Saponin derivatives that improve therapeutic window
A derivative and saponin technology, applied in the field of saponin derivatives, can solve the problems of limiting the therapeutic index to affect the therapeutic window, saponin cell rupture activity contributing to side effects, etc.
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Embodiment 1
[0471] Embodiment 1: the synthesis of saponin derivative
[0472] The following modified SO1861 saponins, saponin derivatives, were synthesized based on naturally occurring SO1861, as outlined in Table A2:
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[0477] Reference is made to the following description of the synthesis of SO1861 derivatives, with reference to Table A2 and accompanying figures.
[0478] Synthesis of SO1861-Ald-EMCH (molecule 2); see Figure 60 , Figure 61 A
[0479] SO1861 (59 mg, 31.7 μmol) from Saponaria and EMCH (301 mg, 888 μmol) were placed in a round flask with a stirrer and dissolved in 13 mL of methanol. TFA (400 μL, approx.) was added to the solution, and the reaction mixture was stirred on a RCT B magnetic stirrer (IKA Labortechnik) at 800 rpm and room temperature for 3 h. After stirring for 3 h, the mixture was diluted with MilliQ water or PBS and dialyzed extensively against MilliQ water or PBS for 24 h using regenerated cellulose memb...
Embodiment 2
[0586]Example 2: Activity of Saponin Derivatives - Preliminary Study
[0587] The saponin modifications described herein were found to substantially not interfere with the ability of a saponin to enhance endosome escape (modified saponin or saponin released from a conjugate free of endosomes). The experimental results are summarized in Table Ex2 below.
[0588] Chemically modified saponin SO1861 did show reactivity in cell-based bioassays, with relative cell viability as readout. HeLa cells were incubated with the following constructs for 72 hours, and cell viability was assessed before and after 72 hours of incubation. In the experiments, cells were exposed to 1.5 pM caryophyllin-EGF conjugate. Negative controls were cells incubated with buffered vehicle and 10 μg / ml saponin without caryophyllin-EGF. The cell viability of the control (in which saponin and EGF-caryophyllin were omitted) was set at 100%. The positive control was 10 μg / ml unmodified saponin SO1861+caryophyll...
Embodiment 3
[0592] Example 3: Activity of Saponin Derivatives - Detailed Study
[0593] Co-administration of various saponins (e.g. SO1861, QS-21) to cells as "free" unconjugated molecules together with ligand-toxin fusions (e.g. EGF-caryophyllin) or antibody-protein toxin conjugates resulted in target The cell killing activity of expressing cells is enhanced.
[0594] The present inventors chemically modified SO1861 (isolated and purified from the root extract of Saponaria officinalis) and QS21 (from Quillaja Saponaria, Desert King), thus providing a series of saponin derivatives listed in Table A2 and A3. Saponin derivatives were tested for 1) endosome escape-enhancing activity of the ligand toxin (modified SO1861 / QS21 titration + 5 pM EGF caryophyllin) on EGFR-expressing cells (HeLa and A431); 2) intrinsic cellularity against HeLa and A431 Toxicity (modified SO1861 / QS21 titration); and 3) human erythrocyte hemolytic activity (modified SO1861 / QS21 titration on human erythrocytes).
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