MOLECULES AS IMMUNOMODULATOR

Technical Field of the Invention

The invention is related to obtaining saponin based novel adjuvants from triterpene glycosides via semi-synthesis studies that can be used in veterinary, prophylactic and therapeutic human vaccines and presenting these to the pharmaceutical/health sector for preclinical and clinical studies after asserting their immunomodulator mechanisms of action.

Known State of the Art (Prior Art)

Saponins that are secondary metabolites, prevalently found in vegetable or marine organisms, that comprise glucose groups on triterpenic or steroidal scaffolds, have found use in several commercial applications in the pharmaceutical or food sector. Saponins are amphiphilic molecules as they contain lipid soluble aglycones and water soluble glucose units and they are generally used as surface active agents. They have biological activities such as anti inflammatory, adjuvant, anti-fungal, antiparasitic, anti-viral, haemolytic activities.

The potentials of saponins as adjuvants have started with studies carried out on Quillaja saponins. Quil-A which is the water extract of the Quillaja saponaria plant, has been used in veterinary vaccines for many years due to its properties to augment humoral and cellular immune responses effectively, however its side effects have restricted its usage in human vaccines. QS-21 has been purified from Quil-A which comprises QS-7, QS-17, QS-18, QS-21 fractions, using reverse phase chromatography. QS-21 is structurally consist of four units: central triterpene, branched trisaccharides extending from C3 and C28 positions and an unstable acyl group with a sugar residue, which is susceptible to hydrolytic cleavage, on the linear C-28 tetrasaccharide section. QS-21 has been subjected to advanced activity tests as it exhibited lower toxic profile and higher abundance in the plant material in comparison to other sub-fractions. As a result of these studies, it has been reported that QS-21 adjuvant generally induced a balanced IgG1/IgG2a antibody response, a Thl mediated cellular response such as IFN-g, IL-2 and cytotoxic T cell response. As it could induce cellular immune response in comparison to alum which is an adjuvant that has been used for many years in human vaccine, QS-21 has been accepted as a valuable adjuvant.

The mechanism of action of QS-21 is generally explained with the formation of pores in the membrane after it binds to a cholesterol in the cell membrane and thereby increased the antigen uptake and presentation. However in the prior art, it can be seen that the activity is linked with inflammasome activation, lysosomal destabilization, cathepsin B release, and lipid body formation. Mouse BMDC and BMDMs have been treated with QS-21 (10 mg/ml and 2 mg/ml) and QS-21+MPLA (5 mg/ml) (monophosphoryllipid A) for 6 hours and IL-Ib secretions have been examined. While QS-21 did not increase IL-Ib production in dendritic cells and macrophages on its own, when applied with TLR4 agonist MPLA it has provided caspase1/11 and NLPR3 dependent IL-Ib secretion. Moreover, saponins such as QS-21, digoxin, sapindoside A, hedaracoside C and b-escin have been applied to macrophages in the presence of MPLA in order to investigate if inflammasome activation is a general phenomenon for saponins. Only QS-21 has produced IL-Ib response and it has been emphasized that inflammasome activation could be specific to Quillaja saponins.

The disadvantages of the QS-21 molecule: it leads to toxicity, it has haemolytic activity, it has low chemical stability as it undergoes hydrolysis in aqueous systems and it has low isolation yield from plant. Saponin formulations and semi-synthesis studies have been carried out in order to reduce toxicity of QS-21 and increase its stability.

ISCOM and ISCOMATRIX are cage like particles of 40 nm in sized containing saponins, cholesterol and phospholipids. ISCOMATRIX can induce humoral and cellular immune responses. Subcutaneous injection increases the expressions of pro-inflammatory cytokines such as IL-6, IL-8, and IFN-g . It has been reported that when human dendritic cells were treated with ISCOMATRIX adjuvants, CD86 and MHC II were weakly expressed, however, during in vivo application, the expression of these markers increased in dendritic cells within lymph nodes. The parenteral application of ISCOMATRIX to mice provided balanced Th1/Th2 cytokine response (IL-2, IL-4, IFN-g) and IgG1 and IgG2a antibody response. Moreover, ISCOMATRIX induces CD4 ⁺ and CD8 ⁺ T cell response. Moreover, in vivo applications of HPV16 have been carried out by being formulated with E6 and E7- ISCOMATRIX. NY-ESO-1 protein has been given to mouse and human monocyte derived dendritic cells and it has effectively induced NY-ESO-1 specific CD4 ⁺ and CD8 ⁺ T cell response. In the patent document numbered US20070190072A1 in which the in vivo activity of NY-ESO-1 antigen with ISCOMATRIX is evaluated, it has been reported that ISCOMATRIX induced CD4 and CD8 T cell response. The company GSK is developing adjuvant systems coded as ASOx series containing immunostimulant agents (MPL- monophosphoryllipid A, CpG) and QS-21. Adjuvant systems that comprise QS-21 are AS01 (liposome, QS-21, MPL), AS02 (QS-21, MPL, squalene), AS15 (liposome, QS-21, MPL, CpG). AS01 system has been used in assays in order to increase effective CD8 T cell response in vaccines such as malaria, tuberculosis, HIV, dengue fever, and influenza; and AS02 has been used in research tuberculosis, malaria, HIV, melanoma in relation to inducing strong humoral and T cell mediated immune response; AS 15 system, on the other hand, has been used in clinical trials for research related to melanoma, breast, prostate, lung and bladder cancer.

Several semi-synthesis studies have been carried out on the QS-21 molecule in the recent years besides adjuvant systems, semi-synthesis products have been given to mice subcutaneously with different antigens (MUC1, ganglioside GD3, KLH, OVA etc.) and activities have been evaluated over antibody response. GPI-0100, which is an analogue of QS-21, is a molecule that has been synthesized through elimination of the acyl group on QS- 21 molecule and binding of dodecylamine to glucuronic acid via carboxylic acid. In an in vivo study where OVA antigen is used, GPI-0100 has provided IL-2, IFN-g, IgG2a production that is a Thl mediated immune response and cytotoxic T lymphocyte (CTL) response. The effect of the GPI-0100 molecule in the pandemic influenza vaccine (H1N1) has been examined and it has been shown that it creates a Thl mediated immune response together with the antibody response. By means of this approach, a molecule that has antibody, T cell and CTL response, lower toxicity than QS-21 and that is more stable has been obtained. Some molecules, which lipophilic groups are conjugated when obtaining the semi-synthetic analogs of triterpene saponins, and some patents related to their immunomodulator /adjuvant effects can be listed as follows: analogs containing lipophilic groups on bidesmosidic saponin derivatives having a triterpene backbone (US6262029B1), immunomodulator effects of triterpene saponin analogues carrying lipophilic groups (US5977081A), effect of GPI-0100 adjuvant on M109 tumour cells (US20030198643A1), the effect of GPI-0100 adjuvant in influenza recombinant vaccine (US20070042002A1), effect of GPI-0100 adjuvant in Ebola vaccine (US20070082011A1), effect of GPI-0100 adjuvant in West nil subunit vaccine (WO2006115548A2), effect of gypsogenin amide derivative in Marburg virus

(US8883170B2).

At the state-of-art, Astragaloside IV used as lead compound and its carboxylic acid form obtained by TEMPO reaaction has been investigated in terms of its cardioprotective activity. Utilization of QS-21, golden standard of saponin based adjuvants, in human vaccines is limited due to its chemical instability in aqueaous environment, difficult and low isolation yield from the plant material, hemolytic activity, dose-dependent toxicity. In large scale productions, insufficient supply of QS-21 is caused problems despite its high activity. Therefore, it is necessary to develop of saponin-based adjuvants, which are stable, cost- effective, producible in large scale, having low toxicity, inducing antigen specific humoral and cellular immune responses and, clarify the immunomodulator action mechanisms of Astragalus saponins.

At the state-of-art, there is no saponin compound isolated form Astragalus or its analogs whether used in clinical trials as adjuvant or immunomodulatory agent nor investigating their immunomodulatory action mechanisms in terms of dendritic cell maturation and T cell activation.

Brief Description of the Invention and its Aims

The AST VII, which is a lead compound, is more advantageous adjuvant in comparison to QS-21 as AST VII can be easily dissolved in physiological water, it has high stability, low haemolytic effect even at high concentrations, as it can be lyophilised, it has low molecular weight (946 g/mol), it has an isolation yield of 0.3% and it has exhibited cellular and humoral immune responses in previous studies.

The molecule, and/or pharmaceutically acceptable salts and/or solvates, illustrated in Formula I that has been synthesized from Astragaloside VII (AST VII) constitute the subject matter of the invention.

W: =O

Y group is selected from O-H or C12H5N or C4-C30 straight or branched alkyl chains or C ₄ - C ₃₀ straight or branched alkenyl groups substituted with 1-4 hydroxy groups, or C ₁ -C ₁₆ alcoxy, carboxy or mercapto groups or C ₆ -C ₂ 4 fatty acids or C ₇ -C ₁₈ fatty acids or saturated fatty acids such as lauric, myristic, palmitic, stearic, arachide, behenic acid, or unsaturated fatty acids such as palmitoleic, oleic, linoleic, linolenic and arachidonic acid or aliphatic amines or aliphatic alcohols or aliphatic mercaptanes or aliphatic groups having a carbon atom between 6-24 saturated or unsaturated carbons with straight or branched chains, or aliphatic groups having a carbon atom of 6-20 or aliphatic groups having a carbon number of 8-16 carbons.

Z group is selected from O-H or C ₁₂ H ₅ N or C ₄ -C ₃₀ straight or branched alkyl chains or C ₄ - C ₃₀ straight or branched alkenyl groups substituted with 1-4 hydroxy groups, or C ₁ -C ₁₆ alcoxy, carboxy or mercapto groups or C ₆ -C ₂₄ fatty acids or C ₇ -C ₁₈ fatty acids or saturated fatty acids such as lauric, myristic, palmitic, stearic, arachide, behenic acid, or unsaturated fatty acids such as palmitoleic, oleic, linoleic, linolenic and arachidonic acid or aliphatic amines or aliphatic alcohols or aliphatic mercaptanes or aliphatic groups having a carbon atom between 6-24 saturated or unsaturated carbons with straight or branched chains, or aliphatic groups having a carbon atom of 6-20 or aliphatic groups having a carbon number of 8-16 carbons.

In a preferred structure of the invention, the Y and Z groups are OH or C ₁₂ H ₅ N groups. Y and Z can be OH or C ₁₂ H ₆ N at the same time or they can be different.

AST VII and analogs (DC-AST VII and DAC-AST VII) can potentially be used as immunomodulator agents against diseases such as cancer, tuberculosis, malaria and AIDS that cellular immune response is important and, there is no effective treatment, by increasing IL- 1b and IL-17A cytokine level, inducing the markers like MHC II, CD86, CD80 that are role in dendritic cell maturation and T cell activation and stimulating of CD4 ⁺ / CD8 ⁺ T cell response.

DAC-AST VII can be used as cytotoxic and immunomodulatory agent in cancer immunotherapy because of inducing high amount of IL-Ib secretion, effective CD8 ⁺ T cell response and causing cytotoxicity on tumor cell lines.

Not only more potent compounds can be obtained by applying advanced modifications on DC-AST VII, but also advanced studies can be carried out in order to reveal the relation of structure-activity and to identify specific gene/transcription factors/pathways that target molecules.

Definition of the Figures of the Invention

The figures that have been prepared in order further describe the chemical structures of AST VII and analogs and, their immunomodulator mechanisms of action have been defined below.

Figure 1: Chemical structures of AST VII and DC- AST VII

Figure 2: Chemical structures of Dicarboxylic AST VII (DC-AST VII) and Dodecylamine Conjugated AST VII (DAC-AST VII)

Figure 3: IL-Ib response obtained following the co-stimulation of PMA-ionomycin with AST VII and analogs in the first trial

Figure 4: IL-17A response obtained following the co-stimulation of PMA-ionomycin with AST VII and analogs in the first trial

Figure 5: IL-Ib response obtained following the co-stimulation of PMA-ionomycin with AST VII and analog in the second trial

Figure 6: IL-2, IFN-g and TNF-a cytokine responses obtained following the co-stimulation of PMA-ionomycin with AST VII and analogs in the second trial

Figure 7: IL-Ib secretion following the co-stimulation of LPS with AST VII and analogs in BMDCs

Figure 8: IL-Ib secretion following the co-stimulation of LPS with AST VII and analogs in BMDMs

Figure 9: The maturation markers of CD11c ⁺ MHC II ^high BMDCs treated with LPS (10 ng/ml) and AST VII (2, 5, 10 mM). a) MHC II expression, b) CD86 expression c) CD80 expression d) IL-12 production

Figure 10: The maturation markers of CD11c ⁺ MHC II ^high BMDCs following treatment with DC-AST VII (2, 5, 10 mM) and DAC-AST VII (2, 5, 10, 20 pM). a) MHC II expression, b) CD86 expression c) CD80 expression

Figure 11: CD44 activation marker expressions in CD8 ⁺ and CD4 ⁺ T cells Definitions of the parts/aspects/sections forming the invention

The parts and sections provided in the figures that have been prepared in order to further describe the chemical structures and synthesis of the analogs (DC-AST VII and DAC-AST VII) that have been synthesized from AST VII which is the lead compound that has been developed by this invention, have each been numbered and the description of each reference number has been given below.

1. Astragaloside VII (AST VII)

2. Dicarboxylic AST VII (DC- AST VII)

3. Dodecylamine Conjugated AST VII (DAC- AST VII)

Detailed Description of the Invention

1. Synthesis of Dicarboxylic AST VII (DC-AST VII) (2) (Figure 1):

1-2 mmol AST VII (1) (preferably 1 mmol) and 0.5-4 mmol (preferably 1.06 mmol) NaBr were dissolved in 5-10 mL (preferably 10 mL) distilled water, which is adjusted pH to 11 (with 1 N NaOH). 0.1-4 mmol (preferably 0.2 mmol) TEMPO reagent was added and mixed under nitrogen and, 1-10 mmol (preferably 4.66 mmol) NaOCl was added dropwise in reaction mixture. The reaction was continued under reflux at 0°C for 6 hours and the progress in the reaction was followed by thin layer chromatography (TLC). The reaction that was neutralized with 1 M HC1 was evaporated in rotary evaporator at 50°C. The reaction mixture was applied as a liquid form into reverse phase (C-18) vacuum liquid chromatography conditioned with water and, methanol (MeOH): water elution was maintained at ratios of 15:85; 20:80; 35:65; 40:60; 50:50; 55:45. The further purification of DC-AST VII (2) enriched fraction was continued in water conditioned reverse phase (C-18) vacuum liquid chromatography and, the column was maintained with acetonitrile (ACN): water elution of 5:95, 10:90. DC-AST VII (2) was obtained in 40-70% yield.

The chemical structure of DC-AST VII (2) was elucidated using mass spectroscopy and NMR. HR-ESI-MS m/z 995.38897 ([M+Na-2H]-) C ₄₇ H ₇₂ O ₂₁ Na = 995.444. ¹ H-NMR (400 MHz, D ₂ O) d 4.72 (1H, dd, J= 6, 4.5 Hz , H-16), 4.70 (1 H, d, J= 8.1 Hz , H-1’”), 4.56 (1 H, d, J= 7.9 Hz , H-1”), 4.50 (1 H, d, J= 7.8 Hz , H-1’), 3.97 (1 H, dd, J= 15.5, 6.4 Hz, H-24), 3.96 (1 H, d, J= 5.8 Hz, H-5’), 3.73 (2 H, s, H-5”, H-5’”), 3.69 (1 H, d, J= 5.3 Hz, H-6), 3.68 (1 H, d, J= 9.1 Hz , H-4’), 3.56 (1 H, d, J= 4.1 Hz , H-4”), 3.54 (1 H, d, J= 2.7 Hz, H-3’), 3.52 (1 H, d, J= 2.9 Hz, H-3”), 3.50 (1 H, s, H-3’”), 3.39 (2 H, d, J= 4.4 Hz, H- 2’, H-3), 3.33 (1 H, s, H-5’), 3.31 (1 H, d, J= 6.2 Hz, H-2”), 3.30 (1 H, d, J= 8.5 Hz, H-2’”), 3.56 (1 H, d, J= 4.1 Hz, H-4’”), 2.46 (2 H, d, J= 8 Hz, H-17, H-22), 2.16 (1 H, s, H-23), 2.12 (2 H, d, J= 9.2 Hz, H-23, H-11), 2.08 (1 H, m, H-15), 1.97 (1 H, d, J= 7.7 Hz, H-2), 1.95 (1 H, d, J= 7.7 Hz, H-7), 1.84 (1 H, d, J= 12.2 Hz, H-8), 1.75 (1 H, d, J= 12.5 Hz, H-22), 1.74 (1

H, d, J= 12.5 Hz, H-12), 1.68 (1 H, s, H-5), 1.67 (2 H, s, H-1, H-2), 1.64 (1 H, s, H-12), 1.45 (1 H, d, J= 10.1 Hz, H-7), 1.44 (1 H, d, J= 10.1 Hz, H-15), 1.39 (3 H, s, H-26), 1.32 (3 H, s, H-28), 1.30 (3 H, s, H-27), 1.29 (6 H, s, H-21, H-18), 1.22 (1 H, s, H-1), 1.16 (1 H, s, H-11),

1.06 (3 H, s, H-29), 1.01 (3 H, s, H-30), 0.7 (1 H, s, H-19), 0.4 (1 H, s, H-19).

¹³ C-NMR (100 MHz, D ₂ O) d 176.1 (s, C-6”), 175.7 (s, C-6’”), 105.5 (d, C-1’), 102.5 (d, C- 1”), 96.8 (d, C-1”’), 89.3 (d, C-3), 87.9 (s, C-20), 81.6 (d, C-24), 80.3 (d, C-6), 79.7 (s, C- 25), 76.6 (d, C-5”), 76.3 (d, C-5’”), 76.1 (d, C-3’), 75.9 (d, C-3”, C-3’”), 73.8 (d, C-16), 73.7 (d, C-2’”), 73.6 (d, C-2’), 73.1 (d, C-2”), 71.8 (d, C-4”, C-4’”), 69.4 (d, C-4’), 65.1 (t, C-5’), 57.0 (d, C-17), 51.6 (d, C-5), 46.3 (d, C-8), 45.6 (s, C-13), 44.8 (s, C-14), 44.2 (t, C- 15), 41.5 (s, C-4), 34.6 (t, C-7), 34.3 (t, C-22), 32.9 (t, C-12), 31.9 (t, C-1), ), 30.7 (t, C-19), 29.2 (t, C-2), 29.1 (s, C-10), 27.4 (q, C-28), 27.3 (q, C-21), 25.7 (t, C-11), 25.6 (t, C-23), 21.2 (q, C-18), 20.4 (s, C-9), 24.7 (q, C-26), 21.5 (q, C-27), 19.3 (q, C-30), 15.7 (q, C-29).

2. Synthesis of Dodecylamine Conjugated AST VII (DAC-AST VII) (3) (Figure 2):

0.01-1 mmol DC-AST VII (2) (preferably 0.05 mmol) was dissolved in 3-5 mL (preferably 5 mL) pyridine. DIPEA 0.1-2 mmol (preferably 0.2 mmol), HOBt 0.1-2 mmol (preferably 0.1 mmol), EDC 0.05-3 mmol (preferably 0.15 mmol) were added and the reaction medium was mixed at room temperature for 1-2 hours (preferably 1 hour). After 1 hour, dodecylamine 0.01-2 mmol (preferably 0, 123 mmol) was added dropwise and, the temperature of the reaction was increased to 60 °C. The progress of the reaction that continues for 4-10 hours (preferably 6 hours) under reflux is tracked with thin layer chromatography. The reaction mixture was quenched with excess amount of water and, liquid-liquid extraction was carried out with 25 mL ethyl acetate (EtOAc) in 3 times. EtOAc phase was evaporated in rotary evaporator at 50°C. EtOAc phase was purified with Sephadex (100% MeOH) column and then silica gel (30 g) column chromatography with 80:20:2 (CHCI ₃ : MeOH: Water) system, respectively. DAC-AST VII (3) was obtained with a yield of 10-40%. The chemical structure of DAC-AST VII (3) was elucidated using mass spectroscopy and NMR. HR-ESI-MS m/z 1331.89196 ([M+Na] ⁺ ) C ₇₁ H ₁₂₄ O ₁₉ Na =1331.88.

¹ H-NMR (400 MHz, d5) d 8.13 (1H, t, J = 6.2 Hz, H- 1 ^V ), 8.09 (1H, t, J= 6.1 Hz, H-1 ^1V ), 5.12 (1H, d,J= 7.8 Hz, H-1’”), 4.99 (1H, s, H-16), 4.95 (1H, d, J= 7.8 Hz, H-1”), 4.86 (1H, d, J = 7.4 Hz, H-1’), 4.36 (1H, dd, J = 11.0, 4.7 Hz, H-5’), 4.35 (1H, s, H-5”), 4.35 (1H, d, J= 5.3 Hz, H-5”’), 4.26 (2H, dd, J = 7.8, 4.7 Hz, H-3”, H-4”), 4.26 (1H, m, H-2’”), 4.22 (1H, m, H-4’), 4.21 (2H, d, J= 4.9 Hz, H-3”’, H-4’”), 4.15 (1H, t, H-3’), 4.04 (2H, m, H-2’, H-2”), 3.95 (1H, dd, J = 8.5, 6.1 Hz, H-24), 3.83 (1H, m, H-6), 3.72 (1 H, d,J= 10.7 Hz, H-5’), 3.64 (2H, m, H-2 ^V ), 3.53 (1H, dd, J = 11.8, 4.4 Hz, H-3), 3.46 (2H, m, H-2 ^1V ), 2.76 (1H, t, J = 9.9 Hz, H-22),2.53 (1H, m, H-17), 2.39 (1H, d,J= 12.4 Hz, H-11), 2.34 (1H, s, H-23), 2.26 (1H, dd, J = 12.3, 7.9 Hz, H-15),2.15 (1H, dd, J = 8.5, 3.5 Hz, H-7), 2.01 (1H, s, H-8), 1.99 (3H, s, H-28), 1.95 (1H, m, H-23), 1.95 (1H, s, H-11), 1.88 (1H, d, J= 8.8, 4.4 Hz, H-5), 1.84 (1H, d, J = 10.1, H-7), 1.80 (1H, m, H-1), 1.78 (1H, d, J= 6.4 Hz, H-15), 1.74 (1H, d, J= 7.3 Hz, H- 12), 1.73 (1H, m, H-2), 1.65 (1 H, s, H-22), 1.64 (3H, s, H-26), 1.62-1.82 (36H, m, H-3 ^1V -11 ^V , H-3 ^V - 11 ^V ), 1.56 (2H, d, J= 12.1 Hz, H-1, H-12), 1.44 (3H, s, H-27), 1.42 (3H, s, H-18), 1.37 (3H, s, H-29), 1.32 (3H, s, H-21), 1.29 (4H, m, H-12 ^1V , H-12 ^V ), 1.26 (1H, m, H-2), 1.12 (3H, s, H-30), 0.88 (6H, s, H-13 ^1V , H-13 ^V ), 0.61 (1H, d, J= 3.9 Hz, H-19), 0.25 (1H, d, J= 4.1 Hz, H- 19).

¹³ C-NMR (100 MHz, d5) d 171.5 (s, C-6”), 171.4 (s, C-6’”), 108.2 (d, C-1’), 105.2 (d, C- 1”), 99.1 (d, C-1’”), 88.9 (d, C-3), 87.7 (s, C-20), 82.5 (d, C-24), 79.7 (s, C-25), 79.5 (d, C- 6), 79 (d, C-3’), 78.9 (d, C-3”, C-2’”), 78.3 (d, C-3’”), 76.6 (d, C-5”), 76.4 (d, C-5’”), 75.4 (d, C-2”), 74.9 (d, C-2’), 74.4 (d, C-4’”), 74.2 (d, C-4”), 74.1 (d, C-16), 71.8 (d, C-4’), 67.6 (t, C-5’), 58.6 (d, C-17), 52.9 (d, C-5), 46.8 (s, C-13), 46.7 (t, C-15), 45.9 (d, C-8), 45.9 (s, C- 14), 43.2 (s, C-4), 39.8 (t, C-2 ^V ), 35.7 (t, C-22), 34.7 (t, C-7), 33.9 (t, C-12), 32.7 (t, C-1,C- 12 ^1V ,C-12 ^V ), 30.2 (t, C-2), 30-31 (t, C-3 ^V - C-11 ^V ), 29.5 (d, C-10), 28.9 (t, C-19), 28.2 (q, C- 28), 27.8 (q, C-21), 27.8-31 (t, C-3 ^1V - C11 ^1V ), 26.7 (t, C-11), 26.5 (t, C-23), 25.4 (q, C-26), 23.5 (q, C-27), 21.6 (q, C-18), 21.5 (s, C-9), 20.6 (q, C-30), 17.1 (q, C-29), 14.6 (q, C- 13 ^1V , 13 ^V ) .

3. Screening Studies for Biological Activity :

The first ever screening study regarding the immunomodulator effects of AST VII (1) and semi-synthesis products has been carried out by searching the cytokine release in human whole blood. 1 :20 diluted heparinized human blood with RPMI 1640 media was cultured in 24 well plate and, stimulated with PMA (50 ng/ml)-ionomycin (400 ng/ml). AST VII (1), DC- AST VII (2) and DAC-AST VII (3) molecules were treated with cells at the doses of 2, 4, 8, 16, 32 mg/ml and, culture plates were incubated for 48 hours. The supernatants from each well was collected and IL-2, IFN-g, IL-Ib, TNF-a, IL-17A and IL-4 cytokine titres were determined via ELISA method. The results were evaluated according to two different trial sets. In the first trial results where PMA-ionomycin stimulation was insufficient, AST VII and analogs induced IL-Ib response. Based on the most effective concentration, AST VII (4 mg/mL) 2.24 fold (p<0.01), DC-AST VII (8 mg/mL) 2.52 fold (p<0.01) and DAC-AST VII (16 mg/mL) 3.32 fold (p<0.001) increased IL-Ib titre in comparison to PMA-ionomycin. When we look at IL-Ib release of molecules at the concentrations of 4 mg/mL, a difference was not observed between AST VII and DC-AST VII, whereas DAC-AST VII, was more active compared to AST VII (Figure 3). In the first trial set, as AST VII at each concentrations induced IL-17A secretion, it increased IL-17A titres at 2 mg/mL 5.05 fold (p<0.01), DC-AST VII 2 mg/mL 5.69 fold (p<0.05), DAC-AST VII 16 mg/mL 3.15 fold (p<0.05) compared to PMA-ionomycin (Figure 4). In the second trial, following the stimulation of whole blood cell with PMA-ionomycin and DAC-AST VII (16 mg/mL) increased IL-Ib cytokine response 4.2 fold more than PMA-ionomycin (p<0.001) (Figure 5). In the second trial, IL-2, IFN-g and TNF-a cytokine responses were inhibited following compound treatments compared to PMA-ionomycin (Figure 6). The inhibitions caused by molecule treatments according to PMA-ionomycin were: AST VII (32 mg/mL) 2.16 fold (p<0.001), DC-AST VII (32 mg/mL) 3.82 fold (p<0.001), DAC-AST VII (16 mg/mL) 3.67 fold (p<0.001) for IL-2; AST VII (32 mg/mL) 1.25 fold (p<0.05), DC-AST VII (32 mg/mL) 1.63 fold (p<0.01), DAC-AST VII (32 mg/mL) 1.36 fold (p<0.01) for IFN-g; AST VII (32 mg/mL) 4.21 fold (p<0.001), DC-AST VII (32 mg/mL) 8.46 fold (p<0.001), DAC-AST VII (32 mg/mL) 2.75 fold (p<0.001) for TNF-a, respectively. The obtained cytokine responses showed that AST VII and its analogs triggered the pro-inflammatory and Thl7 mediated immune responses.

The high IL-Ib induction obtained in the human whole blood assay, makes us think that AST VII and its analogs can be effective on innate immune cells. The bone marrow collected from mice were differentiated into dendritic cells and macrophages by means of GM-CSF and M- CSF and, molecule treatment has been carried out on these cells and the IL-Ib secretions of these cells have been examined. AST VII and its analogs (0.5, 2.5 and 10 mM) were co- stimulated with lipopolysaccharide (LPS) to bone marrow derived dendritic cells (BMDC) and bone marrow derived macrophages (BMDM) and, IL-Ib concentration in the supernatant was determined by ELISA. At the concentrations of 0.5 mM, in BMDCs, AST VII (11 fold) (p<0.01), DC-AST VII (7.2 fold) (p<0.001) and DAC-AST VII (8.2 fold) (p<0.01) increased the release of IL-Ib compared to LPS (Figure 7). In BMDMs, treatment of AST VII (0.5 pM), DC-AST VII (10 pM) in comparison to LPS increased IL-Ib secretions 2.5 fold (p<0.05) and 3 fold (p<0.01), respectively (Figure 8). Response has not been obtained in BMDMs for DAC-AST VII.

Dendritic cells as potent antigen presenting cells ensure the activation and differentiation of naive T cells against specific antigen at lymph node. Following the internalization of antigen, the immature dendritic cells become mature and they express markers such as MHC II, CD86, CD80. These markers strengthen the response and the activation of T cells that are antigen specific. Within this scope, the effect of AST VII and its analogs on dendritic cell maturation and activation has been investigated. BMDCs were added into a 96 well cell culture plate and then, AST VII, DC-AST VII and DAC-AST VII were treated at the concentrations of 2, 5, 10 pM. After 24 hours, the supernatants for the production of IL-12 by ELISA and, the cell pellet in terms of the expression of MHC II, CD86 and CD80 markers using flow cytometry were analyzed. AST VII alone did not cause an induction/inhibition in terms of IL-12 production and expression of MHC II, CD86, CD80 markers in BMDCs. Based on the IL-Ib results obtained by co-stimulation of LPS with saponin compounds, AST VII was treated with LPS for maturation of BMDCs. Following co-stimulation with LPS, AST VII at the concentration of 10 pM increased the expressions of MHC II (1.17 fold, p<0.5), CD86 (1.44 fold, p<0.001), CD80 (1.1 fold, p<0.5) markers on dendritic cells and IL-12 release (1.1 fold, p<0.01) compared to LPS (Figure 9). While DC-AST VII and DAC-AST VII did not make a difference in MHC II expression without an LPS treatment, CD86 and CD80 expressions were induced as follows: for DC-AST VII at the concentration of 5 mg/mL 1.31 fold CD86, at the concentration of 5 mg/mL 1.24 fold CD80 (p<0.05); for DAC-AST VII at the concentration of 2 mg/mL 1.15 fold CD86 (p<0.05), at the concentration of 10 mg/mL 1.14 fold CD80 (p< 0.01) (Figure 10).

T cell activation, proliferation and polarization could be investigated in co-culture systems that enable direct evaluation of the effect of adjuvants on antigen presentation. Within this scope, studies are carried out on the mixed leukocyte reaction (MLR) in vitro model. Bone marrow isolated from Balb/c mice is differentiated into bone marrow derived dendritic cells with GM-CSF. BMDCs were treated with LPS (10 ng/mL), AST VII (5 pM), DAC-AST VII (10 mM), DC-AST VII (10 mM) and, left to incubate for 24 hours. The next day, naive CD4 ⁺ and CD8 ⁺ T cells were isolated from spleens of C57BL/6 mice by negative selection kits. Naive T cells were added onto BMDCs in a ratio of 1 :5 as BMDC: T cells and, incubated for 3 days. CD4 ⁺ and CD8 ⁺ T cell activation was evaluated by looking for the expression of CD44 marker on the cell surfaces. In CD8 ⁺ T cells, AST VII (5 mM) 1.29 fold, LPS+AST VII (5 mM) 1.26 fold (p<0.05), DAC-AST VII (10 mM) 1.49 fold (p<0.05), DC-AST VII (10 mM) 1.26 fold increased the expression of CD44 in comparison to negative control (group treated with only medium). While AST VII alone created a similar response with LPS in CD8 ⁺ T cells, an increase was not observed in treatment carried out with LPS. One of the analogs, DAC-AST VII, was more active than DC-AST VII in CD8 ⁺ T cell response. In CD4 ⁺ T cells, AST VII (5 mM) 1.87 fold, LPS+AST VII (5 mM) 2.37 fold (p<0.01), DAC-AST VII (10 mM) 1.50 fold (p<0.05), DC-AST VII (10 mM) 2.6 fold (p<0.05) increased CD44 expression in comparison to negative control (group treated with only the medium) (Figure 11). In CD4 ⁺ T cell response, co-treatment of AST VII with LPS increased CD44 expression in comparison to treatment of AST VII alone. It was revealed that one of AST VII analogs, DC-AST VII, was more active in comparison to DAC-AST VII in CD4 ⁺ T cell response.

Beside CD4 ⁺ T cells play a role in the activation of CD8 ⁺ T cells, they also help B cells for the production of antibodies. The activation of CD4 ⁺ and CD8 ⁺ T cells initiate a process that plays a role in clearance of the pathogens, infiltration of immune cells to site of infection and the production/release of cytokines, chemokines for induction of an immune response. The number of adjuvants that can induce the cellular and humoral activity of antigens, which are weak immunogens, are low. For this reason, AST VII and AST VII analogs demonstrating CD4 ⁺ /CD8 ⁺ T cell activation, can be used in vaccines for malaria, AIDS, tuberculosis, cancer, which need strong cellular immune response.

Cytotoxicity of DC-AST VII and DAC-AST VII molecules, has been evaluated using MTT reactive in HeLa (human cervical cancer cell line), DU145 (metastatic prostate cancer), MCF- 7 (Human breast adenosarcoma), A549 (Human lung carcinoma), MRC-5 (Human lung fibroblasts) and HCC-1937 (human primary ductal carcinoma) cells. While cytotoxicity was not observed even at the dose of 32 mM for the DC-AST VII molecule, IC ₅₀ values belonging to DAC-AST VII treatment were 11.1; 7.82; 9:42; 29.7; 12.6 and 16.7 mM for HeLa, DU145, MCF-7, A549, HCC-1937 and MRC-5 cells, respectively. The cytotoxicity exhibited by DAC-AST VII indicated that this molecule can be used both as a cytotoxic and immunomodulatory agent, especially in cancer immunotherapy.