NEW IMMUNOSTIMULATORY COMPOUNDS

Abstract

The present invention relates to novel immunostimulatory molecules which are derived from the intestinal protozoan Entamoeba histolytica. The compounds have been found to be useful for enhancing and/or inducing an immune response in a subject in need thereof. Specifically, the compounds have been found to be useful for the treatment of cancer diseases, such as breast cancer, and parasitic diseases, such as leishmaniasis. The invention also provides pharmaceutical compositions comprising the novel compounds.

Claims

1. A method of treating a cancer disease or an infection with an intracellular pathogen in a subject in need thereof comprising administering to the subject a compound having the structure of the following formula (I): ##STR00012## wherein R.sup.1 is Y or Z, R.sup.2, R.sup.3 and R.sup.5 are independently selected from —H, —C(O)Y, —C(O)Z, and groups of formula (II) ##STR00013## and R.sup.4 and R.sup.6 are independently selected from —H, —C(O)Y, —C(O)Z, and groups derived from glycosides, wherein Y is CH.sub.3(CH.sub.2).sub.m—, and m=0-35, and Z is CH.sub.3(CH.sub.2).sub.n—(CH═CH).sub.p(CH.sub.2).sub.q—, and n and q are independently 0-35, and p=1-4 or a pharmaceutically acceptable salt, solvate, prodrug, or protected derivative thereof.

2. The method of claim 1, wherein said subject is a human.

3. The method of claim 1, wherein said cancer disease is selected from the group of breast cancer, colon cancer, pancreatic cancer, stomach cancer, brain cancer, lung cancer, kidney cancer, liver cancer, ovarian cancer, cervical cancer, and thymus cancer.

4. The method of claim 3, wherein said cancer is a breast cancer carcinoma selected from the group of invasive ductal carcinoma, invasive lobular carcinoma, papillary carcinoma, mucinous carcinoma, medullary carcinoma and tubular carcinoma.

5. The method of claim 1, wherein said infection with an intracellular pathogen is an infection with a bacterial or protozoan microorganism.

6. The method of claim 5, wherein said microorganism is a bacterium from the genus Mycobacterium, Rickettsia, Brucella, Chlamydia, Listeria, Legionella, Coxiella, or Francisella, or a protozoa from the genus Leishmania.

7. The method of claim 6, wherein said method is for treating cutaneous leishmaniasis or tuberculosis.

8. The method of claim 1, wherein said compound is selected from the group of: ##STR00014## ##STR00015## ##STR00016## and pharmaceutically acceptable salts, solvates, prodrugs, and protected derivatives thereof.

9. The method of claim 1, wherein the treatment comprises the ex vivo pre-incubation of the compound with antigen presenting cells (APCs) of the subject and the subsequent re-infusion of the APCs into the subject.

10. Method for preparing a composition for inducing and/or enhancing an immune response, said method comprising (a) separating CD14+ monocytes from a blood sample obtained from a cancer (b) differentiating said CD14+monocytes into APCs; (c) incubating the APCs with a compound having the structure of the following formula (I): ##STR00017## wherein R.sup.1 is Y or Z, R.sup.2, R.sup.3 and R.sup.5 are independently selected from —H, —C(O)Y, —C(O)Z, and groups of formula (II) ##STR00018## and R.sup.4 and R.sup.6 are independently selected from —H, —C(O)Y, —C(O)Z, and groups derived from glycosides, wherein Y is CH.sub.3(CH.sub.2).sub.m—, and m=0-35, and Z is CH.sub.3(CH.sub.2).sub.n—(CH═CH).sub.p(CH.sub.2).sub.q—, and n and q are independently 0-35, and p=1-4 or a pharmaceutically acceptable salt, solvate, prodrug, or protected derivative thereof; and (d) optionally, purifying the APCs.

11. Method of claim 10, wherein said CD14+ monocytes are differentiated into dendritic cells (DCs) by the addition of IL-4 and/or GM-CSF.

12. Method of claim 10, wherein said incubation in step (c) is performed for a period of 2-5 hours.

13. Method of claim 10, wherein the incubation in step c) is performed with 10-100 μg/ml of said compound.

14. Compound selected from the group consisting of: ##STR00019## ##STR00020## ##STR00021## and pharmaceutically acceptable salts, solvates, prodrugs, and protected derivatives thereof.

15. Pharmaceutical composition comprising a compound according to claim 14:

16. Method of claim 10, wherein said cancer patient has breast cancer.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0078] FIG. 1 shows the intracellular production of cytokines IFN-γ, TNF-α, IL-17A and IL-4 in human NKT cells following stimulation with αGalCer and EhLPPG.

[0079] FIG. 2 shows the intracellular production of cytokines IFN-γ and IL-4 in human NKT cells following stimulation with αGalCer, EhLPPG, C30-EhPIa-cis and C30-EhPIb-cis. (A) Percentage of IFN-γ positive NKT cells. (B) Percentage of IL-4 positive NKT cells.

[0080] FIG. 3 shows the production of IFN-γ in murine spleen cells and sorted murine NKT cells following co-incubation with αGalCer, EhLPPG, C30-EhPIa-cis, C30-EhPIb-cis, C28-EhPIb and C30-EhPIb-trans pulsed BMDCs. (A) IFN-γ production in culture supernatant of stimulated spleen cells. (B) IFN-γ production in the culture supernatant of sorted murine NKT cells. IFN-γ was determined by ELISA.

[0081] FIG. 4 shows the results from liver toxicity measurements of EhLPPG und αGalCer in C57BL/6 mice. ALT levels measured in blood following four-time intraperitoneal injection of different amounts of αGalCer (A) und EhLPPG (B) are depicted.

[0082] FIG. 5 shows the influence of EhLPPG and αGalCer on the infection of macrophages with Leishmania major in vitro. (A) Percentage of L. major infected macrophages following addition of EhLPPG and αGalCer (4 μg/ml) (8 μg/ml). (B) Percentage of L. major infected macrophages following addition of EhLPPG (8 μg/ml), αGalCer (4 μg/ml) and spleen cells. (C) Leishmania counts per macrophage following addition of EhLPPG and αGalCer. (D) Leishmania counts per macrophage following addition of EhLPPG (8 μg/ml), αGalCer (4 μg/ml) and spleen cells. (E) Influence of EhLPPG and αGalCer on the expression of iNOS-mRNA. MΦ=macrophages; (*)=significant difference to uninfected macrophages; p=0.053 value to untreated L. major infected macrophages (statistics: unpaired Students t-test).

[0083] FIG. 6 shows the influence of EhLPPG and αGalCer on the infection of mice with L. major. (A) Infection and application scheme. (B) Increase of foot-pad size over time after local injection of EhLPPG. (C) Ulceration-free survival time after local injection of EhLPPG.

[0084] FIG. 7 shows the influence of EhLPPG- or αGalCer-pulsed BMDCs on tumor development in a mouse model for mammary cancer. (A) Induction of mammary cancer and therapeutic application scheme using EhLPPG- or αGalCer-pulsed BMDCs. (B) Kaplan-Meier curve for determining the therapeutic influence of EhLPPG- or αGalCer-pulsed BMDCs on the tumor free survival time. (C) Influence of therapeutic application of EhLPPG- or αGalCer-pulsed BMDCs on tumor growth (statistics: Mantel Cox T test).

[0085] FIG: 8 to 15 are referred to in the synthesis part below.

[0086] FIG. 16 shows the haemolytic activity of EhLPPG and the synthetic analogues C30-EhPIa-cis, C30-EhPIb-cis, C30-EhPIb-trans and C28-EhPIb.

[0087] FIG. 17 shows the specific actin expression of L. major in treated vs. untreated mice (time at ulceration/scarification). (A) Parasite burden in lymph nodes independent of ulceration and (B) at time point of ulceration.

[0088] FIG. 18 shows the reduction in actin expression of L. major relative to an untreated control (control=1) after treatment with EhLPPG and the synthetic analogues C30-EhPIa-cis, C30-EhPIb-cis, C30-EhPIb-trans and C28-EhPIb.

[0089] FIG. 19 shows the relative expression of actin of L. major in macrophages (1.5×10.sup.6 per well) infected with L. major promastigotes (1.2×10.sup.7).

EXAMPLES

[0090] Synthesis of EhPIa and EhPIb

[0091] Following the scheme in FIG. 8, the inositol phosphate part and the monoacyl glycerol part were independently prepared and both parts were linked together by using the Mitsunobu reaction similar to Xu et al. (2006). Acid removable protecting groups were used as semi-permanent protection for the hydroxy and phosphate groups, and the last step of the synthesis was their acidic cleavage. The inositol phosphate part was synthesized starting from the myo-inositol via regioselective phosphorylation of the inositol moiety with utilizing chiral phosphorylating reagents such as chiral binaphtol as chiral auxiliaries. C26 saturated fatty acid and C30 unsaturated fatty acids (30:1, cis and trans) were synthesized using a Ni catalyzed sp.sup.3-sp.sup.3 cross coupling reaction described by Iwasaki, T. et al. (2013); Chem. Eur. J. 2013, 19, 2956-2960.

[0092] Regioselective Phosphorylation

[0093] Regioselective phosphorylation was first investigated by using triol 3, which was derived from myoinositol with 4 steps (Table 1 in FIG. 9). To obtain perfect selectivity for asymmetric phosphorylation of myo-inositol derivatives, and the separation of enantiomers, the regioselective phosphorylation was adopted using chiral phosphorylating reagents. The methods may afford a mixture of diastereomers, which can be readily separated.

[0094] At first, R-BINOL was used as a chiral auxiliary. The phosphorylation using (R)-BPC (5) did not afford a good result in yield and selectivity (entry 1). Next, phosphoramidite method was used for phosphorylation. The reaction using (R)-BPA (6) in the presence of 1H-tetrazole as an activating reagent preferentially afforded the desired mono-phosphate 4 in 74% with regioselectivity 79:21 (desired 1-phosphate: undesired 3-phosphate) (entry 2). Reaction using highly reactive activating reagent HOBt-CF.sub.3 decreased the yield (entry 3). Reaction with phosphorylating reagent 7 having octahydroBINOL as a chiral auxiliary also decreased the yield (entry 4). In both cases, the amounts of di- or tri-phosphorylating by-products were increased. The structure of the desired 1-phosphate was confirmed by the X-ray crystallographic analysis with using the p-bromobenzoylated derivative (see FIG. 10).

[0095] Product 4 was a diastereomeric mixture. After introduction of Alloc groups on two hydroxy groups in 4, the desired 8 was readily obtained as a single diastereomer by simple silica-gel chromatography. After removal of Alloc groups of 8 by Ru-catalyst 9, Tanaka, S. et al. (2004), inositol phosphate ester 11 was synthesized via silylation and transesterification of BINOL to the benzyl ester (see Scheme 2 in FIG. 11).

[0096] Synthesis of Long-Chain Fatty Acids using a sp.sup.3-sp.sup.3 Coupling Reaction

[0097] Long-chain fatty acids in EhPIa and EhPIa were synthesized according to the method shown in Scheme 3 in FIG. 12. The sp.sup.3-sp.sup.3 coupling reaction of t-butyl ester of brominated fatty acid (C12) 12 with Grignard reagent 13 prepared from oleic acid (C18, cis) was carried out by using NiCl.sub.2, 1, 3-butadiene, NMP in THF to give long-chain fatty acid t-butyl ester 14 having cis-double bond Iwasaki, T. et al. (2013) Chem. Eur. J., 19, 2956-2960. After removal of the t-butyl ester in 14 under acidic condition, the product was coupled with glycerol derivative 15 and the subsequent removal of the silyl group gave monoacyl glycerol 16.

[0098] Synthesis of C30-EhPIa-Cis

[0099] One of the benzyl groups of inositol phosphate ester 11 was then removed with LiBr. The diester obtained was then coupled with monoacyl glycerol 16 by using Mitsunobu reaction to afford 17. Finally, removal of silyl protecting groups with TBAF and then p-methoxybenzyl (PMB), methoxymethyl (MOM), and benzyl (Bn) groups gave the total synthesis of C30-EhPIa-cis (la) (Scheme 4 in FIG. 13).

[0100] Synthesis of EhPIb (2a, 2b, 2c) Based on a New Allyl-Protecting Strategy

[0101] EhPIb, which shows selective cytokine induction, have a long-chain fatty acid and palmitic acid at 2-position on inositol. The selection of protecting groups on hydroxy functions therefore became more difficult. A new protecting group strategy was therefore employed for the synthesis of EhPIb. The cleavage of Allyl and Alloc groups on hydroxy functions using Ru catalyst 9 under mild neutral condition was reported previously. Here, a new strategy was developed in which Allyl and Alloc groups are used as semi-permanent protecting groups and Allyl and Alloc groups are simultaneously removed by using 9 at the final deprotection step.

[0102] Ru-catalyzed dehydrative allylation (Saburi, H. et al. (2005) Angew. Chem. Int. Ed., 44, 1730-1732) of a common synthetic intermediate 4 using 9 gave di-allylated 18. Transesterification of 18 afforded benzyl phosphate 19. Selective removal of PMB with DDQ and subsequent introduction of Alloc groups to the liberated hydroxy groups yielded 20. Afterwards, the 2-O-MOM group of 20 was cleaved by 50% TFA/CH.sub.2Cl.sub.2, and palmitic acid was introduced into the 2-position of the inositol moiety to give acylated inositol phosphate 21 (Scheme 5 in FIG. 14).

[0103] After removal of one of the benzyl groups on the phosphate in 21, the resulting compound was coupled with Allyl-protected monoacyl glycerol derivative 22 under Mitsunobu conditions to give compound 23. After removal of the other benzyl group on the phosphate in 23, final deprotection of all the allyl-type protecting groups was elucidated by using Ru catalyst 9. Initially, the reaction was carried out under neutral conditions. Ally groups were, however, not cleaved at all, whereas Alloc groups were readily removed under neutral conditions. Lithium phosphate formed after the removal of benzyl group might quench the proton required for the activation of catalyst 9 and hamper the cleavage reaction of ally group. Final deprotection of all the allyl-type protecting groups of 23 was thus carried out by using Ru catalyst 9 in the presence of TFA to successfully afford the desired C30-EhPIb-cis 2a in 88% yield. C30-EhPIb-trans 2b and C28-EhPIb 2c were also achieved in a similar manner (Scheme 6 in FIG. 15).

Example 1

Measuring Cytokine Production in Human NKT

[0104] This NKT cell assays uses whole PBMCs. The complete cells are activated by the stimulating compounds and FACS analysis allows detecting at the same time (a) the different NKT cell subpopulations and (b) intracellular cytokine production by these cells. This differs from the mouse NKT cell assay, where BMDCs are stimulated and then NKTs are added.

[0105] PBMCs from buffy coats or fresh blood samples from blood donors were used for intracellular cytokine analysis of NKT cells following stimulation with a phosphatidylinositol compound of the invention, for example the analogs C30-EhPIa-cis and C30-EhPIb-cis, by flow cytometry (Sandberg et al. 2003). Buffy coats for the isolation of PBMCs were kindly provided by the Department of Transfusion Medicine of the University clinic Hamburg-Eppendorf. All experiments were approved of by the ethical review committee of the Hamburger Arztekammer (PV3551).

[0106] In short, blood was diluted 1:2 with PBS at room temperature (RT). Biocoll (Biochrom AG) was carefully overlayed with the blood/PBS mixture and centrifuged for 30 min, RT, 1500 rpm without break. After centrifugation the leukocyte ring was removed and transferred into a new falcon tube. Here, two fractions were pooled. The leukocytes were washed with sterile PBS and centrifuged for 20 min, 4° C. and 1200 rpm. The supernatant was discarded, thrombocytes removed. Then, the cell pellets were once again pooled and washed with PBS for 12 min, 4° C., 1200 rpm. Subsequent to centrifugation, PBMCs were resuspended in 1 ml X-VIVO 15 supplemented with Pen/Strep (LONZA) or sterile PBS. PBMC were then used in the human iNKT cell assay.

[0107] For the human iNKT cell assay, a modified iNKT cell cytokine expression assay by Sandberg et al. (2003) was used. In brief, 1×10.sup.6 human PBMC/well were cultured in quadruplicates in 96-well round bottom plates with X-VIVOTM 15 supplemented with Pen/Strep (LONZA). Cells were pulsed with 1 μg/ml αGalCer, 10 μg/ml purified EhLPPG or 1-10 μg/ml synthetic EhPI anchors. As a co-stimulant, 3 μg/ml purified αCD28 was added per well for optimal iNKT cell activation without additional APC. Cells were incubated for 15h at 37° C., 5% CO.sub.2. After one hour, 10 μg/ml Brefeldin A was added to the culture to stop golgi transport.

[0108] After incubation cells were harvested and iNKT cells were analyzed for their cytokine production of IFNγ-PE/Cy7, TNFα-FITC, IL-4-PE and IL-17A-BV421. iNKT cells were stained with CD3-PerCP and TCR Vα24-Jα18-APC (all antibodies were obtained by BioLegend). FACS was performed using a FACS LSRII device (BD Bioscience).

[0109] The results are shown in FIGS. 1 and 2. It can be seen that stimulation of human NKT cells with αGalCer or EhLPPG resulted in considerable amounts of CD4+NKT cells positive for IFN-γ and IL-4. However, in contrast to EhLPPG, αGalCer led to the production of tumor necrosis factor (TNF) α and IL-17, cytokines that may exhibit immunopathological functions when used for immunotherapeutic purposes (see FIG. 1).

[0110] When using the two synthetic EhPI analogs C30-EhPIa-cis and C30-EhPIb-cis for stimulation, even higher percentages of CD4+NKT+IFNα+ cells were obtained compared to stimulation with αGalCer. In contrast to αGalCer, and similar to EhLPPG, the synthetic analogs induced more CD4+NKT+IL-4+ cells compared to αGalCer (see FIG. 2).

Example 2

Measuring Cytokine Production in Murine NKT

[0111] To test whether not only human cells, but also murine cells are stimulated by EhLPPG or the synthetic EhPI analogs, an NKT cell assay was performed with murine cells.

[0112] For the isolation of murine spleen cells 8 to 10-week-old mice were sacrificed and the spleens removed. The spleen was rinsed with RPMI 1640 (10% FCS, 1% L-Glutamine, 1% Sodium pyruvate, 50 μg/ml gentamycin) until it became translucent. The cell suspension was transferred to a falcon tube and centrifuged for 8 min, 4° C., 1200 rpm. Following centrifugation, cell pellets were adjusted to the appropriate number. For the isolation of T cells, spleen cells were subjected to magnetic bead sorting using the Pan T cell isolation kit II (MACS Miltenyi). Isolated T cells were directly used for the murine iNKT cell assay or for sorting of iNKT cells with αGalCer-CD1d-Tetramer-PE (kindly provided by the NIH-tetramer facility).

[0113] For cell sorting of murine iNKT cells, T cells isolated from the spleen as described above were stained with αGalCer-CD1d-Tetramer-PE for 30 min, 4° C. Washed twice with PBS at 1200 rpm, 4° C., 4 min and sorted with FACS AriaTM II (BD Biosciences).

[0114] BMDCs were used as antigen presenting cells (APC). In brief, 2-5×10.sup.4 cells/well were cultured in duplicates or triplicates in 96-well round bottom plates with RPMI 1640 (10% FCS, 1% L-Glutamine, 1% Sodium pyruvate, 50 μg/ml gentamycin). Cells were pulsed with 0.1-1 μg/ml αGalCer, 20 μg/ml purified EhLPPG or 0.001-1 μg/ml synthetic EhPI anchors. iNKT enriched lymphocytes or sorted iNKT cells were then added to the pulsed APC with 2×10.sup.4-1×10.sup.5 cells/well and incubated for 48 h. IFN-γ was measured by ELISA (R&D Systems).

[0115] The results are shown in FIG. 3. It was observed that also murine NKT cells are stimulated with the synthetic EhPI analogs. IFN-γ production was induced by all four synthetic analogs in spleen cell preparations in a dose dependent manner (see FIG. 3A). IFN-γ production was likewise observed in sorted murine NKT cells (see FIG. 4B). The results clearly demonstrate that the use of murine models is permissible to investigate the therapeutic potential of EhLPPG and its synthetic analogs.

Example 3

Measuring Liver Toxicity

[0116] It was examined whether the intraperitoneal injections of EhLPPG and αGalCer are toxic for the liver of test animals. Different amounts of EhLPPG (1, 10, 25 and 50 μg/ml) and αGalCer (0.1, 1 and 10 μg/ml) were administered intraperitoneally into C57BL/6 mice. Subsequently, the alanine aminotransferase (ALT) levels were determined in the mouse serum (diluted 1:10 in H20) using the Cobas Integra® 400 plus Analyzer, Roche.

[0117] FIG. 4 shows the results obtained from ALT determination. It can be seen that even high and frequent doses of EhLPPG do not induce an increase of the liver ALT enzyme in liver cells of mice. In contrast, a significant increase in the ALT levels was detected in mice that were treated with αGalCer.

Example 4

In Vitro Leishmania Assay

[0118] It was investigated whether EhLPPG is able to influence intracellular infection of macrophages with Leishmania major parasites directly or indirectly by additionally adding immune cells to an in vitro infection assay.

[0119] Bone marrow derived macrophages (BMM) were harvested from tibias and femurs of 6 to 10 week-old female BALB/c mice and cultured for 10 days in Iscoves Modified Dulbecco's Medium (IMDM) supplemented with 10% FCS, 5% horse serum, 30% supernatant of L929 cells and antibiotics. In 8-well chamber slides (Nunc®) 2×10.sup.5 BMM/well were cultured in duplicates in IMDM without L929 cell supernatant and infected with promastigote L. major ASKHS in a 1:10 ratio for 4h 37° C., 5% CO.sub.2. After infection, BMMs were washed twice with warm PBS and 1 μg/ml αGalCer or 4 μg/ml purified EhLPPG was added with and without 4×10.sup.5 spleen cells in IMDM. The assay was incubated for 48 h, 37° C., 5% CO.sub.2. Following incubation, cells were washed twice with warm PBS and stained with DAPI for calculating the parasite burden of the BMMs. BMMs were washed with wash buffer (1× PBS, 0.01 Triton-X-100), permeabilized (50 nM NH.sub.4Cl, 1× PBS, 0.1% Triton-X-100) and stained for 1 h, RT with 1 μg/ml DAPI diluted 1:50 in blocking solution (2% w/v BSA in 1× PBS, 0.1% Triton-X-100). After staining, BMMs were washed with wash buffer and a cover slip was fixed with Mowiol (25% glycerol, 0.1 M Tris-HCl (pH 8.5), 10% w/v Mowiol 4-88) on the chamber slides. Parasite burden was estimated by counting kinetoplasts per macrophage.

[0120] As shown in FIG. 5, it was found that both EhLPPG and αGalCer significantly influenced the intracellular L. major infection rate. Addition of EhLPPG to the culture led to a considerable reduction in the percentage of infected macrophages (see FIG. 5A) that became significant when spleen-derived lymphocytes were co-incubated (see FIG. 5B). The percentage of infected macrophages was significantly reduced in both cases following treatment of the cell culture with αGalCer (see FIGS. 5A and 5B).

[0121] Regarding the influence of EhLPPG treatment on the parasite load per macrophage, it was found that treatment with EhLPPG, as with αGalCer, significantly reduced the amount of parasites within the macrophages (see FIG. 5C). Addition of spleen cells led to reduction in the amounts of intracellular parasites when EhLPPG was added to the culture and a significant reduction when αGalCer was added to the culture (see FIG. 5D). From the literature it is known that the inducible nitric oxide synthase (iNOS) plays a crucial role in the growth-control of intracellular Leishmania parasites. Preliminary results now suggest that EhLPPG and presumably also αGalCer lead to increase in the expression of iNOS thus leading to a decrease in the parasite load of macrophages (data not shown).

Example 5

Animal Model for Cutaneous Leishmaniasis

[0122] The influence of EhLPPG and αGalCer treatment was investigated in an animal model for cutaneous leishmaniasis. The infection and application scheme is shown in FIG. 6A.

[0123] 10 to 14-week old female BALB/c mice were infected subcutaneously (s.c.) into the foot pad of the right hind leg with 2×10.sup.5 promastigote L. major ASKHS in 50 μl PBS. With developing foot pad swelling, mice were treated s.c. once a week with either 4 μg/ml or 8 μg/ml purified EhLPPG in 25 μl PBS or with PBS only as a control. Foot pad swelling was estimated twice a week with a caliper. According to the guidelines of the ethic commission, mice were sacrificed at the time point of lesion ulceration.

[0124] The results are shown in FIG. 6B and 6C. Beginning from the second application on, treated mice showed a significant (4 μg/ml: p<0.05, 8 μg/ml: p<0.05) reduction and deceleration in the size of the footpads that became significantly different from PBS treated mice at day 46 after infection. At this time point, all PBS treated mice had to be sacrificed due to ulceration of Leishmania lesions. In the following, mice treated with 4 or 8 μg/ml purified EhLPPG showed a continuous increase in the size of the footpads without signs of ulceration. FIG. 6C shows that the onset of the footpad swelling was significantly delayed in EhLPPG treated mice (4 μg/ml: p<0.005, 8 μg/ml: p<0.005) compared to PBS treated control mice.

Example 6

Animal Model for Breast Cancer

[0125] It was examined whether EhLPPG- or αGalCer-pulsed BMDCs could influence tumor growth in a mouse model for mammary cancer. Female BALB/c mice (WAP-T-NP8) were injected with the tumor cell line H8N8 resulting which induces the development of a singular tumor within 4-6 weeks. See Wegwitz et al., 2010. Determination of a therapeutic effect can be estimated by the time of the tumor free survival as well as growth rate and size of a tumor. The treatment scheme is shown in FIG. 7A. The mice were injected intraperitoneally with BMDCs pre-incubated for 3 hours with either 4 μg EhLPPG/ml or 1 μg αGalCer/ml on day 7 and 14 post implantation. As control, two groups of mice received either PBS or BMDCs alone (FIG. 7A).

[0126] The results are shown in FIG. 7B. It can be seen that the treatment with EhLPPG-pulsed BMDCs, but not αGalCer-pulsed BMDCs, significantly increased the tumor free survival time compared to mice that had received PBS or BMDCs alone. Furthermore, the tumor growth was significantly reduced in mice that had been treated with EhLPPG-pulsed BMDCs compared to PBS controls within the first 7 days following a second therapeutic intervention. However, treatment with BMDCs alone also resulted in a decrease of the tumor growth within the same period. At later time points, again no difference in the tumor growth was observed between EhLPPG-pulsed and non-stimulated BMDCs but the tumor sizes were reduced compared to PBS treated controls. Most interestingly, treatment with αGalCer-pulsed BMDCs initially reduced the tumor growth in comparison to PBS controls but tumor growth was enhanced compared to EhLPPG-pulsed BMDCs and BMDCs at later time points (FIG. 7C).

Example 7

Hemolytic Activity of EhLPPG and Synthetic Analogues C30-EhPIa-Cis, C30-EhPIb-Trans and C28-EhPIb

[0127] EhLPPG and the synthetic analogues C30-EhPIa-cis, C30-EhPIb-trans and C28-EhPIb were diluted in PBS buffer and distributed in 96-well plates in concentrations ranging from 0.1-10 μg/ml. An equal volume of red blood cells (RBC) obtained from EDTA blood from healthy blood donors was added.

[0128] After 1 h of incubation at 37° C., the RBC were centrifuged at 800× g for 10 min The absorbance of the supernatant was measured at 540 nm (reference filter 620 nm). The percentage hemolytic activity of each analogue at different concentrations was estimated as % hemolytic activity=(A-A.sub.0/A.sub.max-A.sub.0)×100, where A.sub.0 is the background hemolysis obtained by the incubation of RBC with PBS and A.sub.max is the 100% hemolysis achieved upon incubation of RBCs in distilled water. The results are shown in FIG. 16.

Example 8

Specific Actin Expression of L. Major in Treated vs. Untreated Mice

[0129] 10 to 14-week old female BALB/c mice were infected subcutaneously (s.c.) into the foot pad of the right hind leg with 2×10.sup.5 promastigote L. major ASKHS in 50 μl PBS. With developing foot pad swelling, mice were treated s.c. once a week with either 1, 4 or 8 μg/ml purified EhLPPG in 25 μl PBS or with PBS only as control. According to the guidelines of the ethic commission, mice were sacrificed at the time point of lesion ulceration. Lymph nodes were dissected and submitted to gDNA isolation. To measure the parasite load in the lymph nodes, a Probe-PCR was performed to estimate the relative L. major actin expression. The results are shown in FIG. 17.

Example 9

Reduction in Actin Expression of L. Major Relative to an Untreated Control (Control=1) After Treatment with EhLPPG and Synthetic Analogues C30-EhPIa-Cis, C30-EhPIb-Trans and C28-EhPIb

[0130] Bone marrow derived macrophages (BMM) were harvested from tibias and femurs of 6 to 10 week-old female BALB/c mice and cultured for 10 days in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 10% FCS, 5% horse serum, 30% supernatant of L929 cells and antibiotics. In 8-well chamber slides (Nunc®) 2×10.sup.5 BMM/well were cultured in duplicates in IMDM without L929 cell supernatant and infected with promastigote L. major ASKHS in a 1:8 ratio for 4 h, 37° C., 5% CO.sub.2. The assay was incubated for 48 h, 37° C., 5% CO.sub.2. Following incubation, cells were washed twice with warm PBS and were submitted to gDNA isolation. To measure the parasite load in BMM a Probe-PCR was performed to estimate the relative L. major actin expression. Moreover, the reduction in actin expression of L. major relative to the untreated control (control=1) after treatment was calculated. The results are depicted in FIG. 18.

Example 10

Relative Expression of Actin of L. Major in Macrophages (1.5×10.SUP.6 .per well) Infected with L. Major Promastigotes (1.2×10.SUP.7.)

[0131] Bone marrow derived macrophages (BMM) were harvested from tibias and femurs of 6 to 10 week-old female BALB/c mice and cultured for 10 days in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 10% FCS, 5% horse serum, 30% supernatant of L929 cells and antibiotics. In 8-well chamber slides (Nunc®) 2×10.sup.5 BMM/well were cultured in duplicates in IMDM without L929 cell supernatant and infected with promastigote L. major ASKHS in a 1:8 ratio for 4h 37° C., 5% CO.sub.2. The assay was incubated for 48 h, 37° C., 5% CO.sub.2. Following incubation, cells were washed twice with warm PBS and were submitted to gDNA isolation. To measure the parasite load in BMM a Probe-PCR was performed to estimate the relative L. major actin expression. The results are depicted in FIG. 19.

LITERATURE

[0132] Arias J L (2013), Liposomes in drug delivery: a patent review (2007-present), Expert Opin Ther Pat. 23(11):1399-414.

[0133] Barros N B, et al. (2013) Liposomal-lupane system as alternative chemotherapy against cutaneous leishmaniasis: macrophage as target cell. Experimental parasitology 135: 337-343.

[0134] Blum J, et al. (2012) Local or systemic treatment for New World cutaneous leishmaniasis? Re-evaluating the evidence for the risk of mucosal leishmaniasis. International health 4: 153-163.

[0135] Ishikawa A, et al. (2005) A phase I study of α-galactosylceramide (KRN-7000)-pulsed dendritic cells in patients with advanced and recurrent non-small cell lung cancer. Clinical Cancer Research 11(5): 1919-7.

[0136] Iwasaki, T. et al. (2013); Chem. Eur. J. 19, 2956-2960.

[0137] Laurent X, et al. (2014) Switching Invariant Natural Killer T (iNKT) Cell Response from Anticancerous to Anti-Inflammatory Effect: Molecular Bases. Journal of medicinal chemistry 57: 5489-5508.

[0138] Lotter H, et al. (2009), Natural killer T cells activated by a lipopeptidephosphoglycan from Entamoeba histolytica are critically important to control amebic liver abscess. PLoS Pathog 5: e1000434.

[0139] Lotter H, et al. (2013), Testosterone increases susceptibility to amebic liver abscess in mice and mediates inhibition of IFNgamma secretion in natural killer T cells. PloS one 8: e55694.

[0140] Neumayr A L, et al. (2013) Clinical aspects and management of cutaneous leishmaniasis in rheumatoid patients treated with TNF-alpha antagonists. Travel medicine and infectious disease 11: 412-420.

[0141] Sandberg J K, et al. (2003) Dominant effector memory characteristics, capacity for dynamic adaptive expansion, and sex bias in the innate Valpha24NKT cell compartment. Eur J Immunol 33(3): 588-96.

[0142] Sosa N, et al. (2013) Randomized, double-blinded, phase 2 trial of WR 279,396 (paromomycin and gentamicin) for cutaneous leishmaniasis in Panama. The American journal of tropical medicine and hygiene 89: 557-563.

[0143] Tanaka, S. et al. (2004) Org. Lett., 4, 1873-1875.

[0144] Uchida T, et al. (2008) Phase I study of alpha-galactosylceramide-pulsed antigen presenting cells administration to the nasal submucosa in unresectable or recurrent head and neck cancer, Cancer Immunol Immmunother. 57: 337-345.

[0145] Wegwitz F, et al. (2010) Tumorigenic WAP-T mouse mammary carcinoma cells: a model for a self-reproducing homeostatic cancer cell system. PloS one 5: e12103.

[0146] Xu, Y J et al (2006) J. Org. Chem., 71, 4919-4928.