MACROCYCLIC LACTONE ANTHELMINTICS AGAINST NEMATODES

Abstract

The present invention relates to a milbemycin for use in control, treatment and/or prevention of infections with nematodes, preferably filariae, more preferably Dirofilaria immitis which are resistant to at least one other macrocyclic lactone anthelmintic. The present invention further relates to the use of milbemycins for stimulating attachment of polymorphonuclear neutrophils (PMNs) and/or peripheral blood mononuclear cells (PBMCs) to larvae of nematodes, as well as to a method for stimulating attachment of PMNs and/or PBMCs to larvae of nematodes. In a further aspect, the present invention relates to an in vitro assay for determining and/or characterizing an agent for control, treatment and/or prevention of an infection with nematodes.

Claims

1. A method for the control, treatment and/or prevention of infections with nematodes which are resistant to at least one other macrocyclic lactone anthelmintic comprising administering a milbemycin to a subject in need thereof.

2. The method according to claim 1, wherein the milbemycin is selected from the group consisting of milbemectin, milbemycinoxim, moxidectin, nemadectin, milbemycin-D, and combinations thereof.

3. The method according to claim 1, wherein the infections with nematodes are selected from infections with filariae or larvae of filariae.

4. The method according to claim 1, wherein the at least one other macrocyclic lactone anthelmintic is selected from the group consisting of ivermectin, selamectin, doramectin and abamectin.

5. The method according to claim 1, wherein a resistant nematode is defined as exhibiting an EC.sub.50 value of the at least one other macrocyclic lactone which is increased by at least 15% as compared to the EC.sub.50 value of the wild-type nematode.

6. The method according to claim 1, wherein the subject in need thereof is infected with nematodes which are resistant to at least one other macrocyclic lactone anthelmintic that was previously administered to the subject to treat nematodes.

7. The method according to claim 1, wherein the milbemycin is administered to the subject in need thereof in a dose adjusted to give a plasma concentration in the subject of 0.1-100 nM of milbemycin.

8. The method according to claim 1, wherein the milbemycin is administered to the subject in need thereof every month, every 6 months, or every 12 months.

9. The method according to claim 1, wherein milbemycin is administered orally, topically, or parenterally.

10. (canceled)

11. A pharmaceutical composition comprising at least one milbemycin and a second agent selected from the group consisting of a macrocyclic lactone, peripheral blood mononuclear cells (PBMCs), polymorphonuclear neutrophils (PMNs), and a combination thereof.

12. (canceled)

13. A method for stimulating attachment of polymorphonuclear neutrophils (PMNs) and/or peripheral blood mononuclear cells (PBMCs) to nematodes, or filariae, or larvae thereof, comprising adding a composition comprising at least one milbemycin and PBMCs and/or PMNs to a composition comprising nematodes, or filariae or larvae thereof.

14. (canceled)

15. (canceled)

16. An in vitro assay for determining or characterizing an active agent for control, treatment and/or prevention of an infection with nematodes comprising (i) providing larvae of an isolated nematode, (ii) contacting the larvae of (i) with PBMCs and/or PMNs and an agent to be determined, (iii) incubating the mixture obtained in (ii) for a predetermined time, (iv) measuring the percentage of motile larvae having at least one PBMC or PMN attached, optionally at predetermined concentrations of the agent, and optionally including at least one of the following: (v) comparing the percentage obtained in (iv) with percentage of motile larvae having at least one PBMC or PMN attached in a control sample, (vi) selecting the active agent which has an increased percentage over the control sample by at least 20%, and (vii) determining an EC.sub.50 value of the agent.

17. (canceled)

18. The in vitro assay according to claim 16, wherein a control sample is made in accordance with (i)-(iv) except that (ii) is performed in the absence of the agent.

19. (canceled)

20. The method according to claim 2, wherein the milbemycin is moxidectin.

21. The method according to claim 3, wherein the filariae or larvae thereof are selected from the group consisting of Dirofilaria immitis, Brugia malayi, Wuchereria bancrofti, Loa loa, Mansonella spp., Dirofilaria repens and Onchocerca volvulus.

22. The method according to claim 7, wherein the milbemycin is administered to the subject in need thereof in a dose adjusted to give a plasma concentration in the subject of 0.1-10 nM of milbemycin.

23. The method according to claim 22, wherein the milbemycin is administered to the subject in need thereof in a dose adjusted to give a plasma concentration in the subject of 0.5-3 nM of milbemycin.

24. The method according to claim 9, wherein the administration is subcutaneously, topically, or orally.

25. The method according to claim 11, wherein the milbemycin is moxidectin.

26. The method according to claim 13, wherein the milbemycin is moxidectin.

Description

FIGURES

[0067] FIG. 1: Effect of ivermectin and moxidectin on PMN and PBMC attachment to larvae of the D. immitis isolates Missouri (MO) and Georgia-2 (GA-2). A) Effect of ivermectin on the attachment of PMNs. B) Effect of moxidectin on the attachment of PMNs. C) Effect of ivermectin on the attachment of PBMCs. D) Effect of moxidectin on the attachment of PBMCs. For each panel the bars represent the percentage of Mf with at least one cell attached at varying concentrations of the drug; from left to right these were 0, 1, 3, 10, 30, 100, 300 and 1000 nM. MO=Missouri, GA-2=Georgia-2.

[0068] FIG. 2: Effect of ivermectin and moxidectin on PMN and PBMC attachment to larvae of D. immitis isolates with suspected resistance against at least one macrocyclic lactone. A) Effect of ivermectin on the attachment of PMNs. B) Effect of moxidectin on the attachment of PMNs. C) Effect of ivermectin on the attachment of PBMCs. D) Effect of moxidectin on the attachment of PBMCs. For each panel the bars represent the percentage of Mf with at least one cell attached at varying concentrations of the drug; from left to right these were 0, 1, 3, 10, 30, 100, 300 and 1000 nM. YAZ=Yazoo-2013, MET=Metairie-2014.

EXAMPLES

Methods

1. Parasites

[0069] The Missouri isolate of D. immitis was provided by the NIH/NIAID Filariasis Research Reagent Resource Center. The Georgia-2 isolate was provided by TRS Labs Inc., Athens, Ga. The Yazoo-2013 and Metairie-2014 strains have been described previously (Maclean et al., 2017).

[0070] For microfilariae isolation blood from infected dogs was received in heparinized tubes and centrifuged for 30 minutes at 1200×g at room temperature. The top layer of plasma was removed, and the mass of red blood cells and Mf was brought back to its original volume with 4:1 3.8% (w/v) saline-citrate (38 mg sodium citrate/100 mL physiological saline). 15% (w/v) (1.5 g) saponin was mixed with deionized water (10 mL) and was added (1 mL) for every 15 mL of original volume and the tube was shaken for 30 seconds. The mixture was centrifuged for 30 minutes at 1200×g. The supernatant was discarded, and sodium-citrate was used to bring back to the original volume (15 mL). The mixture was then centrifuged for 4 minutes at 1200×g. The worm mixture was transferred to a new conical tube and mixed with 1× PBS (10 mL). The PBS and Mf mixture was centrifuged for 5 minutes at 1200×g to pellet the Mf and the supernatant was removed. The pellet was resuspended in Roswell Park Memorial Institute (RPMI) cell culture medium prior to assessment of worm numbers.

2. Cell Isolation

[0071] Blood from an uninfected dog was drawn from jugular punctures and put into heparinized tubes. Blood (10 mL) was transferred from the heparinized tube into a sterile, endotoxin free conical tube (50 mL) with 1:1 PBS. The blood was then underlaid with Histopaque® 1077 (5-10 mL) with an 18-gauge needle and a sterile syringe. The gradient was then centrifuged at 400×g at room temperature for 25 minutes with the brake off. The top layer of plasma was discarded and the middle layer, the PBMC layer, was placed in a separate sterile conical tube. 40 ml ACK buffer (155 mM ammonium chloride, 10 mM potassium hydrogen carbonate, 0.1 mM EDTA, pH 7.3) was added to the red blood cell/PMN mixture and gently mixed by inverting the tube. The blood mixture was set to rest for 5 minutes at room temperature in order for the red blood cells to lyse. The mixture was centrifuged for 5 minutes at 400×g to pellet PMNs. The PMN pellet was washed with PBS and re-suspended in 10 ml PBS-2% BSA then centrifuged again at 400×g for 5 minutes. After removing the supernatant, the pellet was re-suspended in 500 μl RPMI and 500 μl serum.

3. Cell Attachment Assays

[0072] Assays were set up in triplicate in a 96-well plate with a minimum of 5 biological replicates for each strain. Each biological replicate is defined as independent Mf isolations in different weeks from the same dog. Each well contained 100 Mf of the strain under test, 20,000 cells (PBMC or PMN) and 10% uninfected dog serum in RPMI. The drug concentrations tested were 1, 3, 10, 30, 100, 300 and 1,000 nM, plus a vehicle (1% DMSO) control. The assays were incubated at 37° C. in a 5% CO.sub.2 atmosphere for 24 h (PMN) or 40 h (PBMC), before being visually scored. Attachment in this assay was defined by a motile Mf having at least one cell attached. Static worms were considered to be dead and were not counted.

4. Data Analysis

[0073] Data were analyzed using Graphpad Prism®, v5 (GraphPad Software, INC., San Diego, Calif.). Cell attachment within each strain was compared using 2-way ANOVA and Tukey's post-hoc test.

Results

Example 1

PMN and PBMC Attachment to Mf in the Presence of Ivermectin

[0074] When purified canine PMNs and PBMCs isolated from uninfected dogs are cultured with D. immitis Mf a low percentage of the parasites had cells attached to them after 16 h (PMNs) or 40 h (PBMCs). The addition of ivermectin to the cultures increased the proportion of the Mf with both PMNs and PBMCs attached in a concentration-dependent manner for nearly all the strain/cell type combinations tested, though the concentration at which a statistically significant increase over the no-drug controls was observed did vary between strains (cf. FIGS. 1 and 2).

[0075] For the Missouri and Georgia-2 isolates, both of which are susceptible to macrocyclic lactone anthelmintics, 1-3 nM ivermectin was sufficient to cause a significant increase in attachment of both PMNs and PBMCs. For the resistant Metairie-2014 and Yazoo-2013 isolates, higher drug concentrations were required, 100-300 nM for Yazoo-2013 and 1 μM or greater for Metairie-2014 (cf. Table 1); there was no significant increase in attachment of PBMCs to the Metairie-2014 Mf at any concentration of ivermectin. It was found that the maximum percentage of the Mf with cells attached varied between the strains, even at the highest concentration tested (1 μM) (Table 2). 13% of the Metairie-2014 Mf had PMNs attached compared to 48% of the Missouri Mf; for the PBMCs the range was 10% of Metairie-2014 to 71% of the Georgia-2 Mf having cells attached.

Example 2

PMN and PBMC Attachment to Mf in the Presence of Moxidectin

[0076] The attachment experiments were repeated using moxidectin instead of ivermectin. Likewise, a concentration dependent increase in attachment to the Missouri and Georgia-2 with both PMNs and PBMCs was found (cf. FIGS. 1 and 2). However, a concentration-dependent increased cell attachment to the Metairie-2014 Mf was also observed with moxidectin, in contrast to ivermectin. The concentration of moxidectin at which a significant increase in attachment was observable with this strain was lower than with ivermectin and the maximum level of attachment was higher; both were similar to the values of the susceptible strains. The concentration of moxidectin at which increased attachment was observed for Yazoo-2013 was similar to all the other strains at 3-10 nM (Table 1), but the proportion of the Mf with cells attached tended to be lower (Table 2).

TABLE-US-00001 TABLE 1 The lowest drug concentration [ivermectin (IVM) or moxidectin (MOX)] at which a statistically significant increase (p = <0.05), judged by 2-way ANOVA, in cell attachment was observed compared to the no-drug control. Strain Missouri Georgia-2 Yazoo-2013 Metairie-2014 Drug IVM MOX IVM MOX IVM MOX IVM MOX PMN 3 nM 3 nM 1 nM 3 nM 100 nM 3 nM 1 μM 10 nM  PBMC 3 nM 1 nM 1 nM 1 nM 100 nM 10 nM  100 nM  1 nM

TABLE-US-00002 TABLE 2 Percentage (±SEM) of Mf with cells attached after incubation with 1 μM ivermectin (IVM) or moxidectin (MOX), rounded to the nearest whole number. Strain Missouri Georgia-2 Yazoo-2013 Metairie-2014 Drug IVM MOX IVM MOX IVM MOX IVM MOX PMN 48 ± 6% 65 ± 5% 41 ± 6% 54 ± 6% 25 ± 5% 28 ± 5% 13 ± 3% 42 ± 6% PBMC 60 ± 3% 63 ± 5% 71 ± 5% 63 ± 5% 18 ± 4% 19 ± 3% 10 ± 2% 49 ± 8%

[0077] If the leukocyte attachment is relevant to the drug's in vivo anthelmintic efficacy, then we would predict that attachment would be reduced in the presence of the drug to Mf of resistant strains as opposed to those of susceptible ones. In general those predictions were supported by the data we obtained. Ivermectin and moxidectin both increased cellular attachment to the Missouri and Georgia-2 Mf at very low concentrations (<10 nM) which correspond to those reported to be present in the plasma of treated dogs. The effects of ivermectin on attachment to the resistant Metairie-2014 and Yazoo-2013 isolates were much less marked (FIG. 2). These results suggest that the in vitro drug-promoted leukocyte attachment is indeed relevant to the full in vivo potential of the macrocyclic lactone anthelmintics and indicates that the host immune response is required for the prevention provided by these drugs. They also suggest that the Mf are a suitable surrogate life-cycle stage for studying resistance in D. immitis, supporting the utility of the present in vitro Mf suppression assay as a diagnostic tool for resistance in infected subjects.

[0078] Moxidectin was more effective at promoting attachment to the Metairie-2014 and Yazoo-2013 Mf than was ivermectin (Table 1).

CITED DOCUMENTS

[0079] Blagburn et al., Parasites & Vectors 9, 191, 2016.

[0080] Bourguinat et al., Vet. Parasitol 176, 374-381, 2011.

[0081] Franks et al., J. Parasitol 31, 158-162, 1945.

[0082] Geary et al., Top. Companion Anim. Med. 26, 186-192, 2011.

[0083] Maclean et al., Parasites & Vectors 10 (Suppl 2), 480, 2017.

[0084] Zahner et al., Experimental Parasitology, 86(2), 110-117, 1997.