Free nonparticulate saponins of various origin including QS fractions exert cytotoxic activity on cancer cells.7
However, QS interacts with cholesterol present in the cell membranes on both normal and cancer cells, thereby trapping the free non-particulate saponin at the site of administration,10
causing membrane damage, cell lysis, and subsequent necrosis resulting in a low therapeutic index. Possibly the free saponin binds more efficiently to cancer cells containing more cholesterol than normal cell, but that is not enough to give adequate therapeutic index to be of any clinical value.21
By formulating ASAP and DSAP with cholesterol into KGI and BBE nanoparticles respectively, the cell lytic toxicity is abolished. To note, no release of free ASAP or DSAP from the particles is recorded and the mode of action is connected to the particulate form. In contrast the concept for most particles intended for cancer treatment, eg, various polymer particles including PLG particles is to gradually release the free active components.11
The cancer cell-killing effect of ASAP11
is related to toxicity manifested by hemolysis, cell membrane damage causing leakage of TK, decreased cell metabolism, and necrosis. The release of intracellular compounds by cell membrane damage including both normal and cancer cells causes even severe toxic effects in vivo
Thus, the therapeutic effect of free QA like ASAP is limited and out of the question for cancer treatment. Notably, free ASAP at 25 μg/mL induced a more rapid cell death of normal cells, ie, at 30 minutes vs one hour for U937 cells.
ASAP in the KGI particulate form caused U937 cancer cell death at low concentrations; 30-fold lower than what was required to kill the normal human monocytes or DCs, ie, a high therapeutic index was achieved. The high therapeutic index is explained by several factors. Firstly, fast replicating cancer cells are generally more sensitive than normal cells being the concept for cytostatic drugs. Secondly, by taking the cancer cells out of the cell cycle the essential recruitment/replacement of sensitive cancer cells is abolished. Thirdly, by guiding the cancer cells to differentiation manifested by, eg, IL-8, leads eventually to programmed cell death. All together, those are factors that result in the evasion of side effects. The different modes of retarding the cancer cell growth by KGI and BBE from that of cytostatic drugs predispose for synergistic effects as demonstrated between docetaxel/KGI and fludarabine/BBE (). KGI has in vivo studies shown high bioavailability after fast transport from the site of injection. In contrast the free form, being lytic, interacts with cell membranes causing local reactions and reduced bioavailability. KGI caused a late cell death by apoptosis requiring 12 hours or more to be observed. No reversal to cell replication was noticed during 12 days of culture even when KGI was removed after three days. The lytic effect of ASAP on red blood cells and nucleated cells was virtually abolished when formulated to KGI particles, and the cell death was confined to apoptosis. The apoptotic effect of KGI was preceded by exit from the cell cycle, G1 arrest (as shown in ) followed by activation and differentiation observed as production of IL-8. KGI as well as BBE cause maturation effects on immature normal human dendritic cells with downregulation of CD14, production of a number of pro-inflammatory cytokines, eg, IL-12, and expression of CD80, CD83, and CD86 (data not shown), which reinforces the concept that KGI and BBE stimulate cancer cells to apoptosis like they do with normal cells by cell differentiation rather than a cytostatic effect.
Different methods were used to measure apoptosis (annexin V staining, apoptag, and Hoechst 33342 staining, DNA fragmentation and caspase 3/7 activation). All methods clearly demonstrate that KGI induces apoptosis. The annexin V staining reveals that KGI induces apoptosis and no necrosis is observed in the early phase. Over time the number of necrotic cells increases as shown with PI double staining with annexin V. The explanation is that apoptotic cells in vitro over time undergo necrosis while in vivo phagocytosed. Higher concentrations of KGI induced apoptosis earlier than lower concentrations. Apoptotic cells are prone to necrosis and become, therefore, stained by PI over time (double stained with both PI and annexin V), leading to reduction of the proportion of annexin V single positive cells (). In addition, the killing activity of KGI was not confined to U937 cells but was also observed on other leukemia cell lines as reflected in .
DSAP formulated into BBE particles had no cytotoxic effect on the U937 cells, but blocked the cancer cell killing effect of KGI in a concentration dependent manner, approaching complete blocking in a BBE: KGI ratio of 10 to 1. This blocking indicates that BBE and KGI compete for one receptor and that KGI induced apoptosis probably requires a second receptor not targeted by DSAP and possibly related to the acyl-chain involving the terminal sugars arabinos and/or rhamnos. The indicated receptor activity has to be further elucidated in future studies.
KGI and BBE particles show high bioavailability and are virtually nontoxic, not causing local or systemic adverse effects in laboratory animals.11
A product similar to the KGI particle, ie, the ISCOM particle has been tested in more than 1000 humans as an adjuvant for vaccines and is in human phase III clinical trials for this indication.11
Thus, the combination of low toxicity, very high bioavailability, and high stability already observed in preclinical studies and in humans (Dr Karin Lövgren-Bengtsson, Isconova AB) indicates a favorable therapeutic index and the possibility of a rapid advancement to clinical testing in cancer.
In addition to the potential use of nanoparticulate QS as stand alone anticancer drug, there are prospects for synergy between KGI and BBE as well as a number of registered cancer drugs eg, docetaxel and fludarabine (). Besides direct pharmacodynamic interactions at the level of the tumor cell, the well documented adjuvant immunological effect of the QS particles11
might also augment the overall anticancer effect. Furthermore the KGI and BBE nanoparticles should also be explored as potential carriers for established anticancer drugs as they can be incorporated into the particles by various techniques24
as being successfully used commercially for delivery of vaccine antigens.11
There are other potential clinical applications of the particulate saponins. The nanoparticles may be targeted to the tumor cells by inserting tumor-targeting molecules, potentially increasing the selectivity of drug delivery, hence increasing the therapeutic index. This principle is illustrated by incorporating envelope proteins from respiratory viruses into particles, rendering enhanced delivery to the common mucosal immune system after intranasal mode of administration as described by Hu and colleagues.13
ISCOMs supplied with the DD portion of protein A of Staphylococcus aureus
are targeting B cells.11
KGI supplied with the DD portion should target B cell lymphoma. The use of monoclonal anti-bodies attached to the particles recognizing specific surface structures on tumor cells is a device for targeting.
In conclusion, QS saponin formulated into nanoparticles represents a potential new mechanistic category of anticancer drugs fundamentally different from standard cytotoxic drugs by being considerably more selective and also by stimulating differentiation. Furthermore, as drug carriers, the particles with inborn cancer cell-killing and immune-stimulating properties create interesting prospects for synergism with integrated standard anticancer compounds.