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═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.

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═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═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═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 ═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

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® at 5 μM (VSP, grey column), ivermectin at 5 μM (IVM, white column) and ivermectin aglycone at 10 μM (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 μM). 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 μg/ml (black square), or with vinblastine from 0 to 1 μg/ml and ivermectin (IVM) at 2.5 μM (open square), or with vinblastine from 0 to 1 μg/ml and ivermectine aglycone (IVM-Agly) at 2.5 μM (open circle), or with vinblastine from 0 to 1 μg/ml and ivermectine aglycone (IVM-Agly) at 5 μM (black circle). CEM cells were incubated 4 days with vinblastine alone from 0 to 1 μg/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 μM (open square), or with actinomycin from 0.01 to 10 μM and ivermectin (IVM) at 5 μM (grey square) or with actinomycin from 0.01 to 10 μM and ivermectin aglycone (IVM-Agly) at 5 μM (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 μM (-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 μm column (150×4.6 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 2×10.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° 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® 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 μM, 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 μM. After the 2-h incubation period, the cells were lysed and lysates were stored at −20° 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 α1, β2 and γ.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 μg/ml streptomycin and 100 μg/ml kanamycin, 5 mM HEPES pH 7.6] at 18° 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 μM GABA and the normalized relative potentiation of 2 μM GABA-evoked currents by increasing concentration of ivermectin, moxidectin, ivermectin aglycone, or ivermectin monosaccharide is determined as:

[(I.sub.MLs+2 μM GABA/I.sub.2 μM GABA alone)/(I.sub.(MLs+2 μM GABA)Max/I.sub.2 μM GABA alone)]×100%

where I.sub.2 μM GABA is the control current evoked by 2 μM GABA, I.sub.MLs+2 μM GABA is the current evoked by each drug concentration in co-applications with 2 μM GABA, and I.sub.(MLs+2 μM GABA)Max is the maximal current evoked by co-applications of drugs and 2 μM 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 μmol/kg) for ivermectin and 16 mg/kg (27 μmol/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° C. until analysis. The brains is removed, washed in saline solution, and frozen at 20° 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 μM; IVM alone: 0, 1, 2, 4, 6, 8, 10, 20 nM; IVM+VRP 8 μM: 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®, 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® (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 μM (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 (μM) 0.5 1.0 C.sub.max (μM) 5.0 10.0 E.sub.max (% valspodar ®) 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 μM 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 μM 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 μM) and nematode HcPgpA (EC.sub.50=0.6 μM) (Table 2, FIGS. 4A and 4B), while ivermectin aglycone has 5 times higher inhibitory potency for parasite HcPgpA (EC.sub.50=0.5 μM) than for mammalian Pgp (EC.sub.50=2.5 μM). 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 μM 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 μmol/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 μM) with or without IVM at 2.5 μM or ivermectin aglycone (IVM-Agly) at 2.5 and at 5 μM (FIG. 7); or 2 days with actinomycin D with actinomycin (concentration range 0.01-10 μM) with or without ivermectin (IVM) or ivermectin aglycone (IVM-Agly) at 5 μM (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 μM vinblastine while the parental cells are highly sensitive to VBL at concentrations below 0.001 μM. Co-incubation of VBL with IVM at 2.5 μM, or IVM-Agly at 5 μM 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 μM the VBL IC.sub.50 was 0.2 μM and in presence of IVM-Agly at 2.5 and 5 μM, the IC.sub.50 values were 1 and 0.2 μM, 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 μM actinomycin D while when combined with IVM or IVM-Agly at 5 μM 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 (μM) RF CEM/VBL VBL Nd VBL + IVM 2.5 μM 0.2 VBL + IVM-Agly 2.5 μM 1.0 VBL + IVM-Agly 5 μM 0.2 DC-3F/ADX ActD 5.0 ActD + IVM 5 μM 0.11 45 ActD + IVM-Agly 5 μM 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 μM 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 μM) 3.6 ± 0.6 2.1 IVM + IVM-agly (11.4 nM) 5.1 ± 0.3 1.5