherein:
(i) R.sub.1 is chosen from the group constituted of
--CH(CH.sub.3).sub.2, --CH(CH.sub.3)CH.sub.2CH.sub.3, or
cyclohexyl, (ii) X represents --CH.sub.2--CH.sub.2--, or
--CH.dbd.CH--, (iii) R.sub.2 is chosen from the group constituted
of
##STR00003##
or --OH group,
(iv) R.sub.3 is OH or NOH,
[0017] (v) represents a single bond when R.sub.3 is OH, or a double
bond when R.sub.3 is NOH, as an inhibitor of a membrane-bound
protein which transports exogenous compounds out of target
cells.
[0018] When R.sub.2 represents --OH group, the compound of formula
I is an aglycone avermectin.
[0019] When R.sub.2 represents
##STR00004##
the compound of formula I is a monosaccharides of avermectin.
[0020] The Inventors of the present invention have surprisingly
observed that aglycone avermectins or monosaccharide of avermectins
expose a comparable inhibitory potency and efficiency with that of
ivermectin or Valspodar.RTM., the last is one of the most efficient
MDR inhibitors already known. Moreover, the Inventors have observed
that aglycone avecmectins or monosaccharide of avermectins have a
higher inhibitory potency for nematode Pgp than that for murine
Pgp. This particularity enables aglycone avermectin or
monosaccharide of avermectins to be used as adjuvant for
conventional antiparasitic, which suffer from an efficiency
restriction due to efflux pump by intermediate of Pgp of parasite.
The most surprisingly, aglycone avermectins or monosaccharide of
avermectins exhibit a weak agonist for GABA receptors, which means
that aglycone avermectins have weaker neurotoxicity compared to
avermectin, especially ivermectin.
[0021] "A membrane-bound protein which transports exogenous
compounds out of target cells" can be a membrane-bound ATP-binding
cassette (ABC) transporter protein which mediates cellular efflux
of distinct drugs or chemicals of a wide variety of structure and
function. Particularly, such membrane-bound protein can be
P-glycoprotein (ABCB1), mutidrug resistance associated protein
family, including MRP1/ABCC1, MRP2, MRP2, or breast cancer
resistant protein (ABCG2).
[0022] The inhibitor of such membrane-bound protein is a compound
which can bind to said membrane-bound protein and thus reduce the
affinity of said membrane-bound transporter with another substrate.
The inhibitory potency of an inhibitor can be measured according to
any conventional method, such as using a reference fluorescent
substrate (ex: rhodamine 123 for Pgp) of the transporter and by
measuring the intracellular accumulation of this substrate.
[0023] Another aspect of the present invention concerns the use of
a compound of formula I, as adjuvant for increasing bioavailability
of an active ingredient of a drug whose efflux out of target cells
depends on a membrane-bound protein which transports exogenous
compounds out of target cells.
[0024] The term "adjuvant" refers to a molecule which has no
therapeutic potency when it is administrated alone, but can improve
therapeutic potency of another molecule when it is simultaneously
administrated with said another molecule.
[0025] The avermectin derivative compound of the present invention,
which efficiently inhibits a membrane-bound protein, in particular
ABC proteins, enables to improve intracellular concentration of the
active ingredient of a drug, consequently, to restore or improve
efficiency of said drug.
[0026] More particularly, the present invention is related to
avermectin derivative compounds of formula I:
##STR00005##
wherein: (i) R.sub.1 is chosen from the group constituted of
--CH(CH.sub.3).sub.2, --CH(CH.sub.3)CH.sub.2CH.sub.3, or
cyclohexyl, (ii) X represents --CH.sub.2--CH.sub.2--, or
--CH.dbd.CH--, (iii) R.sub.2 is chosen from the group constituted
of
##STR00006##
or --OH group,
(iv) R.sub.3 is OH or NOH,
[0027] (v) represents a single bond when R.sub.3 is OH, or a double
bond when R.sub.3 is NOH, for its use as an adjuvant of a drug.
[0028] In one particular embodiment, the invention concerns an
avermectin derivative compound of formula I for its aforementioned
use, wherein:
(i) R.sub.1 is chosen from the group constituted of
--CH(CH.sub.3).sub.2 and --CH(CH.sub.3)CH.sub.2CH.sub.3, (ii) X
represents --CH.sub.2--CH.sub.2--, (iii) R.sub.2 is --OH, (iv)
R.sub.3 is --OH, said compound corresponding to ivermectin aglycone
of formula I(a):
##STR00007##
[0029] In another particular embodiment, the invention concerns an
avermectin derivative compound of formula I for its aforementioned
use, wherein:
(i) R.sub.1 is chosen from the group constituted of
--CH(CH.sub.3).sub.2, and --CH(CH.sub.3)CH.sub.2CH.sub.3, (ii) X
represents --CH.sub.2--CH.sub.2--, (iii) R.sub.2 is
##STR00008##
(iv) R.sub.3 is --OH, said compound corresponding to monosaccharide
of ivermectin of formula I(b).
##STR00009##
[0030] In another particular embodiment, the invention concerns an
avermectin derivative compound of formula I for its aforementioned
use, wherein:
(i) R.sub.1 is chosen from the group constituted of
--CH(CH.sub.3).sub.2, and --CH(CH.sub.3)CH.sub.2CH.sub.3, (ii) X
represents --CH.dbd.CH--, (iii) R.sub.2 is --OH, (iv) R.sub.3 is
--OH, said compound corresponding to formula I(c):
##STR00010##
[0031] A compound of formula I(c) can be eprinomectin aglycone,
eprinomectin monosaccharide, emamectine aglycone, emamectine
monosaccharide, abamectine aglycone or abamectine
monosaccharide.
[0032] In another particular embodiment, the invention concerns an
avermectin derivative compound of formula I for its aforementioned
use, wherein:
(i) R.sub.1 is cyclohexyl, (ii) X represents --CH.dbd.CH--, (iii)
R.sub.2 is --OH or
##STR00011##
(iv) R.sub.3 is --OH, said compound corresponding to doramectin
monosaccharide or doramectine aglycone of formula I(d):
##STR00012##
[0033] In another particular embodiment, the invention concerns an
avermectin derivative compound of formula I for its aforementioned
use, wherein:
(i) R.sub.1 is cyclohexyl, (ii) X represents
--CH.sub.2--CH.sub.2--, (iii) R.sub.2 is --OH or
##STR00013##
(iv) R.sub.3 is .dbd.NOH, said compound corresponding to selamectin
or selamectin aglycone of formula I(e):
##STR00014##
[0034] In one particular embodiment, the avermectin derivative
compound of the present invention is used as an adjuvant of an
active ingredient chosen from the group comprising an
antiparasitic, an antitumor agent, an antiviral agent, an
anti-epileptic agent, an antibacterial agent, in particular an
antibiotic, an antifungal or any compound which is substrate of
said membrane-bound protein.
[0035] Such active ingredient can be any active ingredient used in
an antiparasitic, antitumor agent, antiviral agent, or
anti-epileptic agent known in the art.
[0036] In one particular embodiment, the antiparasitic is chosen
from the group comprising macrocylic lactones, such as the
avermectins, in particular ivermectin, abamectin, doramectin,
eprinomectin or selamectin, or the milbemycins, in particular
moxidectin, nemadectin, or milbemycin oxime.
[0037] In another particular embodiment, the antitumor agent is
chosen from the group comprising: [0038] antibiotic antitumor of
type anthracycline, such as daunurubicin, doxorubicin, mitocycin C,
mitoxantron, adriamycin, and actinomycin, or [0039] taxanes, such
as docetaxel, paclitaxel, or [0040] alcaloides, such as vinblastin,
vincristin, or [0041] epipodophyllotoxins, such as etoposide,
irinotecan, teniposide, et topotecan.
[0042] In another particular embodiment, the antiviral agent is
chosen from the group comprising: HIV-1 protease inhibitors,
ritonavir, saquinavir, nelfinavir and indinavir and non-nucleoside
reverse-transcriptase inhibitors such as efavirenz.
[0043] In another particular embodiment, the anti-epileptic agent
is chosen from the group comprising: Phenobarbital (PB;
5-ethyl-5-phenyl-2,4,6-trioxohexahydropyrimidine), topiramate,
lamotrigine phenytoin (PHT; 5,5-diphenyl-2,4-imidazolidinedione),
and carbamazepine (CBZ;5H-dibenz[b,f] azepine-5-carboxamide).
[0044] In another particular embodiment, the antibacterial agent
can be an antibiotic, such as loperamide, monensin, or the
macrolides.
[0045] In another particular embodiment, the antifungal agent is
chosen from an azole antifungal, such as itraconazole or
ketoconazole.
[0046] Another aspect of the present invention is to provide a
composition comprising a compound of formula I, in particular I(a),
I(b), I(c), I(d) or I(e) for its use as drug.
[0047] Particularly, the present invention concerns a composition
comprising a compound of formula I, in particular I(a), I(b), I(c),
I(d) or I(e) for its use as drug in the treatment of parasite
infections, viral infections, chemotherapy resistant cancers,
epilepsy, bacterial infections or fungal infections.
[0048] The present invention concerns also a synergic composition
comprising: [0049] a compound of formula I, in particular I(a),
I(b), I(c), I(d) or I(e), [0050] an active ingredient chosen from
antiparasiticide, an antitumor agent, an antiviral agent, an
anti-epileptic agent, an antibacterial agent, in particular an
antibiotic, or an antifungal agent.
[0051] More particularly, the composition of the present invention
comprises: [0052] a compound of formula I, in particular I(a),
I(b), I(c), I(d) or I(e), and [0053] an active ingredient chosen
from an antiparasiticide, an antitumor agent, an antiviral agent,
an anti-epileptic agent, an antibacterial agent, in particular an
antibiotic, or an antifungal agent, for its use as drug in the
treatment of parasite infections, viral infections, chemotherapy
resistant cancers, epilepsy, bacterial infections or fungal
infections.
[0054] The present invention provides also a pharmaceutical
composition comprising: [0055] a compound of formula I, in
particular I(a), I(b), I(c), I(d) or I(e), and optionally [0056] an
active ingredient chosen from an antiparasiticide, an antitumor
agent, an antiviral agent, an anti-epileptic agent, an
antibacterial agent, in particular an antibiotic, or an antifungal
agent, and [0057] a pharmaceutically acceptable carrier.
[0058] A pharmaceutically acceptable carrier can be any
conventional pharmaceutically acceptable carrier.
[0059] The pharmaceutical composition according to the present
invention can be used in the treatment of parasite infections,
viral infections, chemotherapy resistant cancers, epilespsy,
bacterial infections or fungal infections.
[0060] The pharmaceutical composition according to the present
invention can be administrated by oral route, subcutaneous
injection, intravenous injection, or intra-tissue injection.
[0061] The pharmaceutical composition according to the present
invention can be administrated with a diary dose from 0.01 mg/kg to
0.5 mg/kg
[0062] The present invention concerns also a kit which is a product
containing [0063] a compound of formula I, and [0064] an active
ingredient chosen from an antiparasitic, an antitumor agent, an
antiviral agent, an anti-epileptic agent, an antibacterial agent,
in particular an antibiotic, or an antifungal agent, as a combined
preparation for simultaneous, separate or sequential use in the
treatment of parasite infections, viral infections, chemotherapy
resistant cancers, epilepsy, bacterial infections or fungal
infections.
[0065] The present invention is illustrated in detail by following
figures and examples. However, in any way, the figures and the
examples can not be considered as a limitation of the scope of the
present invention.
FIGURES
[0066] FIG. 1A: FIG. 1A represents the HPLC profile of ivermectin
and ivermectin aglycone.
[0067] FIG. 1B: FIG. 1B represents the HPLC profile of ivermectin
aglycone and monosaccharide of ivermectin.
[0068] FIG. 2: FIG. 2 represents the mass spectrometric profile of
aglycone ivermectin.
[0069] FIG. 3: FIG. 3 compares the maximum effective concentration
of Valspodar.RTM. at 5 .mu.M (VSP, grey column), ivermectin at 5
.mu.M (IVM, white column) and ivermectin aglycone at 10 .mu.M (Agly
IVM, black column) in LLCPK1 cells transfected with murine Pgp.
Cells are incubated in a buffer containing rhodamine 123 with or
without increasing concentrations of drugs and intracellular
fluorescence was determined. Y axis represents intracellular
fluorescence expressed as percent of the control value (cell
incubated without drug). Look at also example 2.2.
[0070] FIG. 4A: FIG. 4A compares the inhibition of murine Pgp by
ivermectin (open square) with that of ivermectin aglycone (black
square) in LLC-PK1-mdr1a. X axis represents concentration of
ivermectin or ivermectin aglycone. Y axis represents intracellular
rhodamine accumulation compared to control value. Look at also
example 2.3.
[0071] FIG. 4B: FIG. 4B compares the inhibition of nematode Pgp
(HcPgpA) by ivermectin (open square) with that of ivermectin
aglycone (black square) in LLC-PK1-HcPgpA. X axis represents
concentration of ivermectin or ivermectin aglycone. Y axis
represents intracellular rhodamine accumulation compared to control
value. Look at also example 2.3.
[0072] FIG. 5: FIG. 5 shows concentration-response curves of rat
GABA(A) receptor expressed in Xenopus oocytes.
Concentration-dependent potentiation of the GABA receptor,
presented as the percentage of the GABA-evoked response at
EC.sub.10 (2 .mu.M). Y-axis represents normalized response to GABA
receptor according to the protocol described in the part 1.7 below.
X-axis represents the concentration of moxidectin (MOX), ivermectin
(IVM), ivermectin monosaccharide (IVM Monosaccharide), or
ivermectin aglycone (IVM aglycone). Data were fitted to the Hill
equation and are given as mean.+-.S.D. Look at also example 2.4
[0073] FIG. 6: FIG. 6 illustrates toxicity of ivermectin (open
circle) and ivermectin aglycone (black circle) in Pgp-deficient
mice. X axis represents the dose of ivermectin or ivermectin
aglycone administrated to mice. Y axis represents the percentage of
survival mice after one week administration. Look at also example
2.5.
[0074] FIG. 7: FIG. 7 illustrates the reversion of drug-resistance
by ivermectin aglycone in human lymphoma parental CEM cells and in
vinblastine resistant CEM/VBL cells. CEM/VBL cells were incubated 4
days with vinblastine alone from 0 to 1 .mu.g/ml (black square), or
with vinblastine from 0 to 1 .mu.g/ml and ivermectin (IVM) at 2.5
.mu.M (open square), or with vinblastine from 0 to 1 .mu.g/ml and
ivermectine aglycone (IVM-Agly) at 2.5 .mu.M (open circle), or with
vinblastine from 0 to 1 .mu.g/ml and ivermectine aglycone
(IVM-Agly) at 5 .mu.M (black circle). CEM cells were incubated 4
days with vinblastine alone from 0 to 1 .mu.g/ml (-*-). X axis
represents vinblastine (VBL) concentration. Y axis represents
cytotoxicity determined using the MTT test. Values are
mean.+-.S.E.M. of 2 experiments (3 wells per experiment). Look at
also example 2.6.
[0075] FIG. 8: FIG. 8 illustrates the reversion of drug-resistance
by ivermectin aglycone in multidrug resistant cells DC-3F/ADX which
are resistant to actinomicyne D. Multidrug resistant cells
DC-3F/ADX were incubated 3 days with actinomycin alone from 0.01 to
10 .mu.M (open square), or with actinomycin from 0.01 to 10 .mu.M
and ivermectin (IVM) at 5 .mu.M (grey square) or with actinomycin
from 0.01 to 10 .mu.M and ivermectin aglycone (IVM-Agly) at 5 .mu.M
(black triangle). X axis represents actinomycin D concentration. Y
axis represents cytotoxicity determined using the MTT test. Values
are mean.+-.S.E.M. of 2 experiments (3 wells per experiment). Look
at also example 2.6.
[0076] FIG. 9: FIG. 9 shows reversion of ivermectin-resistance by
ivermectin aglycone in Caenorhabditis elegans resistant to
ivermectin. Ivermectin resistance in C. elegans is determined
according to the protocol described in part 1.9 below. X axis
represents ivermectin concentration. Y axis represents the
percentage of gravidity compared to control. Gravidity was
evaluated in the presence of ivermectine (IVM) alone at 0, 1, 2, 4,
6, 8, 10, 20 ng/ml (open circle), or ivermectin at 0, 1, 2, 4, 6,
8, 10, 20 ng/ml with verapamil at 8 .mu.M (-x-), or ivermectin at
0, 1, 2, 4, 6, 8, 10, 20 ng/ml with ivermectin aglycone (IVM Agly)
at 10 ng/ml (11.4 nM) (black square). Assays were performed in 3
replicates per condition treatment and the experiment was performed
3 times. Mean.+-.S.D. Look at also example 2.7.
EXAMPLES
[0077] 1. Materials and Methods
[0078] 1.1 Ivermectin Aglycone Synthesis
[0079] Ivermectin aglycone (22,23-dihydroavermectin B1 aglycone) is
obtained from ivermectin by acid hydrolysis (1% of sulphuric acid).
Ivermectin aglycone is purified by HPLC according to the method
described by Alvinerie et al. (Ann Rech Vet, (1987), 18,
269-274).
[0080] 1.2 Ivermectin aglycone structure analysis [0081] HPLC
[0082] The protocol of HPLC experiment is as follows: the product
obtained after synthesis reaction is analysed by HPLC according a
modified method routinely used in the INRA laboratory. Briefly a
fluorescent derivative was obtained by dissolving the eluent in
N-methylimidazole and trifluoroacetic anhydride (Aldrich,
Milwaukee, Wis., USA) solutions in acetonitrile. The
chromatographic conditions included a mobile phase of acetic acid
2%, methanol, acetonitrile (4:32:64, v/v/v) pumped at a flow rate
of 1.5 ml/min through a Supelcosil C18, 3 .mu.m column
(150.times.4.6 mm) (Supelco, Bellefonte, Pa., USA). Fluorescence
detection (Detector RF 551, Shimadu, Kyoto, Japan) was performed at
365 nm excitation and 475 nm emission wavelength. The validation of
the technique was performed (Alvinerie at al, 1993, Vet Res 24 (5):
417-21). [0083] Mass Spectrometer
[0084] Structural characterization of the purified products was
conducted on the platform Axiom of INRA/ToxAlim, on a LCQ
quadrupole ion trap mass spectrometer (Thermo Finnigan, Les Ulis,
France) fitted with an electrospray ionization source operated in
the positive mode. The protocol of mass spectrometer assay is as
follows: collected samples were introduced into the ionization
source by infusion at a flow rate of 5 L/min with a syringe
pump.
[0085] 1.3 Cell Culture
[0086] The cells used were LLC-PK1, pig kidney epithelial cell
lines, and LLC-PK1-mdr1a which are recombinant LLC-PK1 cells
overexpressing murine abcb1a gene. All cell lines are available in
INRA laboratory. The transfected cell line LLC-PK1-HcPgpA, which
overexpress nematode Haemonchus contortus PgpA, was developed by R.
Prichard (McGill University). Cells were cultured in medium 199
supplemented with penicillin (100 units/ml), streptomycin (100
g/ml), 10% of foetal calf serum and geneticin G418 (400 mg/1) as
selecting compound for the LLC-PKI-mdr1a and LLC-PK1-HcPgpA cells.
All compounds and medium are from Invitrogen, Cergy Pontoise,
France. Cells were seeded on 24-well plates (Sarstedt, Orsay,
France) at 2.times.10.sup.5 cells/well in G418-free medium until
confluence for transport activity and on 96-well plates for
viability assay.
[0087] Multidrug resistant tumor cells used in the present
invention were Human lymphoma parental CEM and
vinblastine-resistant CEM/VLB (Zordan-Nudpo et al., 1993) and
parental CEM and multidrug resistant cells DC-3F/ADX selected from
spontaneously transformed DC-3F Chinese hamster lung fibroblasts on
the basis of their resistance to actinomycin D (Biedler and Riehm,
1970). Both types of resistant cells overexpressed Pgp.
[0088] 1.4 Animal Model
[0089] Wild-type and the Pgp knock-out mdr1ab.sup.-/- mice with a
FVB genetic background were obtained from Taconic (NY, USA). In
rodents, there are two Pgps encoded by abc1a and abc1b genes and
mdr1ab.sup.-/- mice were deficient for the two gene products. Mice
were housed at INRA's transgenic rodent facility at 22.+-.2.degree.
C. under 12-hour light/dark cycles. Animals sampling was designed
to reduce the influence of interfering parameters such as litter
specificity (seven to nine different litters for a ten animals
group). Mice received a standard chow diet recommended for the
breeding and rearing of rodents (Harlan Teklad TRM Rat/Mouse Diet;
Harlan Teklad, Gannat, France). Water and food were available ad
libitum. In vivo studies were conducted in mice under European laws
on the protection of animals and protocols are performed under
procedure and principal for good clinical practice.
[0090] 1.5 Tested Molecules
[0091] Ivermectin aglycone obtained according to the synthesis
method described in part 1.1 and purified is used in all the
comparative experiments of the present invention.
[0092] Ivermectin purchased from Sigma is used as inhibition
standard in all the comparative experiments of the present
invention.
[0093] Valspodar.RTM. was kindly provided by Novartis and is used
as reference inhibitor of Pgp.
[0094] All the three aforementioned compounds are solubilised in
DMSO.
[0095] 1.6 Transport Tests In Vitro
[0096] Cells were cultured with rhodamine 123 (10 .mu.M, purchased
from Sigma) with or without valspodar (VSP, 504). Compounds of
interest were dissolved in DMSO and diluted in the medium (final
DMSO concentration=0.1%) in a concentration range of 0.1-50 .mu.M.
After the 2-h incubation period, the cells were lysed and lysates
were stored at -20.degree. C. until analysis. To study the Pgp
transport activity, the intracellular accumulation of fluorescent
Rho 123 was determined by reading fluorescence in the cell lysates
with a spectrofluorimeter (PerkinElmer LS50B, max excitation=507
nm; max emission=529 nm). Protein concentration was determined in
lysates with BCA kit using bovine serum albumin as protein standard
(Thermo scientific) Results were expressed as fluorescence
arbitrary units after normalization to cellular protein content per
well.
[0097] 1.7 GABA Receptor Affinity Test
[0098] The ability of ivermectin or moxidectin or ivermectin
aglycone or ivermectin monosaccharide to interact with GABA
receptors is assayed by electrophysiology measurements. Xenopus
laevis oocytes are injected with 46 nl of RNA solution, with RNA
coding for .alpha.1, .beta.2 and .gamma..sub.2 subunits of the GABA
channel at a ratio of 10:10:50 nM. The injected oocytes are
incubated in modified Barth's solution [90 mM NaCl, 3 mM KCl, 0.82
mM MgSO.sub.4, 0.41 mM CaCl.sub.2, 0.34 mM Ca(NO.sub.3).sub.2, 100
U/ml penicillin, 100 .mu.g/ml streptomycin and 100 .mu.g/ml
kanamycin, 5 mM HEPES pH 7.6] at 18.degree. C. for approximately 36
h before the measurements to ensure the expression of a functional
receptor.
[0099] Electrophysiological experiments are performed by the
two-electrode voltage-clamp method. Measurements were done in ND96
medium containing 96 mM NaCl, 2 mM KCl, 1 mM gCl.sub.2, 1.8 mM
CaCl.sub.2 and 5 mM HEPES, pH 7.5, at a holding potential of -80
mV. The control current is evoked by the application of 2 .mu.M
GABA and the normalized relative potentiation of 2 .mu.M
GABA-evoked currents by increasing concentration of ivermectin,
moxidectin, ivermectin aglycone, or ivermectin monosaccharide is
determined as:
[(I.sub.MLs+2 .mu.M GABA/I.sub.2 .mu.M GABA alone)/(I.sub.(MLs+2
.mu.M GABA)Max/I.sub.2 .mu.M GABA alone)].times.100%
where I.sub.2 .mu.M GABA is the control current evoked by 2 .mu.M
GABA, I.sub.MLs+2 .mu.M GABA is the current evoked by each drug
concentration in co-applications with 2 .mu.M GABA, and
I.sub.(MLs+2 .mu.M GABA)Max is the maximal current evoked by
co-applications of drugs and 2 .mu.M GABA. A washout period of 4
min between each GABA application is introduced, allowing receptors
to recover from desensitization. Three different batches of oocytes
are used to collect data for each analysis. The perfusion system is
cleaned between two experiments by washing with 10% DMSO after
application of MLs derivatives to avoid contamination.
[0100] 1.8 In Vivo Toxicity Test
[0101] Toxicity of ivermectin and ivermectin aglycone is measured
in Pgp-deficient mice. Mdr1ab.sup.-/- mice are injected
subcutaneously with increasing doses of ivermectin or ivermectin
aglycone formulated in propylene glycol/formaldehyde (60:40, v/v).
Higher injected doses are 1.5 mg/kg (1.7 .mu.mol/kg) for ivermectin
and 16 mg/kg (27 .mu.mol/kg) for ivermectin aglycone, respectively.
Toxicity is evaluated during 24 h. At the end of the monitoring,
plasma is collected, from the orbital sinus vein under
methoxyflurane anesthesia and the mice are sacrificed for the brain
collection. Blood is centrifuged at 1500 g for 10 min, and plasma
is stored at 20.degree. C. until analysis. The brains is removed,
washed in saline solution, and frozen at 20.degree. C. until
analysis.
[0102] 1.9 Ivermectin Resistance Assay in Caenorhabditis
elegans
[0103] A gravid assay method, based on the development of eggs to
gravid adults over a 96 hr incubation period, was used to determine
the resistance with respect to ivermectin (IVM) in C. elegans. The
eggs were collected through rinsing the C. elegans worms resistant
to IVM (IVR10). Sixty eggs were incubated/well, in standard
conditions for four days (96 hours) in order reach adulthood
(gravid) in the presence of drugs as followed: ivermectin aglycone
(IVM-Agly) alone at 10 ng/ml (11.4 nM); verapamil (VRP) alone at 8
.mu.M; IVM alone: 0, 1, 2, 4, 6, 8, 10, 20 nM; IVM+VRP 8 .mu.M: 0,
1, 2, 4, 6, 8, 10, 20 ng/ml IVM; IVM+IVM-Agly 10 ng/ml: 0, 1, 2, 4,
6, 8, 10, 20 ng/ml (0.114-22.8 nM) IVM. Assays were performed in
triplicates per condition treatment and the experiment was
performed 3 times.
[0104] 2. Results
[0105] 2.1 Ivermectin Aglycone Synthesis
[0106] Ivermectin aglycone is obtained from ivermectin by acid
hydrolysis, which cuts the chemical bond between macrocycle and
disaccharide group. The product obtained after this reaction is a
mixture of about 80% ivermectin aglycone and 20% monosaccharide of
ivermectin, as showed by structure profile performed by HPLC (FIGS.
1A and 1B). Ivermectin aglycone obtained by said synthesis method
is characterised by a hydroxyl group on carbon C13 of macrocycle
(FIG. 1B) and a 3 minutes of retention time in our chromatographic
conditions, shorter than that of ivermectin (5 minutes) or that of
monosaccharide derivative (4 minutes).
[0107] The obtained product is then analysed by mass spectrometry,
which confirms the presence of a mass pick at 609.3 which
corresponds to ionised ivermectin aglycone (FIG. 2), while the mass
pick of native ivermectin aglycone is at 586.8.
[0108] 2.2 Ivermectin Aglycone Inhibitory Potency for Pgp in Cell
Model
[0109] Ivermectin aglycone inhibitory potency for transport
activity of Pgp is assayed in transfected cells LLCPK1-mdr1a
overexpressing murine Pgp (mdr1a). Maximum inhibition has been
obtained with Valspodar.RTM., the most powerful reference inhibitor
of Pgp known in the past. It is confirmed that ivermectin is an
inhibitor of Pgp as powerful as Valspodar.RTM. (FIG. 3). It is also
shown that ivermectin aglycone has a comparable efficacy to inhibit
murine Pgp to that of ivermectin, with maximal effect (Eurax) at
about 10 .mu.M (Table 1, FIG. 3).
TABLE-US-00001 TABLE 1 Inhibitory effect of ivermectin and
ivermectin aglycone in cells overexpression murine Pgp Ivermectin
Ivermectin aglycone EC.sub.50 (.mu.M) 0.5 1.0 C.sub.max (.mu.M) 5.0
10.0 E.sub.max (% valspodar .RTM.) 88.0 80.0 EC.sub.50: effective
concentration for inhibiting 50% of transport of rhodamine 123 by
murine Pgp. C.sub.max: concentration to obtain maximum inhibitory
effect. E.sub.max: maximum effect compared to maximum effect
obtained with 5 .mu.M of valspodar.
[0110] EC.sub.50: effective concentration for inhibiting 50% of
transport of rhodamine 123 by murine Pgp.
[0111] C.sub.max: concentration to obtain maximum inhibitory
effect.
[0112] E.sub.max: maximum effect compared to maximum effect
obtained with 5 .mu.M of valspodar.
[0113] 2.3 Different Inhibitory Potency of Ivermectin Aglycone for
Murine Pgp and Nematode Pgp
[0114] Inhibitory potency of ivermectin aglycone or ivermectin for
murine Pgp or nematode Pgp is respectively measured in cells
LLCPK1-mdr1a, which overexpress murine Pgp (MDR1), or in cell model
developed by R. Prichard, which overexpress nematode Haemonchus
contortus Pgp: hc-pgpA. The results show that ivermectin has
similar potency to inhibit mammalian Pgp (EC.sub.50=0.5 .mu.M) and
nematode HcPgpA (EC.sub.50=0.6 .mu.M) (Table 2, FIGS. 4A and 4B),
while ivermectin aglycone has 5 times higher inhibitory potency for
parasite HcPgpA (EC.sub.50=0.5 .mu.M) than for mammalian Pgp
(EC.sub.50=2.5 .mu.M). These results clearly indicated that
ivermectin aglycone is more potent in inhibiting nematode HcPgpA
than mammalian Pgp.
TABLE-US-00002 TABLE 2 Concentration of half inhibitory effect of
ivermectin and ivermectin aglycone in cells overexpression Pgp EC50
.mu.M Ivermectin Ivermectin aglycone Murine Pgp 0.5 2.5 Nematode
PgpA 0.6 0.5
[0115] 2.4 Ability of Ivermectin Aglycone or Ivermectin
Monosaccharide to Open GABA Receptor in Oresence of GABA.
[0116] of the ability of ivermectin aglycone or ivermectin
monosaccharide to potentiate GABA action on GABA receptor, was
assayed according to the protocol described in aforementioned part
1.7, and was compared with ivermectin.
[0117] The results displayed in table 4 show that ivermectine
monosaccharide (IVM Monosaccharide) and ivermectine aglycone
(IVM-Agly) are a weak agonist (EC.sub.50=122.4 nM for IVM
Monosaccharid and EC.sub.50=215.1 nM for IVM-Agly) compared to
ivermectin (EC.sub.50=29 nM) (Table 3, FIG. 5). This result means
that ivermectin monosaccharide and ivermectin aglycone have a much
weaker neurotoxicity when compared with ivermectin, and a
pharmaceutical use of ivermectin aglycone or ivermectin
monosaccharide is possible.
TABLE-US-00003 TABLE 3 Parameters of interaction of IVM and
derivatives with GABA receptors: EC.sub.50 is the concentration
needed to induce half of the maximal potentiation of GABA effect by
MLs or derivatives. MLs EC.sub.50 (nM) MOX 5.6 .+-. 1.5 IVM 29.3
.+-. 3.4 IVM Monosaccharide 122.4 .+-. 20.3 IVM Aglycone 215.1 .+-.
12.45
[0118] 2.5 In Vivo Toxicity of Ivermectin Aglycone
[0119] In vivo toxicity text in Pgp-deficient mice confirms that
the lethal dose for ivermectin is from 0.6 to 0.8 .mu.mol/kg, as
what is described by Schinket et al. (Cell (1994) 77, 491-502). On
the contrary, ivermectin aglycone does not show any toxicity when
it is administered with a dose till 10 times higher than that of
ivermectin (FIG. 6). This result confirms that ivermectin aglycone
has a much weaker in vivo toxicity compared to ivermectin and a
pharmaceutical use of ivermectin aglycone is possible.
[0120] 2.6 Reversal of Multidrug Resistance by Ivermectin Aglycone
in Multidrug Resistant Tumor Cells
[0121] CEM/VLB cells and DC-3F/ADX cells described in
aforementioned part 1.3 were plated into 96 well plates and allowed
to grow for 24 h. They were then incubated 4 days with vinblastine
(concentration range 0-1 .mu.M) with or without IVM at 2.5 .mu.M or
ivermectin aglycone (IVM-Agly) at 2.5 and at 5 .mu.M (FIG. 7); or 2
days with actinomycin D with actinomycin (concentration range
0.01-10 .mu.M) with or without ivermectin (IVM) or ivermectin
aglycone (IVM-Agly) at 5 .mu.M (FIG. 8). Cytotoxicity was
determined using the MTT test. IC.sub.50 values were graphically
determined and they represent the concentration needed for half
cell survival. Fold reversal of multidrug resistance called
reversion factors were the ratio of IC.sub.50 for toxic drug
alone/IC.sub.50 for toxic drug in the presence of IVM-Agly.
[0122] IVM-Agly was able to reverse drug resistance in tumor cells
overexpressing Pgp. CEM/VBL are highly resistant to VBL and cells
were fully viable in 1 .mu.M vinblastine while the parental cells
are highly sensitive to VBL at concentrations below 0.001 .mu.M.
Co-incubation of VBL with IVM at 2.5 .mu.M, or IVM-Agly at 5 .mu.M
provoke a clear left-shift of the viability cell curve (FIG. 7)
demonstrating that cells are sensitized to VBL in presence of the
tested compounds. In the presence of IVM at 2.5 .mu.M the VBL
IC.sub.50 was 0.2 .mu.M and in presence of IVM-Agly at 2.5 and 5
.mu.M, the IC.sub.50 values were 1 and 0.2 .mu.M, reflecting that
IVM-Agly's has similar inhibitory potency compared to that of IVM
(Table 1). In addition, DC-3F/ADX viability was not altered by 1
.mu.M actinomycin D while when combined with IVM or IVM-Agly at 5
.mu.M actinomycin D became toxic (FIG. 8).
[0123] The results of FIG. 7, FIG. 8 and table 4 showed that the
ability of ivermectin aglycone to reverse vinblastine or
actinomycinD-resistance in tumor cells overexpression Pgp was of
the same order of potency as ivermectin, which is potent inhibitor
of MDR transporters.
TABLE-US-00004 TABLE 4 Comparison of IC50 and resistance factor
(RF) for IVM and IVM Agly in multidrug-resistant cells IC50 (.mu.M)
RF CEM/VBL VBL Nd VBL + IVM 2.5 .mu.M 0.2 VBL + IVM-Agly 2.5 .mu.M
1.0 VBL + IVM-Agly 5 .mu.M 0.2 DC-3F/ADX ActD 5.0 ActD + IVM 5
.mu.M 0.11 45 ActD + IVM-Agly 5 .mu.M 0.08 62 Nd: not
determined
[0124] 2.7 Reversal of anthelmintic resistance by ivermectin
aglycone in C. elegans resistant to ivermectin
[0125] The reversal action of ivermectin aglycone (IVM-Agly) was
studied on the nematode Caenorhabditis elegans resistant to
ivermectin (IVR10). This strain has been previously selected under
IVM pressure and it was shown to overexpressed P-gp homologue genes
(James and Davey, 2009). We measured the ability of IVM-Agly to
restore the development from eggs to adults which has been delayed
by the ivermectin effect on the IVR10 strain, and compared its
effect to that of the verapamil (VRP) reversal effect.
[0126] The resistance with respect to invermectin in C. elegans is
measured according to the protocol described in aforementioned part
1.9.
[0127] IVM blocked the development of C. elegans IVR10 eggs at a
concentration averaging 10 nM confirming that this strain is
resistant to IVM. The IC.sub.50 for IVM was 6.8.+-.0.2 ng/ml
(7.8.+-.0.2 nM). verapamil, a known Pgp-reversing agent, at 8 .mu.M
had no effects on the development of the C. elegans when alone, and
was able to restore the development of worms stopped in the
presence of IVM. The curve of IVM efficacy was thus shifted to the
left with the IC.sub.50 of IVM reduced to 3.2.+-.0.5 ng/ml
(3.6.+-.0.6 nM) when compared to IVM alone (FIG. 9, Table 5).
IVM-Agly at 10 ng/ml was also able to significantly decrease the
EC.sub.50 of IVM to 4.5.+-.0.3 ng/ml (5.1 nM, Table 5), and
IVM-Agly alone at 10 ng/ml had no effects on the development of the
C. elegans suggesting that IVM-Agly also reverse a Pgp-mediated
drug resistance.
[0128] The lower EC.sub.50 for ivermectin efficacy in IVM resistant
C. elegans determined in presence of IVM-Agly testifies that
IVM-Agly is able to partly reverse IVM resistance. Based on the
fact that verapamil are well-known inhibitors of Pgp, their effects
comparable to the one produced by IVM-Agly suggest that the
IVM-Agly reversion also occurs through inhibition of Pgp-like
transporters.
TABLE-US-00005 TABLE 5 Comparison of IC.sub.50 and resistance
factor (RF) for the reference reversal agent valspodar and
verapamil and IVM-Agly in ivermectin-resistant C. elegans IC.sub.50
(nM) RF IVM alone 7.8 .+-. 0.2 IVM + verapamil (4 .mu.M) 3.6 .+-.
0.6 2.1 IVM + IVM-agly (11.4 nM) 5.1 .+-. 0.3 1.5
* * * * *