Immunomodulation
Two recent reports purport to show that PXD modifies the activity of both normal lymphocytes and leukaemic cells, with the suggestion that it might have therapeutic potential in the treatment of certain blood-related disorders including leukaemia and certain autoimmune diseases.
The first report is as follows.
Phenoxodiol, an anticancer isoflavene, induces immunomodulatory effects in vitro and in vivo.
Sylvianna G et al.
J Cell Mol Med (2009) Feb 9
Phenoxodiol (PXD) is a synthetic analogue of the plant isoflavone genistein with improved anticancer efficacy. Various properties and mechanisms of action have been attributed to the drug, the most important being its ability to sensitize resistant tumor cells to chemotherapy, which led to its fast track FDA approval for phase II/III clinical trials. In this study, we examined the effects of PXD on human peripheral blood mononuclear cells (PBMC) and its potential role in regulating immune responses. We show that PXD, at concentrations >/=1 mug/mL (4 muM), inhibited proliferation and reduced the viability of healthy donor-derived PBMC. In contrast, lower PXD concentrations (0.05-0.5 mug/mL) augmented, upon 3-day incubation, PBMC cytotoxicity. Experiments with purified CD56(+) lymphocytes revealed that PXD enhanced the lytic function of natural killer (NK) cells by directly stimulating this lymphocytic subpopulation. Furthermore, in an in vivo colon cancer model, Balb/C mice administered low dose PXD, exhibited significantly reduced tumor growth rates and prolonged survival (in 40% of the animals). Ex vivo results showed that PXD stimulated both NK and tumor-specific cell lytic activity. We conclude that PXD, when administered at low concentrations, can act as an immunomodulator, enhancing impaired immune responses, often seen in cancer-bearing individuals.
In this study, PXD was reported to:
(i)enhance the activity of human peripheral blood CD56+ cells (natural killer or NK cells), that play a role in tumor immunity; and,
(ii)enhance in mice the activity of both NK and tumor-specific cells against tumor cells.The suggestion then being that PXD has the potential to reverse the lowered immune responsiveness that can accompany late-stage cancer. The second report is as follows.
The anti-cancer drug, phenoxodiol, kills primary myeloid and lymphoid leukemic blasts and rapidly proliferating T cells
PM. Herst,et al.
Haematologica (2009) 94, 928-934
Background: The redox-active isoflavene anti-cancer drug, phenoxodiol, has previously been shown to inhibit plasma membrane electron transport and cell proliferation and promote apoptosis in a range of cancer cell lines and in anti-CD3/anti-CD28-activated murine splenocytes but not in non-transformed WI-38 cells and human umbilical vein endothelial cells. Design and Methods: We determined the effects of phenoxodiol on plasma membrane electron transport, MTT responses and viability of activated and resting human T cells. In addition, we evaluated the effect of phenoxodiol on the viability of leukemic cell lines and primary myeloid and lymphoid leukemic blasts.
Results: We demonstrated that phenoxodiol inhibited plasma membrane electron transport and cell proliferation (IC50 46 µM and 5.4 µM, respectively) and promoted apoptosis of rapidly proliferating human T cells but did not affect resting T cells. Phenoxodiol also induced apoptosis in T cells stimulated in HLA-mismatched allogeneic mixed lymphocyte reactions. Conversely, on-proliferating T cells in the mixed lymphocyte reaction remained viable and could be restimulated in a third party mixed lymphocyte reaction, in the absence of phenoxodiol. In addition, we emonstrated that leukemic blasts from patients with primary acute myeloid leukemia (n=22) and acute lymphocytic leukemia (n=8) were sensitive to phenoxodiol. The lymphocytic leukemic blasts were more sensitive than the myeloid leukemic blasts to 10 µM phenoxodiol exposure for 24h (viability of 23±4% and 64±5%, respectively, p=0.0002).
Conclusions: The ability of phenoxodiol to kill rapidly proliferating lymphocytes makes this drug a promising candidate for the treatment of pathologically-activated lymphocytes such as those in acute lymphoid leukemia, or diseases driven by T-cell proliferation such as auto-immune diseases and graft-versus-host disease.
In this study, PXD was reported to:
- inhibit the proliferation of activated T-lymphocytes; and
- be cytotoxic to blast cells from leukaemic patients.The suggestion then being that PXD is a promising drug candidate for the treatment of acute lymphoid leukaemia and certain autoimmune disorders.
As positive as this news sounds, I would caution restraint. We are dealing with these two studies with in vitro results that may have little in common with the function of the drug in the whole body.
Any basic understanding of the biology of isoflavonoids in the body suggests that they are unlikely to show any significant biological activity in a haematological setting, which in plain English means that they are unlikely to work with diseases of the blood. The reason for this statement is that isoflavonoids are transported in blood in a way that makes them poorly available to work in blood. In the test-tube (as per the above studies), PXD has been added in purified form to cell cultures. That is an entirely artificial situation that does not mimic the dynamics of the drug in the body.
How steroids and isoflavonoids are transported in blood
Isoflavonoids, like their close chemical cousins, the steroidal hormones, are highly water-insoluble. In order for isoflavonoids and steroids to be transported
around the body (and ultimately to be eliminated from the body), they need to be made water-soluble. The body does this in two ways – either (a) by attaching them to a larger water-compatible compound (such as a large protein) where the overall mass of the protein is so large as to negate the water-insolubility of its much smaller passenger, or (b) by conjugating (chemically bonding) to a small compound such as a sugar (glucuronide) or a salt such as sulphate in which form it is water-soluble.
glucuronide + estrogen + water
The steroid or the isoflavonoid is only biologically active when it is released in its free form. When it is in its transportable form combined with a protein or as a conjugate, it is non-functional. Activation of the steroid or isoflavonoid requires it to be separated from its carrier molecule. In the case of protein-binding, the steroid molecule attaches itself either to albumin (the most common protein in blood) or to a protein known as sex hormone binding globulin, specifically designed for that task.
A characteristic of this protein transport system is that the chemical bond between the steroid and the protein is relatively weak, allowing the steroid to drop off readily whenever it encounters a target steroid receptor. In this way, steroids are able to perform their normal range of functions within the bloodstream despite their very poor water-solubility. In the case of the conjugation method (glucuronide or sulphate salt) of transport, the bond connecting the steroid to the glucuronide or sulphate is particularly strong and can only be broken by specific enzymes (glucuronidase and sulphatase) that cleave off the salt, freeing the steroid.
These enzymes are widely distributed in the body, meaning that steroids carried in this way must first leave the blood and enter solid tissues before they can be activated. That means that their point of activity is limited to that tissue. The conjugated form appears to be a particularly important form of delivering steroids to specific tissues, as in the case of estrogens being delivered to reproductive tissues such as breast, ovary and prostate where the steroid receptors are located on the nuclear membrane of the cell.
The conjugated form also is more water-soluble than the protein-bound form, thus serving as the main way that steroids and
isoflavonoids are eliminated from the body.
Oral steroids and isoflavonoids – mainly glucuronidated.
Steroids in the normal adult body are predominantly (90-95%) transported in blood in the protein-bound form; the conjugated form accounts for only about 5-10%. The exception occurs when steroids are given orally, as in the case of the oral contraceptive pill. In that situation, the steroid in order to enter the bloodstream needs first to pass through the cells lining the gut wall. One of the roles of those cells is to ensure that chemicals arriving in the body from the outside world are sufficiently water-soluble before being passed onto the blood. The method used in the case of steroids is glucuronidation, and to a lesser extent, sulfation. Steroids, such as those in the oral contraceptive pill given orally, are passed into blood in the glucuronide and sulphate forms. Isoflavones present in foodstuffs are treated by the body in an identical fashion to that of orally-administered steroids. They are conjugated mainly as glucuronide and to a lesser extent as sulphate salts. In other words, isoflavones obtained through the diet are present in a form that requires conversion by enzymes within tissues to an active form, thereby largely precluding their ability to function within the bloodstream.
The Company already has reported that PXD is found in blood predominantly (about 90%) in the glucuronidated form, with the sulphated form accounting for most of the remainder. It also has shown and reported that both forms of conjugates are biologically inactive, and activation only occurs in the presence of a glucuronidase or sulphatase. It is entirely legitimate to conduct laboratory studies with PXD in its purified state for, say, prostate cancer studies, because when the drug reaches the prostate tumour in the body, the PXD is released by enzymatic action to its free state. But if blood lacks any such enzymatic activity, then any test-tube studies where blood-borne cells are the ultimate target needs to take account of that and use a PXD-glucuronide conjugate and not the free form of PXD. I have little doubt that that would deliver an entirely different outcome to that reported in the two studies above.
Therapeutic potential of conjugated isoflavonoids?
If isoflavonoid drugs such as PXD were ever to interact with blood-borne cells such as lymphocytes or leukaemic cells, then all the available evidence points to them needing to be bound in a form other than that of a glucuronide or sulphate conjugate.
It isn’t possible to rule out categorically any transport of isoflavones in blood in a protein-bound form, but I am unaware of any evidence to suggest that this happens, and if it does, then it would appear to be a very small proportion of drug.
There is nothing new about the ability of PXD to kill various forms of human leukaemic cells in the test-tube. This has been reported many times. It even led the Company some years ago to initiate two sighting studies of PXD (intravenous dosage form in one case, and the oral dosage form in the other) in patients with various forms of leukaemia. As already reported, both studies were terminated before enrolment was complete due to lack of any evidence of clinical response.
PXD is a remarkable drug with a considerable future in the treatment of human cancer. But all the available evidence points to its use being limited to solid cancers where the presence of glucuronidase and sulphatase enzymes ensures its activation.